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1
Lasek, R. J., P. Paggi, and M. J. Katz. "Slow axonal transport mechanisms move neurofilaments relentlessly in mouse optic axons." Journal of Cell Biology 117, no. 3 (May 1, 1992): 607–16. http://dx.doi.org/10.1083/jcb.117.3.607.
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Pulse-labeling studies of slow axonal transport in many kinds of axons (spinal motor, sensory ganglion, oculomotor, hypoglossal, and olfactory) have led to the inference that axonal transport mechanisms move neurofilaments (NFs) unidirectionally as a single continuous kinetic population with a diversity of individual transport rates. One study in mouse optic axons (Nixon, R. A., and K. B. Logvinenko. 1986. J. Cell Biol. 102:647-659) has given rise to the different suggestion that a significant and distinct population of NFs may be entirely stationary within axons. In mouse optic axons, there are relatively few NFs and the NF proteins are more lightly labeled than other slowly transported slow component b (SCb) proteins (which, however, move faster than the NFs); thus, in mouse optic axons, the radiolabel of some of these faster-moving SCb proteins may confuse NF protein analyses that use one dimensional (1-D) SDS-PAGE, which separates proteins by size only. To test this possibility, we used a 2-mm "window" (at 3-5 mm from the posterior of the eye) to compare NF kinetics obtained by 1-D SDS-PAGE and by the higher resolution two-dimensional (2-D) isoelectric focusing/SDS-PAGE, which separates proteins both by their net charge and by their size. We found that 1-D SDS-PAGE is insufficient for definitive NF kinetics in the mouse optic system. By contrast, 2-D SDS-PAGE provides essentially pure NF kinetics, and these indicate that in the NF-poor mouse optic axons, most NFs advance as they do in other, NF-rich axons. In mice, greater than 97% of the radiolabeled NFs were distributed in a unimodal wave that moved at a continuum of rates, between 3.0 and 0.3 mm/d, and less than 0.1% of the NF population traveled at the very slowest rates of less than 0.005 mm/d. These results are inconsistent with the proposal (Nixon and Logvinenko, 1986) that 32% of the transported NFs remain within optic axons in an entirely stationary state. As has been found in other axons, the axonal transport system of mouse optic axons moves NFs and other cytoskeletal elements relentlessly from the cell body to the axon tip.
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Zhu, Zhongxin, Xuan Zhou, Yang Wang, Lisha Chi, Dandan Ruan, Yuanhu Xuan, Weitao Cong, and Litai Jin. "Fluorescent staining of glycoproteins in sodium dodecyl sulfate polyacrylamide gels by 4H-[1]-benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide." Analyst 139, no. 11 (2014): 2764–73. http://dx.doi.org/10.1039/c3an02309e.
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A sensitive, specific, economic and MS compatible staining method for gel-separated glycoproteins by using BH was described and demonstrated by 1-D and 2-D SDS-PAGE, deglycosylation, glycoprotein affinity enrichment and LC-MS/MS, respectively.
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Kaeswurm, Julia A. H., Bettina Nestl, Sven M. Richter, Max Emperle, and Maria Buchweitz. "Purification and Characterization of Recombinant Expressed Apple Allergen Mal d 1." Methods and Protocols 4, no. 1 (December 27, 2020): 3. http://dx.doi.org/10.3390/mps4010003.
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Mal d 1 is the primary apple allergen in northern Europe. To explain the differences in the allergenicity of apple varieties, it is essential to study its properties and interaction with other phytochemicals, which might modulate the allergenic potential. Therefore, an optimized production route followed by an unsophisticated purification step for Mal d 1 and respective mutants is desired to produce sufficient amounts. We describe a procedure for the transformation of the plasmid in competent E. coli cells, protein expression and rapid one-step purification. r-Mal d 1 with and without a polyhistidine-tag are purified by immobilized metal ion affinity chromatography (IMAC) and fast-protein liquid chromatography (FPLC) using a high-resolution anion-exchange column, respectively. Purity is estimated by SDS-PAGE using an image-processing program (Fiji). For both mutants an appropriate yield of r-Mal d 1 with purity higher than 85% is achieved. The allergen is characterized after tryptic in gel digestion by peptide analyses using HPLC-MS/MS. Secondary structure elements are calculated based on CD-spectroscopy and the negligible impact of the polyhistidine-tag on the folding is confirmed. The formation of dimers is proved by mass spectrometry and reduction by DTT prior to SDS-PAGE. Furthermore, the impact of the freeze and thawing process, freeze drying and storage on dimer formation is investigated.
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Lee, Jeongmin, Se-ah Jeon, and Sooyoung Lee. "Cross-reactivity of Can f 1 with Syrian hamster and Fel d 1 in children." Allergologia et Immunopathologia 49, no. 4 (July 1, 2021): 155–61. http://dx.doi.org/10.15586/aei.v49i4.206.
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Introduction and objectives: With increasing pet allergies among pediatric patients, the need for precise environmental care is increasing. We investigated the clinical, immunological, and environmental characteristics of pediatric patients sensitized to a dog to evaluate the cross-antigenicity of canine lipocalin Can f 1 with feline lipocalin Fel d 1 and Syrian hamsterextract.Materials and methods: The protein fractions of the processed and commercial Syrian hamster extracts were compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An enzyme-linked immunosorbent assay (ELISA) inhibition test was performed on Can f 1, Fel d 1, and processed Syrian hamster extract, and the antigen-specific immunoglobulin E (IgE)-binding capacity for each antigen was analyzed using serum samples from patients.Results: Twelve of 19 patients with a median age of 40.5 months were symptomatic when exposed to dogs. Eleven (91.7%) patients showed a positive IgE response to Can f 1. Two patients were positive for Fel d 1-specific IgE antibody, and one was positive for hamster-specific IgE antibody. SDS-PAGE confirmed the presence of different patterns of protein bandsbetween the commercial and processed hamster extracts. There was no cross-antigenicity among Can f 1, Fel d 1, and processed Syrian hamster extract.Conclusions: Since the standard commercial hamster extract did not contain Syrian hamster antigens that were diverse enough, caution should be taken when using it. In children allergic to cats and dogs, sensitization to isolated Can f 1 or Fel d 1 is unlikely to cause cross-reactivity to Syrian hamster hair and epithelium.
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Umar, Aisha, Anis Ali Shah, and Muhammad Tajammal Khan. "Phylogenetic analysis of forty Solanum melongena L. accessions by SDS-page." Bangladesh Journal of Botany 49, no. 1 (March 31, 2020): 75–84. http://dx.doi.org/10.3329/bjb.v49i1.49096.
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Work was carried out to resolve the existing intraspecific taxonomic relation and protein richest accessions of Solanum melongena by using SDS-PAGE with the reference of their genetic diversity. Phylogenetic relatedness within samples was studied by cluster analysis using an Unweighted Pair Group Method with Arithmetic Mean (UPGMA) to construct a dendrogram. Electrophoretogram of accessions No. (1-19) 018477, 018482 (Faisalabad), 18484 (Sahiwal), whereas accessions from 20 - 40 from D. I Khan (18504, 18500, 18505, 14466(3), Sahiwal (20344) and Batgram (20509) was unique in protein banding position. Largest dendrogram of cluster 1 divided into 6 (6a,b), 7 (7a,b), 8 (8a,b) and 9 (9a,b) sub clusters including accessions 20425 - 4745(3). The results demonstrated that accessions have low level of genetic diversity and almost similar protein contents. No relationship was found between genetic divergence and genetic status of the samples.
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Zhao, X. C., and P. J. Sharp. "An Improved 1-D SDS–PAGE Method for the Identification of Three Bread Wheat «Waxy» Proteins." Journal of Cereal Science 23, no. 2 (March 1996): 191–93. http://dx.doi.org/10.1006/jcrs.1996.0019.
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Trianah, Yeni. "Uji Aktivitas Lektin Biji Kebiul terhadap Kecepatan Penggumpalan Sel Darah Merah Manusia dalam Kondisi Patologis dan Implementasinya sebagai Modul Pembelajaran Kimia." PENDIPA Journal of Science Education 2, no. 2 (October 5, 2018): 214–21. http://dx.doi.org/10.33369/pendipa.2.3.214-221.
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ABSTRACT[Lectin activities of kebiul seeds to the red blood cell agglutination speed in pathological condition and its implementation as a chemical learning module]. The purposes of this research were to determine: 1) the relative moleculer mass protein that behaves as a lectin in the seed extract kebiul , 2) the velocity of red blood cell clumping influenced by seed lectin kebiul, 3) know the difference student results on protein taught modules and a without modules taught at the College of Teacher Training and Education Teachers Association of the Republic of Indonesia (STKIP-PGRI) Lubuklinggau. Extraction of seeds Kebiul carried out in the a cold buffer solution with pH 7.4 and plus 60% saturated ammonium sulfat (salting out method), and made in four concentrations, namely : 2%, 4%, 6% and 8%. Then tested the activity of seed lectin kebiul to speed clotting of human red blood cells in pathological conditions. To determine the relative molecular mass protein that behaves as a lectin in the seed extract kebiul SDS PAGE electrophoresis performed 1-D. The experiment were then implemented on the material of protein biochemistry using modules. The results of the research showed that on the concentration of 8% the velocity of the clumping of a human red blood cells hypertension most of the highest, the relative molecular mass which behaves as a lectin protein electrophoresis results of 1-D SDS PAGE obtained by a three protein bands in the range of moleculer weights 80, 128 and 144 kDa. The Results of the implementation of the experimental class showed an average `post test value was 95 and the post test control class 69.41. There are differences in students' learning about protein for students who are taught by module and who are taught without module.Keywords: Lectin; agglutination; blood; 1-D SDS PAGE; learning outcomes.
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Andersen, D. O., B. K. Murray, R. K. Robins, and J. A. North. "In vitro Antiviral Activity of Ribavirin against Picornaviruses." Antiviral Chemistry and Chemotherapy 3, no. 6 (December 1992): 361–70. http://dx.doi.org/10.1177/095632029200300606.
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The in vitro antiviral activity of ribavirin (Rby; 1-β-D-ribof uranosyl-1,2,4-triazole-3-carboxamide) against selected members of the Picornaviridae is described. When antiviral activity was determined by reduction of infectious virus progeny, Rbv was active against human rhinovirus type 2 (HRV-2) and poliovirus type 1 (Polio-1) in both a drug concentration and multiplicity of infection-(MOI) dependent response. However, an antiviral activity rating assay based on the reductions of cellular cytopathic effects (CPE) indicated that Rbv was active against human rhinoviruses, but was less active against polioviruses. Prophylactic administration of Rbv significantly improved virus yield reduction, especially in Polio-1. SDS Polyacrylamide gel electrophoresis (SDS-PAGE) of HRV-2- and Polio-1-infected cell lysates demonstrated that Rbv inhibited the synthesis of viral-specific proteins. Although actinomycin D (Act D) did not significantly influence Picornavirus yields, when added concomitantly with Rbv, Act D reversed Rbv's antiviral effect.
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Filho, Edivaldo Ximenes Ferreira. "Purification and characterization of a β-glucosidase from solid-state cultures ofHumicola griseavar.thermoidea." Canadian Journal of Microbiology 42, no. 1 (January 1, 1996): 1–5. http://dx.doi.org/10.1139/m96-001.
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The thermophilic fungus Humicola grisea var. thermoidea produced β-glucosidase activity when grown in a solid-state culture on wheat bran as carbon source. A β-glucosidase was purified to apparent homogeneity by ultrafiltration, gel filtration chromatography on Sephacryl S-100, and ion-exchange chromatography on S-Sepharose, as judged by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) on a 12.5% (w/v) slab gel. The enzyme had a molecular mass of 82 and 156 kDa, as estimated by SDS–PAGE and gel filtration on a high performance liquid chromatographic column, respectively, suggesting that the native enzyme may consist of two identical subunits. The purified enzyme was thermostable at 60 °C for 1 h with a half-life of 15 min at 65 °C, and displayed optimum activity at 60 °C and a pH range of 4.0–4.5. The Kmand Vmaxvalues for p-nitrophenyl β-D-glucopyranoside were determined to be 0.316 mM and 0.459 IU∙mL−1, respectively. D-Glucose, D-gluconic acid lactone, Hg2+, Cu2+, and Mn2+inhibited β-glucosidase activity. The enzyme activity was competitively inhibited by D-glucose (Ki = 0.6 mM). The purified enzyme was very active against cellobiose and p-nitrophenyl β-D-glucopyranoside.Key words: Humicola, β-glucosidase, purification, characterization.
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Dodds, Karen L., Malcolm B. Perry, and Ian J. McDonald. "Alterations in lipopolysaccharide produced by chemostat-grown Escherichia coli O157:H7 as a function of growth rate and growth-limiting nutrient." Canadian Journal of Microbiology 33, no. 5 (May 1, 1987): 452–58. http://dx.doi.org/10.1139/m87-075.
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Escherichia coli O157:H7 was grown in chemostats as continuous cultures at different controlled growth rates and under different nutrient limitations to determine the effects on lipopolysaccharide (LPS) structure. LPS from whole cells and extracted using the hot aqueous phenol method was examined by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) and by gel filtration after hydrolysis with acetic acid. At low growth rates under glucose limitation (D = 0.1 h−1, doubling time (td), approx. 416 min; or D = 0.4 h−1, td, approx. 104 min), E. coli O157 produced high molecular weight LPS identical to that previously characterized from cells grown in batch culture. At a high growth rate (D = 0.8 h−1, td, approx. 52 min), the ratio of high molecular weight LPS to low molecular weight LPS produced greatly decreased. A small amount of high molecular weight LPS, containing O-polysaccharide which lacked amino sugars, and which thus was chemically different from that previously characterized, was produced by the cells at high growth rates. The predominant form of LPS from these cells was of slightly higher molecular weight than rough LPS, probably S–R LPS, and it consistently formed aggregates on SDS–PAGE. This form of LPS was also predominant when E. coli O157 was grown under Mg2+ limitation at an intermediate growth rate (D = 0.4 h−1, td, approx. 104 min).
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Vujanovic, V., S. Vidovic, M. R. Fernandez, P. Daida, and D. Korber. "Whole-cell protein and ITS rDNA profiles as diagnostic tools to discriminate Fusarium avenaceum intraspecific variability and associated virulence." Canadian Journal of Microbiology 55, no. 2 (February 2009): 117–25. http://dx.doi.org/10.1139/w08-103.
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A total of 91 isolates of Fusarium avenaceum were regrouped into 15 phenotypes and 10 vegetative compatibility groups showing specific one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (1-D SDS–PAGE) protein profiles and less-specific internal transcribed spacer rDNA profiles. Each isolate possessed reproducible signature protein bands. Indeed, the unweighted pair group method with arithmetic averages clustering revealed that the protein profile of each group of isolates correlated with fungus virulence. The use of SDS–PAGE offers a simple and sensitive technique for routine differentiation between pathogenic and nonpathogenic isolates within unknown F. avenaceum populations. The discovery has significant implications for risk assessment of cereal yield to ensure food and feed safety. This low-cost approach has the potential to be optimized and extended to a broad spectrum of Fusarium head blight pathogens.
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Yoshida, T., T. Inoue, and E. Ichishima. "1,2-α-d-mannosidase from Penicillium citrinum: molecular and enzymic properties of two isoenzymes." Biochemical Journal 290, no. 2 (March 1, 1993): 349–54. http://dx.doi.org/10.1042/bj2900349.
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Two isoforms of acidic 1,2-alpha-D-mannosidases have been isolated from culture filtrate of Penicillium citrinum. The pI values of the two forms, designated 1,2-alpha-mannosidase Ia and Ib, were 4.6 and 4.7 respectively. Isoenzymes Ia and Ib exhibited the same molecular mass which was determined to be 53 kDa by SDS/PAGE and 54 kDa by gel-permeation chromatography. Enzymes Ia and Ib hydrolysed yeast mannan and 1,2-alpha-linked mannooligosaccharides, but did not hydrolyse p-nitrophenyl alpha-D-mannoside. The optimal pH for the hydrolysis of Man(alpha 1-->2)Man was 5.0 for both isoenzymes. Similar kinetic parameters were determined for the two forms. Activation energy was a little lower for Ia than Ib. There was little difference between the enzymes with regard to their performance at acidic or alkaline pH. The N-terminal amino acid sequences of the two enzymes were identical. Analysis of C-terminal peptides, which were prepared by tryptic digestion and anhydrotrypsin-agarose chromatography, showed that Ia and Ib had the same amino acid sequences in the C-terminal region. Tryptic digestion revealed a slight difference between the isoenzymes in the pattern of cleaved peptides on SDS/PAGE.
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Saito, Katsuichi, Kazuya Kondo, Ichiro Kojima, Atsushi Yokota, and Fusao Tomita. "Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2." Applied and Environmental Microbiology 66, no. 1 (January 1, 2000): 252–56. http://dx.doi.org/10.1128/aem.66.1.252-256.2000.
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ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).
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Rehman, A., N. Evans, M. C. Gianibelli, and R. J. Rose. "Allelic variations in high and low molecular weight glutenins at the Glu-Dt locus of Aegilops tauschii as a potential source for improving bread wheat quality." Australian Journal of Agricultural Research 59, no. 5 (2008): 399. http://dx.doi.org/10.1071/ar07229.
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Aegilops tauschii, the donor of the D genome of hexaploid wheat, is accepted as a major contributor of disease resistance and bread-making quality attributes in cultivated wheat. High molecular weight (HMW) glutenins have a significant effect on the bread-making qualities of cultivated wheat. A large range of allelic variation in 424 Ae. tauschii accessions at the Glu-D1t locus for both x- and y-type glutenin subunits as well as at Glu-D3t was observed with SDS-PAGE using the total endosperm protein fraction. Only 4 accessions revealed more than 2 bands on SDS-PAGE. Seventeen new allelic combinations of both x- and y-type glutenin subunits at GluD-1t and 30 new allelic profiles at Glu-D3 were detected. These combinations comprise some lines with x- or y-type null forms. RP-HPLC analysis of accession Aus 18882, which showed 5 bands when the total endosperm protein fraction was resolved on SDS-PAGE, revealed 2 x-types and 1 y-type subunit banding pattern. RP-HPLC of the gliadin fraction exhibited an omega gliadin-like subunit. SDS-PAGE processing of the gliadin-free fraction of the same accession still exhibited the gliadin-like protein. An N-terminal protein sequence of the first 7 amino acids of the slowest moving novel x-type band of accession Aus18882 indicated its uniqueness, as no entries were found to contain this internal sequence. Demonstration of novel allelic combinations at Glu-D1 and Glu-D3 loci implies the potential for exploiting Ae. tauschii to increase the genetic variability of hexaploid wheat, particularly for bread-making qualities.
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Pitson, S. M., R. J. Seviour, B. M. McDougall, J. R. Woodward, and B. A. Stone. "Purification and characterization of three extracellular (1→3)-β-d-glucan glucohydrolases from the filamentous fungus Acremonium persicinum." Biochemical Journal 308, no. 3 (June 15, 1995): 733–41. http://dx.doi.org/10.1042/bj3080733.
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Three (1-->3)-beta-D-glucanases (GNs) were isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. Homogeneity of the purified proteins was confirmed by SDS/PAGE, isoelectric focusing and N-terminal amino acid sequencing. All three GNs (GN I, II and III) are non-glycosylated, monomeric proteins with apparent molecular masses, estimated by SDS/PAGE, of 81, 85 and 89 kDa respectively. pI values for the three enzymes are 5.3, 5.1, and 4.4 respectively. The pH optimum for GN I is 6.5, and 5.0 for GN II and III. All three purified enzymes displayed stability over the pH range 4.5-10.0. Optimum activities for GN I, II and III were recorded at 65, 55 and 60 degrees C respectively, with both GN II and III having short-term stability up to 50 degrees C and GN I up to 55 degrees C. The purified GNs have high specificity for (1-->3)-beta-linkages and hydrolysed a range of (1-->3)-beta- and (1-->3)(1-->6)-beta-D-glucans, with laminarin from Laminaria digitata being the most rapidly hydrolysed substrate of those tested. K(m) values for GN I, II, and III against L. digitata laminarin were 0.1, 0.23 and 0.22 mg/ml respectively. D-Glucono-1,5-lactone does not inhibit any of the three GNs, some metals ions are mild inhibitors, and N-bromosuccinimide and KMnO4 are strong inhibitors. All three GNs acted in an exo-hydrolytic manner, determined by the release of alpha-glucose as the initial and major product of hydrolysis of (1-->3)-beta-D-glucans, and confirmed by viscometric analysis and the inability to cleave periodate-oxidized laminarin, and may be classified as (1-->3)-beta-D-glucan glucohydrolases (EC 3.2.1.58).
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Costas, M., D. D. Morgan, R. J. Owen, and D. R. Morgan. "Differentiation of strains ofHelicobacter pyloriby numerical analysis of 1-D SDS-PAGE protein patterns: Evidence for post-treatment recrudescence." Epidemiology and Infection 107, no. 3 (December 1991): 607–17. http://dx.doi.org/10.1017/s095026880004930x.
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SUMMARYTwenty-three pre- and post-treatment isolates ofHelicobacter pylorifrom the antral mucosa of eight patients with dyspepsia and gastritis were compared using 1-D SDS PAGE of proteins. The protein patterns were highly reproducible and were used as the basis for two numerical analyses. The first, based on the total protein patterns, showed that a number of the strains did not cluster with their respective patient set. This was thought to be due to differences in both mobility and intensity of proteins in the major band region. The second analysis, based on partial patterns, excluding the major band region (51–68 kDa), divided the clinical isolates into clearly defined groups corresponding to the patient sets. Although there was a degree of heterogeneity with respect to protein pattern between the pre- and post-treatment isolates of some patients, there was nonetheless clear evidence that each patient was harbouring strains of only a single type. These results suggested that patients were not being reinfected with a different strain but that there was recrudescence of the pre-treatment strain. Protein ‘fingerprints’ provided a precise and reproducible means of strain differentiation, and revealed that in each patient the same strain persisted after drug therapy even though there was marked patient-to-patient strain variation.
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Bloy, C., D. Blanchard, P. Lambin, D. Goossens, P. Rouger, C. Salmon, and JP Cartron. "Human monoclonal antibody against Rh(D) antigen: partial characterization of the Rh(D) polypeptide from human erythrocytes." Blood 69, no. 5 (May 1, 1987): 1491–97. http://dx.doi.org/10.1182/blood.v69.5.1491.1491.
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Abstract A human monoclonal anti-Rh(D) antibody produced by an Epstein-Barr virus (EBV)-transformed B-cell line (IgG1(lambda), clone H2D5D2) has been purified on protein A-Sepharose column and used for binding studies and immune precipitation of the blood group rhesus (Rh) antigens. Scatchard plot analyses show that the 125I-labeled antibody (iodo-gen procedure), binds to 1.09 X 10(5), 0.43 X 10(5), and 0.32 X 10(5) antigen sites on each D--/D--, R2R2 and R1R1 RBC, respectively, with an association constant of approximately 0.6 X 10(8) mol/L-1. Immune precipitation studies indicate also that the Rh(D) antigen of the Rh(D)-positive RBCs is carried by a 29 kd polypeptide as deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE). No material could be precipitated from Rh(D)-negative or Rhnull RBCs. These results indicate that the monoclonal and the polyclonal human anti-Rh(D) behave similarly. A sample (Blo., presumed genotype R2r or R0R2) showing an increased number of antigen sites (0.76 X 10(5)/cell) and a high binding constant (5.7 X 10(8) mol/L-1) was used, as well as D--/D-- RBCs, for further purification of the 29-kd component. Extraction by Triton X-100 (0.1% to 5%) of the immune complexes formed between the membrane-bound Rh(D) antigens and the monoclonal antibody as well as a direct quantitative estimate of the 29- kd component, suggest that the Rh(D) polypeptide is loosely bound to the skeleton, since less than or equal to 80% can be solubilized from the membrane. In similar conditions, glycophorin A showed a slight association with the Triton-insoluble residue, whereas glycophorin B was easily and completely extracted. In contrast, both the minor RBC sialoglycoproteins, glycophorin C and glycoprotein gamma, remained predominantly bound to the membrane skeleton. The purified Rh(D) polypeptide obtained from Blo. and D--/D-- RBCs by immunoprecipitation and preparative gel electrophoresis was homogenous as judged by SDS- PAGE. Amino acid composition indicated that the Rh(D) protein contained sulfhydryl groups which are essential for biological activity.
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Bloy, C., D. Blanchard, P. Lambin, D. Goossens, P. Rouger, C. Salmon, and JP Cartron. "Human monoclonal antibody against Rh(D) antigen: partial characterization of the Rh(D) polypeptide from human erythrocytes." Blood 69, no. 5 (May 1, 1987): 1491–97. http://dx.doi.org/10.1182/blood.v69.5.1491.bloodjournal6951491.
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A human monoclonal anti-Rh(D) antibody produced by an Epstein-Barr virus (EBV)-transformed B-cell line (IgG1(lambda), clone H2D5D2) has been purified on protein A-Sepharose column and used for binding studies and immune precipitation of the blood group rhesus (Rh) antigens. Scatchard plot analyses show that the 125I-labeled antibody (iodo-gen procedure), binds to 1.09 X 10(5), 0.43 X 10(5), and 0.32 X 10(5) antigen sites on each D--/D--, R2R2 and R1R1 RBC, respectively, with an association constant of approximately 0.6 X 10(8) mol/L-1. Immune precipitation studies indicate also that the Rh(D) antigen of the Rh(D)-positive RBCs is carried by a 29 kd polypeptide as deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE). No material could be precipitated from Rh(D)-negative or Rhnull RBCs. These results indicate that the monoclonal and the polyclonal human anti-Rh(D) behave similarly. A sample (Blo., presumed genotype R2r or R0R2) showing an increased number of antigen sites (0.76 X 10(5)/cell) and a high binding constant (5.7 X 10(8) mol/L-1) was used, as well as D--/D-- RBCs, for further purification of the 29-kd component. Extraction by Triton X-100 (0.1% to 5%) of the immune complexes formed between the membrane-bound Rh(D) antigens and the monoclonal antibody as well as a direct quantitative estimate of the 29- kd component, suggest that the Rh(D) polypeptide is loosely bound to the skeleton, since less than or equal to 80% can be solubilized from the membrane. In similar conditions, glycophorin A showed a slight association with the Triton-insoluble residue, whereas glycophorin B was easily and completely extracted. In contrast, both the minor RBC sialoglycoproteins, glycophorin C and glycoprotein gamma, remained predominantly bound to the membrane skeleton. The purified Rh(D) polypeptide obtained from Blo. and D--/D-- RBCs by immunoprecipitation and preparative gel electrophoresis was homogenous as judged by SDS- PAGE. Amino acid composition indicated that the Rh(D) protein contained sulfhydryl groups which are essential for biological activity.
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Yanagawa, S., H. Yokozeki, and K. Sato. "Origin of periodic acid-Schiff-reactive glycoprotein in human eccrine sweat." Journal of Applied Physiology 60, no. 5 (May 1, 1986): 1615–22. http://dx.doi.org/10.1152/jappl.1986.60.5.1615.
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To evaluate the possible involvement of ductal blockade with periodic acid-Schiff (PAS)-positive materials in the mechanism of hidromeiosis in humans, skin slices were incubated with methacholine for 2 h and PAS-positive materials localized histologically in the ductal lumen. In 20% of the glands complete ductal blockade with PAS-positive materials was noted. The characteristics and origin of such PAS-positive glycoproteins in human sweat were then studied using various electrophoretic techniques. One-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis (1-D SDS-PAGE) demonstrated considerable individual variation in the electrophoretic pattern; however, four major bands at 45, 28, 20, and 18K shared by different individuals, were PAS positive. Further studies using two-dimensional SDS-PAGE, immunodiffusion and immunoaffinity chromatography demonstrated that the PAS-positive glycoproteins are not derived directly from serum because they are electrophoretically and antigenically distinct from serum proteins, including alpha 1-glycoprotein, alpha 2-HS-glycoprotein, and alpha 1-antitrypsin. Since only dark cell granules are densely stained in the histochemical PAS staining, and because antiserum produced against the PAS-positive band selectively stained cells facing the secretory coil lumen (which are most likely dark cells), it is suggested that PAS-positive sweat glycoproteins are derived predominantly from the dark cells.
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Cong, Wei-Tao, Sun-Young Hwang, Li-Tai Jin, and Jung-Kap Choi. "Sensitive fluorescent staining for proteomic analysis of proteins in 1-D and 2-D SDS-PAGE and its comparison with SYPRO Ruby by PMF." ELECTROPHORESIS 29, no. 21 (November 2008): 4304–15. http://dx.doi.org/10.1002/elps.200800150.
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Kottahachchi, D. U., T. R. Ariyaratne, and G. A. U. Jayasekera. "Mass Spectrometry Based Analysis of Erythrocyte Membrane Associated Proteins in Chronic Myeloid Leukemia Patients in Sri Lanka." International Letters of Chemistry, Physics and Astronomy 38 (September 2014): 74–86. http://dx.doi.org/10.18052/www.scipress.com/ilcpa.38.74.
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The research reported in this paper was conducted to analyze erythrocyte membrane associated proteins (ERMBPs) of some of chronic myeloid leukemia (CML) patients and selected individuals of Sri Lanka employing one dimensional sodium dodecyl sulphate poly acrylamide gel electrophoresis (1D-SDS-PAGE) combined with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI–TOF-MS). Erythrocyte membranes from blood were isolated by osmotic lysis, centrifugation and washings. ERMBPs were separated on 1D-SDS-PAGE, visualized by silver staining and the separated protein bands dissected out from the gel and were subjected to digestion by proteolytic enzyme, trypsin, and the resulting peptide mixture was analyzed by MALDI-TOF-MS. Resulting experimental peptide mass values were analyzed by peptide mass fingerprint (PMF) technique. From this analysis 10 ERMBPs including α and β spectrin, ankyrin, band 3, band 4.1, band 4.2, band 7, dematin, actin, 55 KDa erythrocyte membrane protein were identified accurately with their primary structure information. The study was able to provide some evidence for Cathepsin associated cleavage of Band 3 anion transport protein in CML patients reported previously. In addition we were able to detect changes in gel bands between healthy controls and CML patients around the area of 20 kDa in the 1 D-SDS-PAGE. It was identified as nuclear protein Dbf 4 related factor 1 isoform 2. Although erythrocytes are devoid of nuclei, such unexpected nuclear proteins have been identified in previous research. We were successful in identifying several human ERMBPs with available resources. As the identified proteins were known to be related to pathology of some of the hematological diseases, this methodology could be extended to detect the protein changes in erythrocyte membrane protein associated diseases. Therefore, this initial research would at some point lead to discovery of biomarkers to these hematological diseases in Sri Lanka.
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Filip, Ewa. "Composition of High Molecular Weight Glutenin Subunits in Polish Common Wheat Cultivars (Triticum aestivum L.)." Journal of Food Quality 2018 (2018): 1–8. http://dx.doi.org/10.1155/2018/2473420.
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The main goal of our study was to present research data on genes encoding high molecular weight glutenin subunits (HMW-GS) associated with high flour bread-making quality. This is the leading research objective in our institute in the area of wheat gluten in cultivars that have not been studied so far in that respect, but which can potentially be a valuable source of new information. Identification and characterization of high molecular weight glutenin subunits (HMW-GS) were performed using sequencing and SDS-PAGE and STS-PCR methods. Genes located in the vicinity of the Glu-1 locus have been identified and characterized in 28 Polish cultivars of Triticum aestivum. The results were then analyzed using the following computer programs: Finch TV, BLAST, MEGA 4, Molecular Imager® Gel Doc™ XR, and Quantity One software (Bio-Rad). Three alleles (a, b, c) have been identified in the Glu-A1 locus, 6 alleles (a, b, c, d, e, k) in the Glu-B1 locus, and 2 alleles (a, d) in Glu-D1 using the SDS-PAGE method. The amplification of specific HMW-GS sequences generated one product of 450 bp in 1Dx5 in 13 cultivars of old wheat and of 435 bp in 1Dx2 in 15 cultivars. The amplification products of primers for 1Dy10 and 1Dy12 genes were 422 bp and 552 bp in size, respectively.
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Matsushima, K., C. G. Larsen, G. C. DuBois, and J. J. Oppenheim. "Purification and characterization of a novel monocyte chemotactic and activating factor produced by a human myelomonocytic cell line." Journal of Experimental Medicine 169, no. 4 (April 1, 1989): 1485–90. http://dx.doi.org/10.1084/jem.169.4.1485.
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A novel basic heparin-binding monocyte chemotactic factor (MCF) was purified to homogeneity from the conditioned media of human myelomonocytic cell line THP-1 based on its in vitro monocyte chemotactic activity. The purified MCF was homogenous and estimated to be 15 kD on SDS-PAGE. Purified MCF stimulated normal human monocytes to be growth inhibitory in vitro at 2-3 d for several human tumor cell lines. This represents the first report of the identification and purification of a chemoattractant cytokine that also activates monocytes but is distinct from interferons and other known cytokines.
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MISTRY, Abinash Chandra, Shinji HONDA, and Shigehisa HIROSE. "Structure, properties and enhanced expression of galactose-binding C-type lectins in mucous cells of gills from freshwater Japanese eels (Anguilla japonica)." Biochemical Journal 360, no. 1 (November 8, 2001): 107–15. http://dx.doi.org/10.1042/bj3600107.
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Using a Japanese-eel (Anguilla japonica) gill cDNA subtraction library, two novel β-d-galactose-binding lectins were identified that belong to group VII of the animal C-type lectin family. The eel C-type lectins, termed eCL-1 and eCL-2, are simple lectins composed of 163 amino acid residues, including a 22-residue signal peptide for secretion and a single carbohydrate-recognition domain (CRD) of ∼ 130 residues typical of C-type lectins. The galactose specificity of the CRD was suggested by the presence of a QPD motif and confirmed by a competitive binding assay. Using Ruthenium Red staining, the lectins were shown to bind Ca2+ ions. SDS/PAGE showed that native eCL-1 and eCL-2have an SDS-resistant octameric structure (a tetramer of disulphide-linked dimers). Northern and Western blot analyses demonstrated high-level expression of eCL-1 and eCL-2 mRNAs and their protein products in gills from freshwater eels, which decreased markedly when the eels were transferred from freshwater to seawater. Immunohistochemistry showed that the eel lectins are localized in the exocrine mucous cells of the gill.
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Fujita, Akihiro, Akira Kawashima, Yuuki Mitsukawa, Noriaki Kitagawa, Hikaru Watanabe, Tetsuya Mori, Tomoyuki Nishimoto, Hajime Aga, and Shimpei Ushio. "Purification and characterization of cycloisomaltotetraose-forming glucanotransferases from Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006." Bioscience, Biotechnology, and Biochemistry 85, no. 3 (February 3, 2021): 600–610. http://dx.doi.org/10.1093/bbb/zbaa093.
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ABSTRACT Glucanotransferases that can synthesize cyclo-{→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→} (CI4) from dextran were purified to homogeneity from the culture supernatant of Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006. The molecular mass of both enzymes was estimated to be 86 kDa by SDS-PAGE. The glucanotransferase, named CI4-forming enzyme, from Agreia sp. exhibited the highest activity at pH 6.0 and 40 °C. The enzyme was stable on the pH range of 4.6-9.9 and up to 40 °C. On the other hand, the enzyme from M. trichothecenolyticum exhibited the highest activity at pH 5.7 and 40 °C. The enzyme was stable on the pH range of 5.0-6.9 and up to 35 °C. Both enzymes catalyzed 4 reactions, namely, intramolecular α-1,6-transglycosylation (cyclization), intermolecular α-1,6-transglycosylation, hydrolysis of CI4, and coupling reaction. Furthermore, the CI4-forming enzyme produced CI4 from α-1,6-linked glucan synthesized from starch by 6-α-glucosyltransferase. These findings will enable the production of CI4 from starch.
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Zhu, Hong, K. J. Cheng, and Cecil W. Forsberg. "A truncated β-xylosidase from the anaerobic fungus Neocallimastix patriciarum 27." Canadian Journal of Microbiology 40, no. 6 (June 1, 1994): 484–90. http://dx.doi.org/10.1139/m94-078.
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Two extracellular β-xylosidases, xylosidase I and II, were isolated from the ruminal fungus Neocallimastix patriciarum 27 after growth in a barley straw medium. Xylosidase I was purified 88-fold to apparent homogeneity by ion-exchange, affinity, and gel filtration chromatography. The purified xylosidase I had an isoelectric point (pI) of 4.7 and was a monomelic protein with a molecular mass of 39.5 kDa as estimated by both SDS-PAGE and gel filtration. Xylosidase II was partially purified to approximately 95% purity. Xylosidase II had the same pI (4.7) as xylosidase I, and appeared to be a dimeric enzyme composed of two polypeptides with molecular masses of 85 and 45 kDa, respectively, on SDS-PAGE. Peptide mapping of the three proteins suggested that xylosidase I was a truncated product originating from xylosidase II. Xylosidases I and II had similar pH optima of 6.0, but different temperature optima of 50 and 40 °C, respectively. The Km and Vmax for xylosidase I were 0.59 mM of p-nitrophenyl-β-D-xylopyranoside and 38.04 U∙mg protein−1, respectively, and those for xylosidase II were 0.13 mM and 8.9 U∙mg protein−1, respectively. Both enzymes hydrolysed pNPX and xylobiose with the production of xylose, but only xylosidase I exhibited activity toward p-nitrophenyl-α-L-arabinofuranoside.Key words: xylosidase, Neocallimastix, patriciarum, glycosidase.
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Journet, A., A. Chapel, S. Jehan, C. Adessi, H. Freeze, G. Klein, and J. Garin. "Characterization of Dictyostelium discoideum cathepsin D." Journal of Cell Science 112, no. 21 (November 1, 1999): 3833–43. http://dx.doi.org/10.1242/jcs.112.21.3833.
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Previous studies using magnetic purification of Dictyostelium discoideum endocytic vesicles led us to the identification of some major vesicle proteins. Using the same purification procedure, we have now focused our interest on a 44 kDa soluble vesicle protein. Microsequencing of internal peptides and subsequent cloning of the corresponding cDNA identified this protein as the Dictyostelium homolog of mammalian cathepsins D. The only glycosylation detected on Dictyostelium cathepsin D (CatD) is common antigen 1, a cluster of mannose 6-sulfate residues on N-linked oligosaccharide chains. CatD intracellular trafficking has been studied, showing the presence of the protein throughout the entire endocytic pathway. During the differentiation process, the catD gene presents a developmental regulation, which is also observed at the protein level. catD gene disruption does not alter significantly the cell behaviour, either in the vegetative form or the differentiation stage. However, modifications in the SDS-PAGE profiles of proteins bearing common antigen 1 were detected, when comparing parental and catD(-) cells. These modifications point to a possible role of CatD in the maturation of a few Dictyostelium lysosomal proteins.
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Harris, H., and S. E. Zalik. "Studies on the endogenous galactose-binding lectin during early development of the embryo of Xenopus laevis." Journal of Cell Science 79, no. 1 (November 1, 1985): 105–17. http://dx.doi.org/10.1242/jcs.79.1.105.
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Embryos of the frog Xenopus laevis at cleavage, blastula, gastrula and neurula stages contain a galactose-specific lectin. Extracts of gastrula embryos show the highest specific activity for this lectin compared to the other stages. Haemagglutinating activity of crude extracts is inhibited by lactose, alpha-galactose, beta-galactose, alpha Gal(1----4) beta Gal, beta Gal(1----3) alpha GalNAc, beta Gal(1----3) beta GlcNAc, beta Gal (1----4) beta GlcNAc, and most effectively by the disaccharide alpha Gal(1----3) beta Gal. Lectin from all stages was purified by absorption to galactose-linked immunoadsorbent or by affinity chromatography on a column of p-aminophenyl-beta-D-lactoside coupled to Sepharose 4B. In order to identify a single lectin band under reducing conditions in sodium dodecyl sulphate/polyacrylamide electrophoresis SDS/PAGE, it was necessary to treat aqueous suspensions of the purified lectin with chloroform/methanol (2:1, v/v). The lectin remained in the aqueous layer and gave rise on SDS/PAGE to a distinct band of 65 500 +/− 2780 molecular weight. Aqueous suspensions of the purified lectin that were not subjected to extraction with chloroform/methanol gave rise to several bands. Isoelectric focussing of the purified lectin resulted in two bands that separated at pI 4.3 and 4.5. In aqueous solution in the presence of lactose the chloroform/methanol-treated lectin appears to be an aggregate of apparent molecular weight of 375 000; the non-treated lectin under the same conditions has an apparent molecular weight of 490 000.
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Gupta, M. K., J. M. Jang, J. W. Jung, S. J. Uhm, K. P. Kim, and H. T. Lee. "173 SIMULTANEOUS QUANTIFICATION OF MULTIPLE PROTEINS IN PORCINE PARTHENOTES BY MULTIPLE SELECTED REACTION MONITORING." Reproduction, Fertility and Development 20, no. 1 (2008): 166. http://dx.doi.org/10.1071/rdv20n1ab173.
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Simultaneous quantification of multiple proteins could be a very useful tool in cell signaling studies. Parthenogentic embryos do not develop to term, although their in vitro developmental ability to the blastocyst stage and to form pluripotent stem cells upon culture is comparable to those of sperm- or somatic nucleus-introduced embryos. This study was designed to quantify the expression of eight selected proteins by multiple selected reaction monitoring (mSRM) of trypsinized preparations from porcine zygotes (n = 1500) produced by parthenogenesis. The trypsinized samples were prepared, spiked with isotopically labeled synthetic MAP3K (IPTGTV*HNQAK) peptides as internal standards for quantification (asterisk denotes isotopic amino acid residue), and analyzed by reverse phase LC-MS/MS. Upon the appearance of the target peptide, SRM data were acquired within fragmention mass �1.5 m/z and each SRM transition, with its respective retention time, was validated as indicative of each specific peptide. Data were processed by integrating the appropriate peaks for the native and internal standard peptide, followed by calculation of the ratio of peak areas to estimate the relative abundance of the peptide. Analysis of data showed that the relative abundance of Janus kinase 2 (JAK2), signal transducer and activator of transcription 1 (STAT1), STAT2, calpastatin, complement cytolysis inhibitor, plakoglobin, serotransferrin, and epsilon-globin were 203.72, 138.52, 60.50, 18.63, 31.23, 0.85, 10.33, and 0.12, respectively. These data were consistent with those observed by reverse phase LC-MS/MS combined with 1-dimensional (1-D) sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) of cellular lysates from porcine embryos (n = 6000). The data presented here, therefore, provide mSRM of embryos for the first time in any species and suggest that mSRM in combination with reverse-phase LC-MS/MS combined with 1-D SDS-PAGE could be a powerful tool for proteome analysis of porcine embryos to obtain vital new information for understanding the basic biology of embryonic development. This study was supported by grants from Biogreen 21, RDA, and the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea.
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Cruz Rodríguez, Alberto, Fabiola Anaid Sánchez Esperanza, Eduardo Pérez-Campos, María Teresa Hernández-Huerta, Laura Pérez-Campos Mayoral, Carlos Alberto Matias-Cervantes, Alexis Martínez Barras, et al. "Aggregation and Molecular Properties of β-Glucosidase Isoform II in Chayote (Sechium edule)." Molecules 25, no. 7 (April 8, 2020): 1699. http://dx.doi.org/10.3390/molecules25071699.
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The presence of isoforms of β-glucosidase has been reported in some grasses such as sorghum, rice and maize. This work aims to extract and characterize isoform II in β-glucosidase from S. edule. A crude extract was prepared without buffer solution and adjusted to pH 4.6. Contaminating proteins were precipitated at 4 °C for 24 h. The supernatant was purified by chromatography on carboxymethyl cellulose (CMC) column, molecular exclusion on Sephacryl S-200HR, and exchange anionic on QFF column. Electrophoretic analyzes revealed a purified enzyme with aggregating molecular complex on SDS-PAGE, Native-PAGE, and AU-PAGE. Twelve peptides fragments were identified by nano liquid chromatography-tandem mass spectrometry (nano LC-ESI-MS/MS), which presented as 61% identical to Cucurbita moschata β-glucosidase and 55.74% identical to β-glucosidase from Cucumis sativus, another Cucurbitaceous member. The relative masses which contained 39% hydrophobic amino acids ranged from 982.49 to 2,781.26. The enzyme showed a specificity to β-d-glucose with a Km of 4.59 mM, a Vmax value of 104.3 μM∙min−1 and a kcat of 10,087 μM∙min−1 using p-nitrophenyl-β-D-glucopyranoside. The presence of molecular aggregates can be attributed to non-polar amino acids. This property is not mediated by a β-glucosidase aggregating factor (BGAF) as in grasses (maize and sorghum). The role of these aggregates is discussed.
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Sitasiwi, Agung Janika, Wayan Tunas Artama, Agung Budiyanto, and Edy Dharmana. "MOLECULAR EXPRESSION OF WINGLESS-TYPE MMTV INTEGRATION SITE FAMILY MEMBER 4 GENE USING Escherichia coli BL21." Jurnal Kedokteran Hewan - Indonesian Journal of Veterinary Sciences 11, no. 1 (April 7, 2017): 11–14. http://dx.doi.org/10.21157/j.ked.hewan.v11i1.5891.
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This research was conducted to find out the Wnt4 recombinant proteins which expressed by Escherichia coli (E. coli) BL21 carrying the recombinant DNA wnt4 (E. coli transformation). Research materials were E. coli BL21 transformation and E. coli BL21 non-transformation (negative control). The expression of recombinant protein was conducted by culturing E. coli for 24 hours in Luria-Bertani (LB) media with isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. Recombinant protein was isolated by sonication of pellet bacteria. Protein analysis performed by 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that recombinant protein with a molecular weight of 33 kDa has been expressed by E. coli BL21 transformation successfully.
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Val, J., M. A. Gracia, A. Blanco, E. Monge, and M. Pérez. "Polypeptide Pattern of Apple Tissues Affected by Calcium-related Physiopathologies." Food Science and Technology International 12, no. 5 (October 2006): 417–21. http://dx.doi.org/10.1177/1082013206070217.
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Polypeptides from the apple pulp of Smoothee Golden Delicious and White Renete apples were resolved by 1-D denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). According to the electropherograms, there were lower concentrations of 88, 74, 70.6 and 47.5-42kDa proteins in bitter pit spots. Proteins weighing 30 and 26kDa were rare in sound pulp but frequently appeared in pits and adjacent tissue. Finally, a novel 18kDa protein was found in bitter pit spots in both varieties, and also in chemically induced corky lesions either by magnesium infiltration or ammonium oxalate cortical injections. The available data suggested that the novel protein might be an inhibitor of pectinmethylesterase, a small heat stress protein (smHSP) or a product of the Ypr-10 gene family identified with ‘Mal d 1’, the main allergen of apples. To elucidate the possible smHSP nature of the 18kDa, a set of apples were heated at 40°C for 20h, developing this protein in both the oxidised tissue and in the adjacent.
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Wang, Xiangzhu, Chanchan Chen, Ting Shen, and Jiangying Zhang. "Heterologous expression, purification and biochemical characterization of a glutamate racemase (MurI) from Streptococcus mutans UA159." PeerJ 7 (December 20, 2019): e8300. http://dx.doi.org/10.7717/peerj.8300.
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Background Glutamate racemase (MurI) is a cofactor-independent enzyme that is essential to the bacterial peptidoglycan biosynthesis pathway and has therefore been considered an attractive target for the development of antimicrobial drugs. While in our previous study the essentiality of the murI gene was shown in Streptococcus mutans, the primary aetiologic agent of human dental caries, studies on S. mutans MurI have not yet provided definitive results. This study aimed to produce and characterize the biochemical properties of the MurI from the S. mutans UA159 genome. Methods Structure characterization prediction and multiple sequence alignment were performed by bioinformatic analysis. Recombinant His6-tagged S. mutans MurI was overexpressed in the expression vector pColdII and further purified using a Ni2+ affinity chromatography method. Protein solubility, purity and aggregation state were analyzed by SDS–PAGE, Western blotting, native PAGE and SEC-HPLC. Kinetic parameters were assessed by a circular dichroism (CD) assay. Kinetic constants were calculated based on the curve fit for the Michaelis–Menten equation. The effects of temperature and pH on enzymatic activity were determined by a series of coupled enzyme reaction mixtures. Results The glutamate racemase gene from S. mutans UA159 was amplified by PCR, cloned and expressed in Escherichia coli BL21 (DE3). The 264-amino-acid protein, as a mixture of dimeric and monomeric enzymes, was purified to electrophoretic homogeneity. In the CD assay, S. mutans MurI displayed unique kinetic parameters (Km, d-Glu→l-Glu = 0.3631 ± 0.3205 mM, Vmax, d-Glu→l-Glu = 0.1963 ± 0.0361 mM min−1, kcat, d-Glu→l-Glu = 0.0306 ± 0.0065 s−1, kcat/Km, d-Glu→l-Glu = 0.0844 ± 0.0128 s−1 mM−1, with d-glutamate as substrate; Km, l-Glu→d-Glu = 0.8077 ± 0.5081 mM, Vmax, l-Glu→d-Glu = 0.2421 ± 0.0418 mM min−1, kcat, l-Glu→d-Glu = 0.0378 ± 0.0056 s−1, kcat/Km, l-Glu→d-Glu = 0.0468 ± 0.0176 s−1 mM−1, with l-glutamate as substrate). S. mutans MurI possessed an assay temperature optimum of 37.5 °C and its optimum pH was 8.0. Conclusion The findings of this study provide insight into the structure and biochemical traits of the glutamate racemase in S. mutans and supply a conceivable guideline for employing glutamate racemase in anti-caries drug design.
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Takeyama, Masahiro, Kana Sasai, Shoko Furukawa, Midori Shima, and Keiji Nogami. "Contribution of Factor VIII A2 Domain Residues 400–409 to a Factor X-Interactive Site in the Factor Xase Complex." Thrombosis and Haemostasis 118, no. 05 (April 3, 2018): 830–41. http://dx.doi.org/10.1055/s-0038-1637745.
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AbstractThe link between factor (F)VIII and FX is essential for optimum activity of the tenase complex. The interactive site(s) in FVIII for FX remains to be completely clarified, however. We investigated the FVIII A2 domain-FX association that was speculated from inhibitory mechanism(s) by an anti-A2 autoantibody. SDS-PAGE demonstrated that the purified inhibitor IgG recognizing residues 373–562 blocked FXa cleavage at Arg372 in FVIII, and surface-plasmon resonance (SPR)-based assays showed that intact A2 subunit directly bound to FX (K d; 63 nM). The FVIII structure model indicated possible FX-binding site(s) in residues 400–429 in A2. One peptide corresponding to residues 400–409 competitively inhibited both the A2–FX binding and FVIIIa/FIXa-dependent FXa generation. Covalent cross-linking was observed between this peptide and FX following reaction with EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) using SDS-PAGE. K408 and S409 were not evident in N-terminal sequence analysis of the cross-linked product, suggesting that two residues participated in cross-link formation. SPR-based assays using recombinant FVIII mutants with one or both residues substituted to alanine demonstrated that K408A and K408A/S409A had approximately fourfold high K d values of wild-type (WT-)FVIII. FXa cleavages at Arg372 in both mutants were significantly delayed, suggesting a contribution of K408 for FXa cleavage at Arg372. Furthermore, FXa generation assays with these mutants demonstrated that the K m values were 1.4- to 1.7-fold greater, and overall catalytic efficiency (k cat/K m) was 0.49- to 0.89-fold lower than with WT-FVIII, suggesting a significant contribution of K408 for FVIII–FX interaction in tenase assembly. We concluded that the K408 in the A2 domain provided an interactive-site for FX.
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Ucieklak, Karolina, Sabina Koj, and Tomasz Niedziela. "Bordetella holmesii Lipopolysaccharide Hide and Seek Game with Pertussis: Structural Analysis of the O-Specific Polysaccharide and the Core Oligosaccharide of the Type Strain ATCC 51541." International Journal of Molecular Sciences 21, no. 17 (September 3, 2020): 6433. http://dx.doi.org/10.3390/ijms21176433.
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Whooping cough is a highly contagious disease caused predominantly by Bordetella pertussis, but it also comprises of a pertussis-like illness caused by B. holmesii. The virulence factors of B. holmesii and their role in the pathogenesis remain unknown. Lipopolysaccharide is the main surface antigen of all Bordetellae. Data on the structural features of the lipopolysaccharide (LPS) of B. holmesii are scarce. The poly- and oligosaccharide components released by mild acidic hydrolysis of the LPS were separated and investigated by 1H and 13C NMR spectroscopy, mass spectrometry, and chemical methods. The structures of the O-specific polysaccharide and the core oligosaccharide of B. holmesii ATCC 51541 have been identified for the first time. The novel pentasaccharide repeating unit of the B. holmesii O-specific polysaccharide has the following structure: {→2)-α-l-Rhap-(1→6)-α-d-Glcp-(1→4)-[β-d-GlcpNAc-(1→3]-α-d-Galp-(1→3)-α-d-GlcpNAc-(1→}n. The SDS-PAGE and serological cross-reactivities of the B. holmesii LPS suggested the similarity between the core oligosaccharides of B. holmesii ATCC 51541 and B. pertussis strain 606. The main oligosaccharide fraction contained a nonasaccharide. The comparative analysis of the NMR spectra of B. holmesii core oligosaccharide fraction with this of the B. pertussis strain 606 indicated that the investigated core oligosaccharides were identical.
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Ledesma-Osuna, Ana Irene, Gabriela Ramos-Clamont, and Luz Vázquez-Moreno. "Characterization of bovine serum albumin glycated with glucose, galactose and lactose." Acta Biochimica Polonica 55, no. 3 (September 17, 2008): 491–97. http://dx.doi.org/10.18388/abp.2008_3054.
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The non-enzymatic reaction between reducing sugars and proteins, known as glycation, has received increased attention from nutritional and medical research. In addition, there is a large interest in obtaining glycoconjugates of pure well-characterized oligosaccharides for biological research. In this study, glycation of bovine serum albumin (BSA) by d-glucose, d-galactose and d-lactose under dry-heat at 60 degrees C for 30, 60, 120, 180 or 240 min was assessed and the glycated products studied in order to establish their biological recognition by lectins. BSA glycation was monitored using gel electrophoresis, determination of available amino groups and lectin binding assays. The BSA molecular mass increase and glycation sites were investigated by mass spectrometry and through digestion with trypsin and chymotrypsin. Depending on time and type of sugar, differences in BSA conjugation were achieved. Modified BSA revealed reduction of amino groups' availability and slower migration through SDS/PAGE. d-galactose was more reactive than d-glucose or d-lactose, leading to the coupling of 10, 3 and 1 sugar residues, respectively, after 120 minutes of reaction. BSA lysines (K) were the preferred modified amino acids; both K256 and K420 appeared the most available for conjugation. Only BSA-lactose showed biological recognition by specific lectins.
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Montero, C., and P. Llorente. "Artemia purine phosphoribosyltransferases. Purification and characterization." Biochemical Journal 275, no. 2 (April 15, 1991): 327–34. http://dx.doi.org/10.1042/bj2750327.
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Adenine phosphoribosyltransferase (APRTase) and hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) have been purified from Artemia cysts and nauplii to apparent homogeneity, as determined by SDS-PAGE. The purification includes affinity chromatography on AMP-Sepharose, which binds both enzymes, and they are eluted at different 5-phospho-alpha-D-ribosyl diphosphate (PP-Rib-P) concentrations. The purified enzymes from Artemia cysts were similar to nauplii enzymes with respect to Mr in denaturing gel electrophoresis and gel filtration, pH and cation dependence and kinetic constants for substrates and inhibitors. By Sephadex G-100 filtration, the native Mr of the adenine and hypoxanthine-guanine enzymes was estimated to be Mr 28,000 and 66,000, respectively. Analysis by SDS-PAGE revealed that the APRTase was a dimer of Mr 15,000 sub-units and the HGPRTase, a tetramer of four identical Mr 19,000 sub-units. The pH profile of the HGPRTase shows two apparent buffer-independent pH optima, at 7.0 and 9.5, while the APRTase has just one, at about pH 8-9. The purine phosphoribosyltransferase activity with adenine was highest, about tenfold the HGPRTase activity with hypoxanthine and fivefold that with guanine. Both enzymes exhibited similar requirements for divalent cations, either Mg2+, Mn2+ or Zn2+, while Ca2+ is highly inhibitory. The Km values of APRTase for adenine and PP-Rib-P are 2 and 30 microM, respectively, and the Km values of HGPRTase for hypoxanthine, guanine and PP-Rib-P are less than 1, less than 1 and 15 microM, respectively. Plots of the reciprocal enzyme activities versus reciprocal concentrations of one substrate at several fixed levels of the second one yield a pattern of inhibition by guanine and hypoxanthine. Product-inhibition studies indicated that AMP is a competitive inhibitor with respect to PP-Rib-P in the APRTase reaction, while the HGPRTase shows a mixed inhibition by GMP.
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Romaniec, M. P. M., U. Fauth, T. Kobayashi, N. S. Huskisson, P. J. Barker, and A. L. Demain. "Purification and characterization of a new endoglucanase from Clostridium thermocellum." Biochemical Journal 283, no. 1 (April 1, 1992): 69–73. http://dx.doi.org/10.1042/bj2830069.
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An endoglucanase (1,4-beta-D-glucan glucanohydrolase, EC 3.2.1.4) from the thermophilic anaerobe Clostridium thermocellum was purified to apparent homogeneity without the use of denaturants. No carbohydrate is associated with the endoglucanase. A molecular mass of 76,000 Da was determined by SDS/PAGE. The optimal pH is 7.0 and the enzyme is isoelectric at pH 5.05. The enzyme has a temperature optimum of 70 degrees C and retains approx. 50% of its activity after 48 h at 60 degrees C. Hydrolysis of CM-cellulose takes place with a rapid decrease in viscosity but a slow liberation of reducing sugars, indicating an endoglucanase type of activity. The endoglucanase shows little ability to hydrolyse highly ordered cellulose. Cellobiose inhibits whereas Mg2+ and Ca2+ stimulate the activity. The enzyme is completely inactivated by 1 mM-Hg2+ and is inhibited by a thiol-blocking reagent.
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Babakhin, A. A., A. A. Laskin, V. V. Smirnov, S. M. Andreev, K. K. Babievsky, I. S. Gushchin, and M. R. Khaitov. "MONOMERIC ALLERGOID FROM HOUSE DUST MITE DERMATOPHAGOIDES PTERONYSSINUS: IMMUNOLOGICAL PROPERTIES." Russian Journal of Allergy 13, no. 4-5 (December 15, 2016): 29–36. http://dx.doi.org/10.36691/rja351.
Full textAbstract:
Background. The purpose of this study was to characterize the immunologic properties of the monomeric allergoid obtained from house dust mite Dermatophagoides pteronyssinus (D. pteronyssinus) extract. Methods. To obtain monomeric allergoid the extract from D. pteronyssinus (D1) was chemically modified by succinilation and then the degree of modification and total protein content were determined. Analysis of proteins in native D1 or modified sD1 was performed by SDS-PAGE. Determination of the major allergen Der p 1 in the native extract D1 was done by mass-spectrometry method. For measurement of tertiary structure alterations of protein molecules in D1 or recombinant Der p 1 (rDer p 1) after their modification by succinilation the circular dichroism (CD) method was applied. Allergenicity of sD1 was estimated by inhibition of specific IgE binding in ELISA and chemiluminescent analysis using Phadia ImmunoCAP as well as by detection of histamine released from leukocytes of whole blood of Der p sensitized patients after its incubation with D1 or sD1. To evaluate immunogenicity of D1 and sD1 BALB/c mice were i.p. immunized 4 times in 3 week interval with D1 or sD1 with or without adjuvant Al(OH),. The levels of anti-Der p IgE, IgG1 and IgG2a in pulled mice sera were determined by ELISA. Results. Succinilation of D1 extract led to «unfolding» of allergen molecules in modified sD1. The degree ofmodification was 98,9%. Results of SDS-PAGE demonstrated that native D1 and succinilated sD1 extract showed bands at the same molecular mass confirming that succinilation does not influence the size of modified molecule which thus maintains the monomeric nature of the sD1. Mass-spectrometry analysis revealed the presence of major allergen Der p 1 in the native D1 extract. CD method demonstrated the alteration of tertiary structure of succinilated recombinant rDer p 1 that may be extrapolated on the other allergenic proteins in the sD1 extract. Allergenicity of sD1 was significantly reduced in compare to D1 that was confirmed by inhibition of IgE-binding and histamine release from leukocytes of whole blood, Immunization of BALB/c mice with sD1 (with or without Al(OH)3) induced anti-Der p IgE-response which was substantially lower than that of D1 induced. At the same time anti-Der p IgG1 and IgG2a responses after immunization with sD1 (with or without Al(OH)3) were significantly higher than that ofD1 induced. Conclusion. Modification of the extract from house dust mite D. pteronyssinus by succinilation leads to formation of monomeric allergoid possesses low allergenicity and preserved (or even increased) immunogenicity that may a new approach for creation of allergy vaccines for safe and effective allergen-specific immunotherapy.
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Palumbo, A., M. d'Ischia, G. Misuraca, L. De Martino, and G. Prota. "A new dopachrome-rearranging enzyme from the ejected ink of the cuttlefish Sepia officinalis." Biochemical Journal 299, no. 3 (May 1, 1994): 839–44. http://dx.doi.org/10.1042/bj2990839.
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A melanogenic enzyme catalysing the rearrangement of dopachrome has been identified in the ejected ink of the cuttlefish Sepia officinalis. This enzyme occurs as a heat-labile protein which co-migrates with tyrosinase under a variety of chromatographic and electrophoretic conditions. On SDS/PAGE it shows like a single band with an approx. molecular mass of 85 kDa. The enzyme possesses high substrate specificity, acting on L-dopachrome (Km = 1 mM at pH 6.8) and on L-alpha-methyl-dopachrome, but not on D-dopachrome, L-dopachrome methyl ester, dopaminochrome and adrenochrome. Significant inhibition of the catalytic activity was observed with tropolone and L-mimosine. H.p.1.c. analysis of the enzyme-catalysed rearrangement of L-dopachrome revealed the quantitative formation of the decarboxylated product, 5,6-dihydroxyindole. These results point to marked differences between melanogenesis in cephalopod pigment cells and in melanocytes, which may have important implications in relation to the use of sepiomelanin as a model for studies of mammalian melanins.
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Dong, Yuan, Hanjin Hou, An Chen, Wei Ma, Moli Yin, Fanwei Meng, Chuanmin Hu, Huiyan Wang, and Jianhui Cai. "Generation of a Monoclonal Antibody against D-Dimer Using HTS-Based LiCA." SLAS DISCOVERY: Advancing the Science of Drug Discovery 25, no. 3 (September 27, 2019): 310–19. http://dx.doi.org/10.1177/2472555219878407.
Full textAbstract:
D-dimer is an essential diagnostic index of thrombotic diseases. Since the existing anti-D-dimer antibodies vary in quality and specificity, a search for alternative anti-D-dimer antibodies is required. The present study aimed to screen a novel monoclonal antibody (mAb) against D-dimer using a light-initiated chemiluminescence assay (LiCA). In this work, mice were immunized with antigen prepared from human plasma by enzyme hydrolysis. After screening, a novel mAb, DD 2G11, was obtained. The results of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis indicated that DD 2G11 could be used as a standard marker for D-dimer. The isotype of DD 2G11 was IgG1, the Ka value was 0.646 nM-1, and the Kd value was 50 nM, indicating that the binding affinity to D-dimer was very high. Furthermore, no cross-reactivity between DD 2G11 and other fibrinogen degradation products (FgDPs) was found. Finally, the correlation between DD 2G11 and the reference antibody (commercial antibody) was investigated by analyzing 56 clinical samples using a latex-enhanced turbidimetric immunoassay (LTIA). The R2 value of the linear regression was 0.94538, indicating that DD 2G11 met clinical requirements. In conclusion, the present study provides a more expeditious protocol to screen mAbs and provides a clinically usable mAb against D-dimer.
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Millonig, R., H. Salvo, and U. Aebi. "Probing actin polymerization by intermolecular cross-linking." Journal of Cell Biology 106, no. 3 (March 1, 1988): 785–96. http://dx.doi.org/10.1083/jcb.106.3.785.
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We have used N,N'-1,4-phenylenebismaleimide, a bifunctional sulfhydryl cross-linking reagent, to probe the oligomeric state of actin during the early stages of its polymerization into filaments. We document that one of the first steps in the polymerization of globular monomeric actin (G-actin) under a wide variety of ionic conditions is the dimerization of a significant fraction of the G-actin monomer pool. As polymerization proceeds, the yield of this initial dimer ("lower" dimer with an apparent molecular mass of 86 kD by SDS-PAGE [LD]) is attenuated, while an actin filament dimer ("upper" dimer with an apparent molecular mass of 115 kD by SDS-PAGE [UD] as characterized [Elzinga, M., and J. J. Phelan. 1984. Proc. Natl. Acad. Sci. USA. 81:6599-6602]) is formed. This shift from LD to UD occurs concomitant with formation of filaments as assayed by N-(1-pyrenyl)iodoacetamide fluorescence enhancement and electron microscopy. Isolated cross-linked LD does not form filaments, while isolated cross-linked UD will assemble into filaments indistinguishable from those polymerized from unmodified G-actin under typical filament-forming conditions. The presence of cross-linked LD does not effect the kinetics of polymerization of actin monomer, whereas cross-linked UD shortens the "lag phase" of the polymerization reaction in a concentration-dependent fashion. Several converging lines of evidence suggest that, although accounting for a significant oligomeric species formed during early polymerization, the LD is incompatible with the helical symmetry defining the mature actin filament; however, it could represent the interfilament dimer found in paracrystalline arrays or filament bundles. Furthermore, the LD is compatible with the unit cell structure and symmetry common to various types of crystalline actin arrays (Aebi, U., W. E. Fowler, G. Isenberg, T. D. Pollard, and P. R. Smith. 1981. J. Cell Biol. 91:340-351) and might represent the major structural state in which a mutant beta-actin (Leavitt, J., G. Bushar, T. Kakunaga, H. Hamada, T. Hirakawa, D. Goldman, and C. Merril. 1982. Cell. 28:259-268) is arrested under polymerizing conditions.
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Chandan, Vandana, Susan M. Logan, Blair A. Harrison, Evgenii Vinogradov, Annie Aubry, Jacek Stupak, Jianjun Li, and Eleonora Altman. "Characterization of a waaF mutant of Helicobacter pylori strain 26695 provides evidence that an extended lipopolysaccharide structure has a limited role in the invasion of gastric cancer cells." Biochemistry and Cell Biology 85, no. 5 (October 2007): 582–90. http://dx.doi.org/10.1139/o07-056.
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An ld-heptosyltransferase gene, HP1191 (waaF), involved in biosynthesis of the inner-core region of Helicobacter pylori strain 26695 lipopolysaccharide (LPS), has been cloned and its function established by complementation of Salmonella enterica serovar Typhimurium waaF mutant strain, strain 3789. Insertional inactivation of the HP1191 open reading frame in strain 26695 resulted in the formation of a deeply truncated LPS molecule, as observed using SDS–PAGE. Subsequent compositional and fatty acid analyses, followed by capillary electrophoresis – mass spectrometry and nuclear magnetic resonance studies established its structure as the following: PE→7)-l-α-d-Hepp-(1→5)-α-Kdop-(2→6)-Lipid A, where PE represents a phosphoethanolamine group, ld-Hep represents l-glycero-d-manno-heptose, and Kdo represents 3-deoxy-d-manno-oct-2-ulosonic acid. This structural analysis identifies the activity of HP1191 as a heptosyltransferase and a waaF homolog. In vitro invasion assays using human cultured gastric adenocarcinoma cells as a host cell model confirmed that the level of invasion was unaffected for an H. pylori HP1191::Kan deep-rough mutant strain compared with the wild-type strain 26695 expressing the O-chain polysaccharide, providing evidence that LPS is not a critical factor for invasion.
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Sampaio, Claudio, Reinhard Mentele, Antonio Camargo, Edwin Fink, and Solange Serrano. "A Novel Fibrinogen-clotting Enzyme, TL-BJ, from the Venom of the Snake Bothrops jararaca: Purification and Characterization." Thrombosis and Haemostasis 83, no. 03 (2000): 438–44. http://dx.doi.org/10.1055/s-0037-1613834.
Full textAbstract:
SummaryThree chromatographically distinct forms of a novel fibrinogenclotting serine endopeptidase, TL-BJ 1, 2 and 3, were purified from the venom of Bothrops jararaca by a combination of ammonium sulfate precipitation and chromatographic steps. The three forms of TL-BJ have similar amidolytic and plasma coagulating activities. TL-BJ 1, TL-BJ 2 and TL-BJ 3 cause the specific clotting of fibrinogen with release of fibrinopeptide A, the specific activities are 16.8 NIH U/mg (TL-BJ 1), 16.7 NIH U/mg (TL-BJ 2) and 20.8 NIH U/mg (TL-BJ 3). The most sensitive chromogenic substrates for measuring the amidolytic activity of TL-BJ 3 were D-Pro-Phe-Arg-pNA, D-Phe-pipecolyl-ArgpNA and Z-D-Arg-Gly-Arg-pNA. The amidolytic and coagulant activities of TL-BJ were inhibited by phenylmethylsulfonyl fluoride but not by hirudin. Benzamidine derivatives, which are competitive inhibitors of trypsin-like serine endopeptidases, also inhibited the amidolytic activity of TL-BJ. In SDS/PAGE the main bands of TL-BJ 1, 2 and 3 showed molecular masses of 30 kDa, 31 kDa and 32 kDa. Upon incubation with N-glycosidase F only TL-BJ 3 remained unchanged, whereas TL-BJ 1 and TL-BJ 2 showed products with molecular masses around 23 kDa. Thus, TL-BJ 3 does not seem to be N-glycosylated. The N-terminal amino acid sequences of TL-BJ 2 and TL-BJ 3 are identical while TL-BJ 1 has five substitutions.
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Wang, Jinshui, Yanli Yin, and Feng Jia. "Impact of ultrasound treatment on molecular structures of casein." JOURNAL OF ADVANCES IN BIOTECHNOLOGY 2, no. 2 (December 30, 2012): 106–12. http://dx.doi.org/10.24297/jbt.v2i2.1702.
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Casein was modified by ultrasound treatment (160W, 400 W)and the structural characteristics of casein (control), USC-1 (160W of ultrasound treated casein) and USC-2 (400W of ultrasound treated casein) were investigated. Ultrasound treatment resulted in increase in the numbers of protein bands of casein in region of low molecular weight according to SDS-PAGE pattern. The relative percentage of the proteins with molecular mass (MM) of over 30 kD in USC-1 and USC-2 significantly decreased compared with control sample. Coupled with this is the relative percentage of the proteins with MM of region 20 kD-30 kD for the ultrasonic-treated caseins increased. Contents of α-helix, β-sheet and random coil of casein for USC-1 and USC-2 decreased compared with control. While, β-turn content for USC-1 and USC-2 increased. Ratio of α-helix to β-sheet in USC-1 and USC-2 significantly decreased (from 0.70 to 0.47 and 0.46). Ultrasound treatment had a strong crushing action of surface of the casein micelle and resulted in its structural disruption. Particle size distribution range of casein was approximately 0.5-70 μm, Particle size of USC-1 and USC-2 was less than 30μm. D (50), D (4, 3) as well as D (3, 2) for USC-1 and USC-2 reduced compared with the control. The ratio of D (4, 3) to D (3, 2) also decreased with increase of ultrasonic intensity. This study demonstrated that ultrasound treatment could be an effective method for modification of casein structure. This modification is the basis on improvement in functional properties.
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Cullen, B. M., I. M. Halliday, G. Kay, J. Nelson, and B. Walker. "The application of a novel biotinylated affinity label for the detection of a cathepsin B-like precursor produced by breast-tumour cells in culture." Biochemical Journal 283, no. 2 (April 15, 1992): 461–65. http://dx.doi.org/10.1042/bj2830461.
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In this report we demonstrate how the recently developed biotinylated affinity label biotinyl-Phe-Ala-diazomethane (Bio-Phe-Ala-CHN2) [Cullen, McGinty, Walker, Nelson, Halliday, Bailie & Kay (1990) Biochem. Soc. Trans. 18, 315-316; Walker, Cullen, Kay, Halliday, McGinty & Nelson (1992) Biochem. J. 283, 449-453] can be used for the detection of a precursor form of a cathepsin B-like enzyme produced by breast-tumour cells in culture. Thus the cell lines MDA-MB-436, ZR-75-1 and T47-D produce a soluble protein that can be allowed to react with the biotinylated affinity label to yield an SDS-resistant complex; this can be revealed with a streptavidin/alkaline phosphatase label after PAGE and Western blotting. This protein (molecular mass 47 kDa) can also be detected by immunoblotting using sheep anti-(cathepsin B) antibodies in conjunction with a donkey anti-sheep IgG label. None of the cell lines studied produced any mature cathepsin B-like activity, as gauged by the lack of turnover of the fluorogenic substrate benzyloxycarbonyl-Arg-Arg-4-methylcoumarin-7-ylamide (Cbz-Arg-Arg-NH-Mec). However, treatment of medium samples with pepsin resulted in the generation of such activity. When the pepsin-catalysed activation step was analysed by SDS/PAGE, the protein of 47 kDa was completely converted into two species of very similar molecular masses of 30.5 kDa and 29 kDa. Both these proteins can incorporate the biotinylated probe and, in common with the 47 kD species, they can be detected with the streptavidin/alkaline phosphatase label and immunoblotting. We propose that the 47 kD form is the pepsin-activable proform of these lower-molecular-mass species. The release of the proform from the oestrogen-receptor (ER)-positive breast-tumour cell lines ZR-75-1 and T47-D is stimulated 5-10-fold when these cells are grown in medium containing epidermal growth factor (EGF) at a concentration of 10 ng/ml. In contrast, there is no modulation in the amount of proform released by the ER-negative cell line MDA-MB-436, over a range of EGF concentrations from 0 to 100 ng/ml.
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Souza, Thaís T. S., Maria J. B. Bezerra, Maurício F. van Tilburg, Celso S. Nagano, Luciana D. Rola, José M. B. Duarte, Luciana M. Melo, Arlindo A. Moura, and Vicente J. F. Freitas. "Protein profile of the ovarian follicular fluid of brown brocket deer (Mazama gouazoubira; Fisher, 1814)." Zygote 28, no. 2 (December 2, 2019): 170–73. http://dx.doi.org/10.1017/s0967199419000741.
Full textAbstract:
SummaryThe aim of this study was to characterize the protein profile of ovarian follicular fluid (FF) of brown brocket deer (Mazama gouazoubira). Five adult females received an ovarian stimulation treatment and the FF was collected by laparoscopy from small/medium (≤3.5 mm) and large (>3.5 mm) follicles. Concentrations of soluble proteins in FF samples were measured and proteins were analyzed by 1-D SDS-PAGE followed by tryptic digestion and tandem mass spectrometry. Data from protein list defined after a Mascot database search were analyzed using the STRAP software tool. For the protein concentration, no significant difference (P > 0.05) was observed between small/medium and large follicles: 49.2 ± 22.8 and 56.7 ± 27.4 μg/μl, respectively. Mass spectrometry analysis identified 13 major proteins, but with no significant difference (P > 0.05) between follicle size class. This study provides insight into elucidating folliculogenesis in brown brocket deer.
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Figueiredo, Camila Soares, Suzana Cristina Marucci, Renata Izabel Dozzi Tezza, Manoel Victor Franco Lemos, and Janete Apparecida Desidério. "Caracterização do gene vip3A e toxicidade da proteína Vip3Aa50 à lagarta-do-cartucho e à lagarta-da-soja." Pesquisa Agropecuária Brasileira 48, no. 9 (September 2013): 1220–27. http://dx.doi.org/10.1590/s0100-204x2013000900005.
Full textAbstract:
O objetivo deste trabalho foi caracterizar o gene vip3A de Bacillus thuringiensis e verificar a toxicidade da proteína Vip3Aa50 a larvas da lagarta-do-cartucho (Spodoptera frugiperda) e da lagarta-da-soja (Anticarsia gemmatalis). O gene vip3A foi amplificado por PCR, com iniciadores específicos, e gerou um fragmento de 2.370 pb. Esse fragmento foi clonado em vetor pGEM-T Easy e, em seguida, sequenciado, subclonado em vetor de expressão pET-28a (+) e inserido em células de Escherichia coli BL21 (DE3). A expressão da proteína Vip3Aa50 foi induzida por isopropil-β-D-1-tiogalactopiranosídeo (IPTG), visualizada em SDS-PAGE e detectada por "Western blot". Os ensaios de toxicidade revelaram alta atividade da proteína Vip3Aa50 contra as larvas neonatas da lagarta-da-soja e da lagarta-do-cartucho, com CL50 de 20,3 e 79,6 ng cm-2, respectivamente. O gene vip3Aa50 é um novo gene da classe vip3A.
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Sung, Nackmoon, and Michael T. Collins. "Variation in Resistance of Mycobacterium paratuberculosis to Acid Environments as a Function of Culture Medium." Applied and Environmental Microbiology 69, no. 11 (November 2003): 6833–40. http://dx.doi.org/10.1128/aem.69.11.6833-6840.2003.
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ABSTRACT Acid resistance of Mycobacterium paratuberculosis was examined as a function of growth conditions (i.e., in vitro growth medium and pH). M. paratuberculosis was cultured in either fatty acid-containing medium (7H9-OADC) or glycerol-containing medium (WR-GD or 7H9-GD) at two culture pHs (pHs 6.0 and 6.8). Organisms produced in these six medium and pH conditions were then tested for resistance to acetate buffer at pHs 3, 4, 5, and 6 at 20°C. A radiometric culture method (BACTEC) was used to quantify viable M. paratuberculosis cell data at various acid exposure times, and D values (decimal reduction times, or the times required to kill a 1-log10 concentration of bacteria) were determined. Soluble proteins of M. paratuberculosis grown under all six conditions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to identify proteins that may be associated with acid resistance or susceptibility. The culture medium affected growth rate and morphology: thin floating sheets of cells were observed in 7H9-OADC versus confluent, thick, waxy, and wrinkled pellicles in WR-GD. Culture medium pH affected growth rate (which was highest at pH 6.0), but it had little or no effect on D values for M. paratuberculosis at any test pH. When grown in 7H9-OADC, M. paratuberculosis was more acid resistant at all test pHs (higher D values) than when grown in WR-GD. Glycerol appeared to be the culture medium component most responsible for lower levels of M. paratuberculosis acid resistance. When glycerol was substituted for OADC in the 7H9 medium, D values were significantly lower than those of 7H9-OADC-grown M. paratuberculosis and were approximately the same as those for M. paratuberculosis grown in WR-GD medium. Comparison of the SDS-PAGE protein profiles for M. paratuberculosis cultures grown in 7H9-OADC, WR-GD, or 7H9-GD medium revealed that increased expression of 34.2- and 14.0-kDa proteins was associated with higher levels of acid resistance of M. paratuberculosis grown in 7H9-OADC medium and that 56.6- and 41.3-kDa proteins were associated with lower levels of acid resistance. This is the first report showing that in vitro culture conditions significantly affect growth characteristics, acid resistance, and protein expression of M. paratuberculosis, and the results emphasize the importance of culture conditions for in vitro susceptibility studies.
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Raykovska, Vessela, Pavlina Dolashka-Angelova, Donka Paskaleva, Stanka Stoeva, Juri Abashev, Lubo Kirkov, and Wolfgang Voelter. "Isolation and characterization of a xylose-glucose isomerase from a new strain Streptomyces thermovulgaris 127, var. 7-86." Biochemistry and Cell Biology 79, no. 2 (April 1, 2001): 195–205. http://dx.doi.org/10.1139/o00-100.
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A thermostable D-xyloseglucose isomerase was isolated from the thermophilic strain Streptomyces thermovulgaris 127, var. 7-86, as a result of mutagenic treatment by γ-irradiation of the parent strain, by precipitation and sequential chromatographies on DEAESephadex A50, TSK-gel, FPLC-Mono Q/HR, and Superose 12 columns. The N-terminal amino acid sequence and amino acid analysis shows 7392% homology with xyloseglucose isomerases from other sources. The native molecular mass, determined by gel filtration on a Superose 12 column, is 180 kDa, and 44.6 and 45 kDa were calculated, based on amino acid analysis and 10% SDS-PAGE, respectively. Both, the activity and stability of the enzyme were investigated toward pH, temperature, and denaturation with guanidine hydrochloride. The enzyme activity showed a clear pH optimum between pH 7.2 and 9.0 with D-glucose and 7.4 and 8.3 with D-xylose as substrates, respectively. The enzyme is active up to 6085°C at pH 7.0, using D-glucose, and up to 5060°C at pH 7.6, using D-xylose as substrates. The activation energy (Ea = 46 kJ·mol1) and the critical temperature (Tc = 60°C) were determined by fluorescence spectroscopy. Tc is in close coincidence with the melting temperature of denaturation (Tm = 59°C), determined by circular dichroism (CD) spectroscopy. The free energy of stabilization in water after denaturation with Gdn.HCl was calculated to be 12 kJ·mol1. The specific activity (km values) for D-xylose-glucose isomerase at 70°C toward different substrates, D-xylose, D-glucose, and D-ribose, were determined to be 4.4, 55.5, and 13.3 mM, recpectively.Key words: D-xylose-glucose isomerase, protein sequencing, protein stability, protein denaturation.