Academic literature on the topic '1-D SDS-PAGE'
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Journal articles on the topic "1-D SDS-PAGE"
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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).
Dissertations / Theses on the topic "1-D SDS-PAGE"
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Mahlatsi, Tladi Abram. "Characterization of heavy metal tolerant bacterial plasmids isolated from a platinum mine tailings dam / by Tladi Abram Mahlatsi." Thesis, North-West University, 2012. http://hdl.handle.net/10394/9780.
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The development of metal-tolerance and antibiotic resistance in bacteria may be caused by metals polluting a particular environment. During mining and mineral processing activities, large quantities of metals are deposited into the soil. These high concentrations of metals are evolutionary pressures selecting for microorganisms tolerant to these metals. Metaltolerance maybe conferred to these organisms by mobile genetic elements such as plasmids. This study describes the characteristics of plasmids isolated from various bacteria that displayed an ability to withstand high metal concentrations. The isolated plasmids were individually transformed into Escherichia coli JM109. Transformants were then evaluated for metal-tolerant capabilities using a microdilution approach. Plasmids were then isolated from the transformants and the concentration of the plasmid DNA ranged between 11.75 – 118.06 ng/μl. These plasmids were of the same size as the original ones. This demonstrated that successful transformations with plasmid DNA were conducted. In order to determine the compatibility group, plasmids were subjected to PCR amplification using IncQ, IncP-9 and IncW specific primers. Only the IncW provided positive results. To demonstrate that the plasmids were free of genomic DNA, a 16S rDNA PCR test was included. The plasmids that were positive for IncW PCRs were all negative for the rDNA PCRs. Plasmids were stably inherited and at least three, isolated from three different Gram positive species, belonged to the Inc W group of plasmids. These were originally isolated from Paenibacillus ginsingari, Paenibacillus lautus and Bacillus cereus. Minimum inhibition concentrations (MICs) were carried out to determine the ability of transformed E. coli JM109 to tolerate metals at varying concentrations. Results indicated that transformed E. coli JM109 developed ability to grow in the presence of several heavy metals. Some strains were resistant to high concentrations (+10 mM) of Ni2+/Al3+, Pb2+ and Ba2+. The order of metal resistance was Ni/Al=Pb>Ba>Mn>Cr>Cu>Co=Hg. All the x transformants were sensitive to 1 mM of Co2+ and Hg2+. Moreover, protein profiling was used to determine the impact of plasmids on E. coli JM109. Proteins were extracted from both transformed and un-transformed E. coli JM109 using acetone-SDS protocol and subjected to one-dimensional (1D) and two-dimensional (2D) Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS- PAGE). Transformed E. coli JM109 were grown under the metal stress. One dimension SDS-PAGE illustrated general similarity of the profiles except for two banding positions in the 30 to 35 kDa region where bands were present in the transformants that were grown in the Ni/Al alloy containing media. Twodimensional electrophoresis PAGE analysis showed that some of the proteins were upregulated while others were down-regulated. The largest numbers of proteins were from 15 – 75 kDa. The majority of these proteins had isoelectric points (pI) between 5 and 6. It was concluded that plasmids isolated from various heavy metal-tolerant bacterial species were successfully transformed into E. coli JM109 rendering various new metal-tolerant E. coli JM109 strains. Furthermore, the study showed that metal resistance was due to the presence of the plasmids. Two-dimensional SDS-PAGE resolved more differences in the protein expression profiles. Since the plasmids rendered the E. coli JM109 tolerant to metals tested, it also can be concluded that the change in the protein profiles was due to the effects of the plasmids. Furthermore, plasmids were also re-isolated from the transformants and these plasmids were of the same size as the original ones.. All the plasmids in this study were also stably inherited, a feature associated with IncW plasmids. More detailed genetic characterization of these plasmids is required. Plasmids isolated and characterized in this study may hold biotechnology potential. Such features should be exploited in follow-up experiments.Thesis (Master of Environmental Sciences)--North-West University, Potchefstroom Campus, 2013.
Book chapters on the topic "1-D SDS-PAGE"
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Sultana, Rukhsana, Shelley F. Newman, Quanzhen Huang, and D. Allan Butterfield. "Detection of Carbonylated Proteins in 2-D SDS Page Separations." In Redox-Mediated Signal Transduction, 149–59. Totowa, NJ: Humana Press, 2008. http://dx.doi.org/10.1007/978-1-59745-129-1_11.
Full textConference papers on the topic "1-D SDS-PAGE"
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Lund-Hansen, T., and L. C. Peterson. "COMPARISON OF ENZYMATIC PROPERTIES OF HUMAN PLASMA FVIIa AND HUMAN RECOMBINANT FVIIa." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643787.
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Human plasma FVIIa (pFVIIa) and human recombinant FVIIa (rFVIIa) were both purified by immune adsorption chromatography using a calcium dependent monoclonal antibody. The FVII obtained is highly purified and contains only trace contaminants as revealed by SDS-PAGE and reverse phase HPLC chromatography. FVII was fully activated during the purification procedure. A FVIIa activity assay has been developed in microplates using human FX as a substrate and methoxycarbonyl-D-cyclohexal-alanyl-glycyl-arginine-pNA as a chromogenic substrate for the FXa generated. The assay was linear at FVIIa concentrations between 0.5 and 10 nM. The concentration of the chromogenic substrate was 0.5 mM. A pH optimum at about 8 was found. An apparent Km=0.2 μM for FX was found for both pFVIIa and rFVIIa.The results suggest that the kinetics of human FX activation by pFVIIa and rFVIIa are identical. The FVIIa activity was found to be calcium dependent with maximal activity at about 0.25 mM, while the activities at 1 and 2 mM were 20% and 3%, respectively. When rabbit brain extract is used, the well-known dramatic enhancement effect of thromboplastin could be demonstrated with both FVII preparations. Also this reaction is calcium-dependent; however, the profile of the curve is distinctly different. Poly-D-lysine (MW 160,000) was found to enhance the FVIIa activity in a concentration dependent manner. Maximum stimulation (fivefold) was obtained at a concentration of about 10 mg/l.
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Orthner, Carolyn L., Prabir Bhattacharya, and Dudley K. Strikland. "PURIFICATION AND CHARACTERIZATION OF A PROTEIN C ACTIVATOR FROM THE VENOM OF AGKISTRODON CONTORTRIX CONTORTRIX." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643813.
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There are two recent reports on the purification and properties of a protein C activator (PCA) from the venom of the Southern copperhead snalce. The purification of a 37,000 Mr nonenzymatic PCA (Martinoli and Stocker, Thrcmb. Res. 43, 253, 1976) as well as of a 20,000 Mr thrombin-like enzyme (Klein and Walker, Biochem. ,25, 4175, 1986) have been described. The purpose of this investigation was to purify and further characterize the PCA(s) from this vencm. A PCA has been isolated by sulphopropyl-Sephadex followed by gel filtration chromatography resulting in approximately a 100-fold purification with a 50% yield. PCA appeared as a single band on SDS-PAGE with an estimated Mr of 32,000 or 37,000 in the absence or presence of β-mercaptoethanol, respectively. High pressure gel permeation cinematography of PCA in Tris-buffered saline, pH 7.5 resulted in a single protein peak with a Mr of 39,000 which was coincident with activity. PCA was a potent activator of human protein C (PC) with a Km for PC of 0.6uM and a Vm of 0.02 sec-1. In addition, PCA catalyzed the arnidolysis of Tosyl-gly-pro-arg-p-nitroanilide (TGPRpNA) with a Km of 1.1 irM and a Vim of 66 sec-1. The rate of arnidolysis of five other pept idyl-arginyl-pNA substrates each tested at 1.0 mM was < 10% that of TGPRpNA. PCA was inhibited by nitrophenylguanidi-nobenzoate (NPGB), phenylmethylsulphonylflouride, D-phe-pro-arg-chloromethyi_ketone (PPACK) and soybean trypsin inhibitor indicating that PCA is a serine protease. The active site concentration of PCA as measured by NPGB titration was 90% that of the protein concentration. Measurement of the rate of PCA inhibition at varying levels of PPACK indicated that it had a Ki of 34uM .and an aUcylation rate constant of 0.09 min-1. PCA activation of PC was completely inhibited by CaC12 with an apparent Ki of 99uM. Since neither PCA arnidolysis of TGPRpNA nor inhibition by PPACK was affected by Ca2+, the effect of this metal was likely on the substrate PC. In summary, a PCA has been purified to homogeneity and has properties which are distinct from those reported. PCA premises to be a useful enzyme in studies of PC and its activation.
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Kuo, Be-Sheng, Gil Korner, and Thorir D. Bjornsson. "ROLE OF POLYAMINES IN THE REGULATION OF SYNTHESIS AND SECRETION OF PLASMINOGEN ACTIVATOR FROM BOVINE AORTIC ENDOTHELIAL CELLS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644655.
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The effects of three polyamines, putrescine (PUT), spermidine (SPD) and spermine (SPM), were investigated on the synthesis and secretion of plasminogen activator (PA) and antiactivator (PAI) activities in confluent bovine aortic endothelial cells. PA activity was determined bythe fibrin plate method, and individual species with PA and PAI activities were separated and visualized using SDS-PAGE with zymography and reverse fibrin autography. Both control cells and cells treated with polyamines secreted PA activity in a time-dependent fashion. After 24-hour incubation, the three polyamines enhanced PA secretion in a dose-dependent manner (10-6 to 2.5 × 10-3 M), with a potency order of SPM > SPD> PUT, as estimated by the fibrin plate method. The maximum PA releases after PUT (0.5 mM), SPD (2.5 mM) and SPM (0.5 mM) were 1.7, 4.5 and 5.4 times control levels, respectively. Concentrations lower than 1 μM had essentially no effects. The enhancement of PA activity by polyamines was blocked by actino-mycin D and cycloheximide, while it was not affected by inhibitors of polyamine biosynthesis except that the enhancement by PUT (0.5 mM) was reduced by methylglyoxal bis(guanylhydrazone). These data suggest that polyamines directly stimulate PA synthesis and secretion through promotion of gene transcription and translation, and that this effect appears to be related to their position in the biosynthetic pathway of polyamines. The kinetic patterns of activities of ornithine decarboxylase and S-adenosy-methionine decarboxylase in confluent endothelial cells stimulated withfresh culture medium suggest that there is rapid turnover of intracellular polyamines. Multiple forms of secreted PA were observed and both tissue- and urokinase-type PA were enhanced by polyamines, while the PAI activity, as evaluted by reverse fibrin autograpy, was apparently reduced. These experimental results suggest that polyamines may play an important role in the regulation of the synthesis and secretion of plasminogen activators, and that this biological function could be modified by disease states and by agents that are associated with altered polyamine metabolism.
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Rånby, M., A. Brändstrôm, L. Hansen, K. Henson, and G. Larsen. "REC. t-PA GENETICALLY MODIFIED AT THE CLEAVAGE SITE OF ONE-CHAIN TO TWO-CHAIN CONVERSION: ENZYMOLOGY AND DIAGNOSTIC APPLICATIONS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644410.
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Native one-chain t-PA is cleaved by plasmin or by trypsin after the Arg in the sequence -Gln-Phe-Arg-Ile-Lys-. Variants of one-chain t-PA where the -Arg- was replaced by a His (Arg to His) or by a Lys (Arg to Lys) or by a Thr (Arg to Thr) were made through genetic modification. The three mutants and the wild type were expressed in animal cells and purified in the one-chain form by affinity chromatography as was t-PA from Bowes melanoma cells. In contrast to wild type and melanoma t-PA the mutants reacted poorly with polyclonal antibodies raised against the peptide -Gln-Pro-Gln-Phe-Arg-Ile-Lys--Gly-Gly- indicating mutation in the sequence. Of these proteins only the Arg to Thr mutant was resistant to plasmin cleavage as evidenced by SDS-PAGE. t-PA antigen values (ELISA) and fibrinolytic activity values (fibrin clot lysis assay) yielded the following specific activities expressed in IU/|μg: 810 (Arg to His), 640 (Arg to Lys), 290 (Arg to Thr), 810 (wild type) and 660 (melanoma t-PA). The amidolytic activities for the one-chain proteins against D-Ile-Pro-Arg-pNA at pH 9.0 and 37°C, expressed in mOD per minute at 1 M-g/mL of enzyme were: 15.8 (Arg to His), 13.6 (Arg to Lys), 8.3 (Arg to Thr), 10.0 (wild type), 9.6 (melanoma t-PA) as compared to 55.2 for two-chain melanoma t-PA.All mutants including the uncleavable Arg to Thr mutant could be used in determination of PAI activity in plasma samples. Only one-chain t-PA reacts selectively with PAI 1. Thus, use of the Arg to Thr mutant represents a theoretical advantage in PAI 1 activity determination since preparations of this mutant most likely is free of contaminating two-chain t-PA.The plasminogen activation rate as measured in a coupled assay in the presence and absence of fibrin at 0.5 jiM plasminogen and 37°C was measured and the stimulation factor calculated. This was about 950 fold for the Arg to Thr mutant wich was considerably higher than that of melanoma one chain t-PA and the other mutants wich all were about 550 fold. The stimulation factor for melanoma two-chain t-PA was in the same experiment about 120 fold. The extra fibrin sensitivity of the Arg to Thr mutant resulted in improved soluble fibrin assay according to Wiman and Renby Thromb. Haemostas, (1986) 55:189-193.In conclusion: the use of a plasmin insensitive protein-engineered mutant of t-PA gives advantages in assays for PAI 1 and soluble fibrin.