Academic literature on the topic 'Biochemistry of proteins'

The mechanism of protein translocation into mitochondria has been investigated by studying properties of precursor proteins destined for the mitochondrial matrix. Characterization of the amphiphilic properties of the signal sequence for pre-ornithine carbamyltransferase has led to the conclusion that precursors can not translocate across the inner membrane via a lipid route alone (i.e. in the absence of proteins). A correlation was established between the rate of precursor import and the degree of hydrophobicity of a short region in the presequence, suggesting that precursor binding to the two-dimensional phospholipid surface of the outer membrane may enhance the rate of diffusion to the translocation apparatus.<br>The conformations of the mature portions of two hybrid proteins, pOCAT and pODHFR, were examined at various steps on the import pathway. The bulk population of these precursors remained in a near-native conformation prior to precursor engagement of the import apparatus. Unfolded polypeptide translocation intermediates, the formation of which requires ATP, an intact signal sequence, and a protease-sensitive component of the outer mitochondrial membrane, have been detected in association with submitochondrial fractions containing sites of contact between the inner and outer mitochondrial membranes.

The transforming growth factor (TGF)-β superfamily mediates a wide range of differentiation and developmental processes across many genera. GDF9 and BMP15 are expressed exclusively in the mammalian ovary and are the only TGF-β ligands that lack the conserved cysteine residue used for dimerisation. As a platform for studying the interactions between GDF9 and BMP15 and their receptors, BMPRII and BMPRIb, a variety of strategies were attempted to produce soluble and active proteins from recombinant systems. Both ligands and receptors showed a tendency to form insoluble aggregates when expressed in prokaryotic systems; however after extensive screening, quantities of biologically active GDF9 were produced using in vitro refolding. When expressed alone, either containing a histidine tag or as an untagged protein, the BMPRII ectodomain was deposited as insoluble inclusion bodies. This protein, subjected to in vitro refolding procedures, exhibited multiple species following anion exchange chromatography and size exclusion chromatography, as visualised on native PAGE. Separation of these species could be achieved using a MonoP matrix. One of these separated fractions, representing about 5% of the starting material, was amenable to crystallisation, and furthermore exhibited activity in a rat granulosa cell thymidine incorporation assay. Two different crystals forms of the extracellular domain of BMPRII were grown from the same protein batch under similar crystallisation conditions. Notably, the tetragonal form that grew more slowly possessed several disordered finger regions, while electron density for the entire molecule was clear in the orthorhombic form. The hydrophobic core of the ligand binding surface of BMPRII , as seen in both structures, resembles that of ActRII bound to BMP2. The A-loop of BMPRII, which is involved in ligand binding, lies in two different conformations in the two structures of BMPRII, mediated by a rearrangement in disulfide Cys94-Cys117. It is proposed here that the tetragonal form represents the ligand-bound receptor structure. Although the majority of the hydrophobic binding surface is shared with ActRII(b), it is likely that His87 and Tyr40 are unique residues that confer specificity in BMPRII ligand binding.

This thesis investigated the nutritional consequences of processing chicken feed, in particular the loss of available amino acids as a result of the Maillard reaction, both in model protein systems and during poultry feed processing. Model systems containing RNase A and pure or feed-type carbohydrates were incubated at 37°C, 50°C or 70°C for up to 8 days. From lysine analysis, reduction in reactive amino groups in RNase A was shown to occur rapidly at room temperature, with no incubation period necessary. However, protein fragmentation that occurred during incubation, especially at higher temperatures, interfered with the quantification of lysine. SDS-PAGE showed crosslinking reactions of RNase A with the tested carbohydrates to be slow below 70°C. Incubation of RNase A with cyclotene or xylose produced the greatest rate of crosslinking, while starch and sucrose produced the least. A modified OPA method was developed such that the level of reactive lysine could be quantitated in barley flour proteins, in a manner that was technically straightforward, inexpensive and allowed good through-put of samples. This method was shown to have good agreement with the published ninhydrin method in the measurement of Maillard reacted lysine in barley flours. Up to 25% loss of reactive lysine was observed in barley flour that had not undergone processing beyond milling. Samples were taken before, during and after the pelleting of chicken feed. Lysine analysis of these samples showed loss in amino group content of at least 18% could occur during processing, although this was dependent on variations between pelleting runs. Protein fragmentation during processing potentially masked further losses. A growth trial assessed a novel method of lysine addition to the feed, via spraying on free lysine solution post-pelleting. Over the 2 week trial period, 10% of the lysine applied by this method remained in the uneaten fines. Lysine eaten by chickens aged day 8-14, as calculated from measurements taken using the OPA method, correlated well with bird growth. Increased feed intake was also seen as the lysine content of the diet increased. In chickens aged day 15-21, lysine addition above the first addition level produced no further significant gain in performance. Therefore, lysine was not growth limiting above this level. Results indicated that over the 2 week period of the trial, equivalent performance for birds on the standard feed could have been achieved with a 50% reduction in free lysine added to the feed formulation. No lysine loss occurred in the standard sample as a result of pelleting. Therefore, while the potential exists for lysine loss to occur during chicken feed pelleting, this thesis has shown that this is not a significant problem. As up to 25% lysine blockage in unprocessed barley flour was observed, obtaining feed ingredients with consistently low levels of Maillard reaction damage may be as important as maintaining ideal processing conditions.

Kyoto University (京都大学)<br>0048<br>新制・論文博士<br>博士(農学)<br>乙第11672号<br>論農博第2555号<br>新制||農||912(附属図書館)<br>学位論文||H17||N4055(農学部図書室)<br>23485<br>UT51-2005-D590<br>京都大学大学院農学研究科応用生命科学専攻<br>(主査)教授 江﨑 信芳, 教授 喜多 恵子, 教授 植田 充美<br>学位規則第4条第2項該当