THE PLASTEIN REACTION REVISITED - EVIDENCE FOR A PURELY AGGREGATION REACTION-MECHANISM

被引:36
作者
ANDREWS, AT
ALICHANIDIS, E
机构
[1] Biotechnology and Enzymology Department, AFRC Institute of Food Research, Reading Laboratory, Shinfield, Reading
关键词
D O I
10.1016/0308-8146(90)90015-V
中图分类号
O69 [应用化学];
学科分类号
081704 ;
摘要
Various aspects of the plastein synthesis reaction were investigated using peptides derived from casein as substrate. With peptides obtained by partial acid hydrolysis a clear requirement for a proteinase to catalyse plastein synthesis was demonstrated and, although enzymes whose hydrolytic activity had been inhibited may act as rather inefficient catalysts, the native active enzymes were preferred. Blockage of peptide NH2 or COOH groups reduced plastein yield but did not prevent synthesis. Results following addition of water-miscible organic solvents to reaction mixtures were more consistent with increased solubility of hydrophobic amino acids and peptides rather than with the influence of viscosity changes. Although plastein separates out of the reaction mixture in the form of a gel or precipitate and can be collected as an insoluble centrifuge pellet, on repeated washing and incubation it was gradually solubilised even in aqueous buffers, showing clearly that plastein formation is a reversible process. This was in effect confirmed by ionexchange chromatography and gel filtration experiments, in which there were some quantitative differences in peptide composition between peptide hydrolysate starting materials and resolubilised plastein pellets produced from them but no qualitative differences. This showed clearly that no appreciable amounts of new peptides were formed and ruled out covalent bond formation in a reversed hydrolysis or a transpeptidation pathway as the reaction mechanism. This conclusion was confirmed by SDS-PAGE and by preliminary small-angle neutron scattering experiments. We therefore conclude that the plastein synthesis reaction is a purely entropy-driven physical aggregation process. © 1990.
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页码:243 / 261
页数:19
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