van den Burg B, Eijsink V G, Stulp B K, Venema G
Department of Genetics, Centre of Biological Sciences, Haren, The Netherlands.
Biochem J. 1990 Nov 15;272(1):93-7. doi: 10.1042/bj2720093.
Autocatalytic degradation of purified Bacillus subtilis neutral proteinase was examined under various conditions. At elevated temperatures, under non-inhibitory conditions, mature protein was rapidly degraded, but no accumulation of specific breakdown products occurred. However, by incubating purified neutral proteinase on ice during extended periods of time, specific peptides accumulated. These peptides were analysed by SDS/PAGE and Western blotting, and the N-terminal sequences were determined for the four major peptides, which had sizes of 30, 22, 20 and 15 kDa. Sequence data identified five fission sites in the neutral proteinase, three of which were identical with autodigestion target sites in thermolysin, a thermostable neutral proteinase. Comparison of the identified fission sites of the B. subtilis neutral proteinase with the known substrate-specificity of the enzyme indicated that they were in agreement, showing a preference for the generation of fissions at the N-terminal side of large hydrophobic residues, such as leucine, isoleucine and methionine. These results suggest a high degree of similarity in the three-dimensional structures of B. subtilis neutral proteinase and thermolysin.
在不同条件下研究了纯化的枯草芽孢杆菌中性蛋白酶的自催化降解作用。在高温且无抑制条件下，成熟蛋白迅速降解，但未出现特定降解产物的积累。然而，通过在冰上长时间孵育纯化的中性蛋白酶，特定肽段得以积累。通过SDS/PAGE和蛋白质免疫印迹法对这些肽段进行了分析，并测定了四种主要肽段（大小分别为30、22、20和15 kDa）的N端序列。序列数据确定了中性蛋白酶中的五个裂解位点，其中三个与嗜热菌蛋白酶（一种耐热中性蛋白酶）的自消化靶点相同。将枯草芽孢杆菌中性蛋白酶已确定的裂解位点与该酶已知的底物特异性进行比较，结果表明二者相符，显示出在大的疏水残基（如亮氨酸、异亮氨酸和蛋氨酸）的N端一侧更倾向于产生裂解。这些结果表明枯草芽孢杆菌中性蛋白酶和嗜热菌蛋白酶在三维结构上具有高度相似性。
