Miyata S, Moriyama R, Miyahara N, Makino S
Department of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University, Japan.
Microbiology (Reading). 1995 Oct;141 ( Pt 10):2643-50. doi: 10.1099/13500872-141-10-2643.
Antiserum was raised against a 31 kDa spore-cortex-lytic enzyme, which is released during germination of Clostridium perfringens S40 spores. Western blotting of dormant spore and vegetative cell fractions separated by SDS-PAGE indicated that the 31 kDa enzyme is spore-specific and that the enzyme in the dormant spore exists as a 36 kDa protein which has no cortex-lytic activity. A gene encoding the 31 kDa enzyme, sleC, was cloned into Escherichia coli using a synthetic oligonucleotide as a hybridization probe and the nucleotide sequence of the entire gene was determined. The N-terminal amino acid sequence of the 36 kDa protein was found in this reading frame, confirming that the 36 kDa protein is a pro-form of the 31 kDa enzyme. The deduced amino acid sequence indicated that the 31 kDa enzyme is produced as a precursor, comprising three portions; an N-terminal prepro-sequence (114 amino acid residues), a pro-sequence (35 amino acid residues) and a mature enzyme (289 amino acid residues). It is suggested that the 36 kDa pro-enzyme is non-covalently attached to the exterior of the cortex layer, and that the proform is processed to release the active enzyme during germination.
针对一种31 kDa的芽孢皮层溶解酶制备了抗血清，该酶在产气荚膜梭菌S40芽孢萌发过程中释放。对经SDS-PAGE分离的休眠芽孢和营养细胞组分进行蛋白质免疫印迹分析表明，31 kDa的酶是芽孢特异性的，并且休眠芽孢中的该酶以一种无皮层溶解活性的36 kDa蛋白质形式存在。利用合成寡核苷酸作为杂交探针，将编码31 kDa酶的基因sleC克隆到大肠杆菌中，并测定了整个基因的核苷酸序列。在该阅读框中发现了36 kDa蛋白质的N端氨基酸序列，证实36 kDa蛋白质是31 kDa酶的前体形式。推导的氨基酸序列表明，31 kDa的酶以前体形式产生，包括三个部分：一个N端前原序列（114个氨基酸残基）、一个原序列（35个氨基酸残基）和一个成熟酶（289个氨基酸残基）。有人提出，36 kDa的酶原通过非共价方式附着在皮层外层，并且在萌发过程中原体被加工以释放出活性酶。
