Kessler E, Safrin M
Maurice and Gabriela Goldschleger Eye Research Institute, Tel Aviv University, Sackler Faculty of Medicine, Sheba Medical Center, Tel Hashomer, Israel.
J Bacteriol. 1988 Nov;170(11):5241-7. doi: 10.1128/jb.170.11.5241-5247.1988.
Three cell-associated elastase precursors with approximate molecular weights of 60,000 (P), 56,000 (Pro I), and 36,000 (Pro II) were identified in Pseudomonas aeruginosa cells by pulse-labeling with [35S]methionine and immunoprecipitation. In the absence of inhibitors, cells of a wild-type strain as well as those of the secretion-defective mutant PAKS 18 accumulated Pro II as the only elastase-related radioactive protein. EDTA but not EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] inhibited the formation of Pro II, and this inhibition was accompanied by the accumulation of Pro I. P accumulated in cells labeled in the presence of ethanol (with or without EDTA), dinitrophenol plus EDTA, or carbonyl cyanide m-chlorophenyl hydrazone plus EDTA. Pro I and Pro II were localized to the periplasm, and as evident from pulse-chase experiments, Pro I was converted to the mature extracellular enzyme with Pro II as an intermediate of the reaction. P was located to the membrane fraction. Pro I but not Pro II was immunoprecipitated by antibodies specific to a protein of about 20,000 molecular weight (P20), which, as we showed before (Kessler and Safrin, J. Bacteriol. 170:1215-1219, 1988), forms a complex with an inactive periplasmic elastase precursor of about 36,000 molecular weight. Our results suggest that the elastase is made by the cells as a preproenzyme (P), containing a signal sequence of about 4,000 molecular weight and a "pro" sequence of about 20,000 molecular weight. Processing and export of the preproenzyme involve the formation of two periplasmic proenzyme species: proelastase I (56 kilodaltons [kDa]) and proelastase II (36 kDa). The former is short-lived, whereas proelastase II accumulates temporarily in the periplasm, most likely as a complex with the 20-kDa propeptide released from proelastase I upon conversion to proelastase II. The final step in elastase secretion seems to required both the proteolytic removal of a small peptide from proelastase II and dissociation of the latter from P20.
通过用[35S]甲硫氨酸脉冲标记和免疫沉淀法，在铜绿假单胞菌细胞中鉴定出三种细胞相关的弹性蛋白酶前体，其近似分子量分别为60,000（P）、56,000（Pro I）和36,000（Pro II）。在没有抑制剂的情况下，野生型菌株的细胞以及分泌缺陷型突变体PAKS 18的细胞都积累Pro II作为唯一的与弹性蛋白酶相关的放射性蛋白。EDTA而非EGTA [乙二醇双（β-氨基乙醚）-N，N，N'，N'-四乙酸]抑制Pro II的形成，并且这种抑制伴随着Pro I的积累。P在存在乙醇（有或没有EDTA）、二硝基苯酚加EDTA或羰基氰化物间氯苯腙加EDTA的情况下标记的细胞中积累。Pro I和Pro II定位于周质，并且从脉冲追踪实验可以明显看出，Pro I以Pro II作为反应中间体转化为成熟的细胞外酶。P定位于膜组分。Pro I而非Pro II被分子量约为20,000的蛋白质（P20）的特异性抗体免疫沉淀，正如我们之前所表明的（Kessler和Safrin，《细菌学杂志》170:1215 - 1219，1988），它与分子量约为36,000的无活性周质弹性蛋白酶前体形成复合物。我们的结果表明，弹性蛋白酶由细胞作为前原酶（P）产生，其包含分子量约为4,000的信号序列和分子量约为20,000的“原”序列。前原酶的加工和输出涉及形成两种周质原酶种类：原弹性蛋白酶I（56千道尔顿[kDa]）和原弹性蛋白酶II（36 kDa）。前者寿命短暂，而原弹性蛋白酶II暂时在周质中积累，最有可能作为与原弹性蛋白酶I转化为原弹性蛋白酶II时释放的20 kDa前肽的复合物。弹性蛋白酶分泌的最后一步似乎既需要从原弹性蛋白酶II中蛋白水解去除一个小肽，又需要后者与P20解离。
