Achstetter T, Emter O, Ehmann C, Wolf D H
J Biol Chem. 1984 Nov 10;259(21):13334-43.
A previous study led to the discovery of new proteinases in yeast (Achstetter, T., Ehmann, C., and Wolf, D. H. (1981) Arch. Biochem. Biophys. 207, 445-454). The search for proteolytic enzymes active in the neutral pH range has been extended. Studies were done on a mutant lacking four well-known proteinases involved in protein degradation, the two endoproteinases A and B and the two carboxypeptidases Y and S. Twenty-nine chromogenic peptides (amino terminally blocked peptidyl-4-nitroanilides) as well as [3H]methylcasein were used as substrates in this search. For the detection of endoproteolytic activity using chromogenic peptide substrates two versions of the assay were used. In one system the direct cleavage of the 4-nitroanilide bond was measured. In the second, the cleavage of the chromogenic peptide at some site other than the 4-nitroanilide bond was measured. Both variations led to the discovery of multiple proteinase activities. Regulation of these proteolytic activities under different growth conditions of cells was observed. Proteolytic activity on [3H]methylcasein was also found. Ion-exchange chromatography and gel filtration were used for the reproducible separation of the multiple proteolytic activities.
此前的一项研究发现了酵母中的新蛋白酶（阿奇施泰特，T.，埃曼，C.，以及沃尔夫，D. H.（1981年）《生物化学与生物物理学文献》207卷，445 - 454页）。对在中性pH范围内具有活性的蛋白水解酶的搜寻范围得到了扩展。研究针对一种缺乏参与蛋白质降解的四种知名蛋白酶的突变体展开，这四种蛋白酶分别是两种内肽酶A和B以及两种羧肽酶Y和S。在此次搜寻中，使用了29种生色肽（氨基端封闭的肽基 - 4 - 硝基苯胺）以及[³H]甲基酪蛋白作为底物。为了使用生色肽底物检测内蛋白水解活性，采用了两种版本的测定方法。在一个系统中，测量4 - 硝基苯胺键的直接裂解。在第二个系统中，测量生色肽在4 - 硝基苯胺键以外的某个位点的裂解。这两种变体均导致发现了多种蛋白酶活性。观察到了这些蛋白水解活性在细胞不同生长条件下的调控情况。还发现了对[³H]甲基酪蛋白的蛋白水解活性。离子交换色谱法和凝胶过滤法被用于对多种蛋白水解活性进行可重复的分离。
