Melloni E, Pontremoli S, Salamino F, Sparatore B, Michetti M, Horecker B L
Arch Biochem Biophys. 1984 Aug 1;232(2):505-12. doi: 10.1016/0003-9861(84)90567-8.
Two Ca2+-requiring proteinases have been purified from rabbit liver cytosol and shown to be present in isolated hepatocytes. They differ in relative molecular mass, with the major and minor forms, Mr = 150,000 and Mr = 200,000, accounting for 75 and 18% of the total cytosolic neutral proteinase activity, respectively. Both are recovered as inactive proenzymes that can be converted to the active, low-Ca2+-requiring proteinases by incubation with Ca2+ and substrate [S. Pontremoli, E. Melloni, F. Salamino, B. Sparatore, M. Michetti, and B. L. Horecker (1984) Proc. Natl. Acad. Sci. USA 81, 53-56. Each proenzyme is composed of two subunits, with molecular masses of 80 and 100 kDa, respectively. Activation of the proenzymes was found to correlate with their dissociation into subunits. The optimum pH for conversion of the proenzymes to the active proteinases in the presence of 5 mM Ca2+ and 2 mg/ml of denatured globin was approximately 7.5, and the same pH optimum was observed for the digestion of denatured globin by the activated proteinases. Following activation, each proteinase was observed to undergo autolytic inactivation at rates that were dependent on the concentration of both Ca2+ and the digestible substrate. A model is proposed for the activation of the proenzymes and the subsequent inactivation of the active proteinases.
已从兔肝细胞溶胶中纯化出两种需要钙离子的蛋白酶，并证实它们存在于分离的肝细胞中。它们的相对分子质量不同，主要形式和次要形式的相对分子质量分别为150,000和200,000，分别占细胞溶胶中性蛋白酶总活性的75%和18%。两者均以无活性的酶原形式回收，通过与钙离子和底物一起孵育可转化为活性的、低钙离子需求的蛋白酶[S. Pontremoli, E. Melloni, F. Salamino, B. Sparatore, M. Michetti, and B. L. Horecker (1984) Proc. Natl. Acad. Sci. USA 81, 53 - 56]。每种酶原由两个亚基组成，分子量分别为80 kDa和100 kDa。发现酶原的激活与其解离为亚基相关。在5 mM钙离子和2 mg/ml变性球蛋白存在下，酶原转化为活性蛋白酶的最适pH约为7.5，激活的蛋白酶消化变性球蛋白时也观察到相同的最适pH。激活后，观察到每种蛋白酶以依赖于钙离子浓度和可消化底物浓度的速率进行自溶失活。提出了一个关于酶原激活和随后活性蛋白酶失活的模型。
