Levy M R, McConkey C L
J Cell Physiol. 1977 Feb;90(2):253-63. doi: 10.1002/jcp.1040900211.
A protease from Tetrahymena pyriformis inactivated eight of nine commercially available enzymes tested, including lactate deyhdrogenase, isocitrate dehydrogenase (TPN-specific), glucose-6 phosphate dehydrogenase, D-amino acid oxidase, fumarase, pyruvate kinase, hexokinase, and citrate synthase. Urate oxidase was not inactivated. Inactivation occurred at neutral pH, was prevented by inhibitors of the protease, and followed first order kinetics. In those cases tested, inactivation was enhanced by mercaptoethanol. Most of the enzyme-inactivating activity was due to a protease of molecular weight 25,000 that eluted from DEAE-Sephadex at 0.3 M KCl. A second protease of this molecular weight, which was not retained by the gel, inactivated only isocitrate dehydrogenase and D-amino acid oxidase. These two proteases could also be distinguished by temperature and inhibitor sensitivity. Two other protease peaks obtained by DEAE-Sephadex chromatography had little or no no enzyme inactivating activity, while another attacked only D-amino acid oxidase. At least six of the enzymes could be protected from proteolytic inactivation by various ligands. Isocitrates dehydrogenase was protected by isocitrate, TPN, or TPNH, glucose-6-dehydrogenase by glucose-6-P or TPN, pyruvate kinase by phosphoenolypyruvate or ADP, hexokinase by glucose, and fumarase by a mixture of fumarate and malate. Lactate dehdrogenase was not protected by either of its substrates of coenzymes. Citrate synthase was probably protected by oxalacetate. Our data suggest that the protease or proteases discussed here may participate in the inactivation or degradation of a least some enzymes in Tetrahymena. Since the inactivation occurs at neutral pH, this process could be regulated by variations in the cellular levels of substrates, coenzymes, or allosteric regulators resulting form changes in growth conditions or growth state. Such a mechanism would permit the selective retention of enzymes of metabolically active pathways.
来自梨形四膜虫的一种蛋白酶使所测试的九种市售酶中的八种失活，这些酶包括乳酸脱氢酶、异柠檬酸脱氢酶（TPN特异性）、葡萄糖-6-磷酸脱氢酶、D-氨基酸氧化酶、延胡索酸酶、丙酮酸激酶、己糖激酶和柠檬酸合酶。尿酸氧化酶未被失活。失活发生在中性pH条件下，可被蛋白酶抑制剂阻止，并且符合一级动力学。在测试的那些情况下，巯基乙醇可增强失活作用。大部分酶失活活性归因于一种分子量为25,000的蛋白酶，该蛋白酶在0.3M KCl条件下从DEAE-葡聚糖凝胶上洗脱下来。另一种分子量相同的蛋白酶，未被凝胶保留，仅使异柠檬酸脱氢酶和D-氨基酸氧化酶失活。这两种蛋白酶在温度和抑制剂敏感性方面也可区分。通过DEAE-葡聚糖凝胶色谱法获得的另外两个蛋白酶峰几乎没有或没有酶失活活性，而另一个仅作用于D-氨基酸氧化酶。至少六种酶可被各种配体保护免受蛋白水解失活。异柠檬酸脱氢酶受到异柠檬酸、TPN或TPNH的保护，葡萄糖-6-脱氢酶受到葡萄糖-6-P或TPN的保护，丙酮酸激酶受到磷酸烯醇丙酮酸或ADP的保护，己糖激酶受到葡萄糖的保护，延胡索酸酶受到延胡索酸和苹果酸混合物的保护。乳酸脱氢酶未受到其任何一种底物或辅酶的保护。柠檬酸合酶可能受到草酰乙酸的保护。我们的数据表明，这里讨论的一种或多种蛋白酶可能参与了四膜虫中至少一些酶的失活或降解。由于失活发生在中性pH条件下，这个过程可能受生长条件或生长状态变化导致的底物、辅酶或变构调节物细胞水平变化的调控。这样一种机制将允许选择性保留代谢活跃途径的酶。
