An inhibitor of cytoplasmic guanine deaminase of rat liver was isolated from liver ;heavy mitochondrial' fraction after freezing and thawing and treatment with Triton X-100. 2. Submitochondrial fractionation revealed that the inhibitor was localized in the outer-membrane fraction. 3. The method of purification of inhibitor, involving precipitation with (NH(4))(2)SO(4) and chromatography on DEAE-cellulose, its precipitability by trichloroacetic acid and the pattern of absorption in the u.v. indicated that the inhibitor was a protein. In confirmation, tryptic digestion of the isolated material resulted in destruction of the inhibitor activity. The inhibitor was stable to acid, but labile to heat. 4. The isolated inhibitor required phosphatidylcholine (lecithin) for activity. Phosphatidylcholine also partially protected the inhibitor against heat inactivation. 5. When detergent treatment was omitted, the inhibitor activity of frozen mitochondria was precipitated by (NH(4))(2)SO(4) in a fully active form without supplementation with phosphatidylcholine, indicating that Triton X-100 ruptured the linkage between inhibitor and lipid. 6. A reconstituted sample of inhibitor-phosphatidylcholine complex was precipitated in a fully active form by dialysis against 2-mercaptoethanol, but treatment of the precipitate with NaCl yielded an extract which was inactive unless supplemented with fresh phosphatidylcholine. 7. We interpret the results as evidence that the inhibitor was present in vivo as a lipoprotein and that once the complex was dissociated by the action of detergent and the protein precipitated, there was an absolute need for exogenous phosphatidylcholine for its activity. The manner in which inhibitor associated with the outer membrane of rat liver mitochondria might regulate the activity of the enzyme in the supernatant has been suggested.
摘要
从大鼠肝脏中分离出一种胞质鸟嘌呤脱氨酶抑制剂；该抑制剂来自经冻融和Triton X - 100处理后的肝脏重线粒体部分。2. 亚线粒体分级分离显示该抑制剂定位于外膜部分。3. 抑制剂的纯化方法包括用硫酸铵沉淀和在DEAE - 纤维素上进行色谱分离，其对三氯乙酸的沉淀性以及紫外吸收图谱表明该抑制剂是一种蛋白质。经证实，对分离得到的物质进行胰蛋白酶消化会导致抑制剂活性丧失。该抑制剂对酸稳定，但对热不稳定。4. 分离得到的抑制剂的活性需要磷脂酰胆碱（卵磷脂）。磷脂酰胆碱也能部分保护抑制剂免受热失活。5. 若省略去污剂处理，冷冻线粒体的抑制剂活性可被硫酸铵以完全活性形式沉淀出来，无需补充磷脂酰胆碱，这表明Triton X - 100破坏了抑制剂与脂质之间的连接。6. 抑制剂 - 磷脂酰胆碱复合物的重构样品通过对2 - 巯基乙醇进行透析以完全活性形式沉淀出来，但用氯化钠处理沉淀物得到的提取物无活性，除非补充新鲜的磷脂酰胆碱。7. 我们将这些结果解释为证据，表明该抑制剂在体内以脂蛋白形式存在，并且一旦复合物因去污剂的作用而解离且蛋白质沉淀，其活性就绝对需要外源性磷脂酰胆碱。有人提出抑制剂与大鼠肝脏线粒体外膜结合的方式可能调节上清液中酶的活性。
