Guanine deaminase inhibitor from rat liver. Isolation and characterization.
1. 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.
D000619:Aminohydrolases / Q000037:antagonists & inhibitors*; D000645:Ammonium Sulfate; D000818:Animals; D002458:Cell Fractionation; D011232:Chemical Precipitation; D002848:Chromatography, DEAE-Cellulose; D003080:Cold Temperature; D004355:Drug Stability; D006147:Guanine; D008074:Lipoproteins / Q000302:isolation & purification / Q000494:pharmacology*; D008297:Male; D008930:Mitochondria, Liver; D010713:Phosphatidylcholines; D051381:Rats; D012965:Sodium Chloride; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D013501:Surface-Active Agents; D013997:Time Factors; D014238:Trichloroacetic Acid; D014357:Trypsin; D014461:Ultracentrifugation
D008074:Lipoproteins; D010713:Phosphatidylcholines; D013501:Surface-Active Agents; D012965:Sodium Chloride; D014238:Trichloroacetic Acid; D006147:Guanine; D014357:Trypsin; D000619:Aminohydrolases; D000645:Ammonium Sulfate