Purification of a liver alkaline protease which degrades oxidatively modified glutamine synthetase. Characterization as a high molecular weight cysteine proteinase.
A nonlysosomal alkaline protease which degrades the oxidatively modified form of Escherichia coli glutamine synthetase has been purified to apparent homogeneity from rat and mouse liver acetone powders. Its molecular weight was determined to be 300,000 by Sephacryl S-300 gel filtration but results of further studies using high pressure liquid chromatography gel filtration suggest a value of 650,000. Examination of the subunit structure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed multiple bands of molecular weights between 22,000 and 34,000. The alkaline protease was inhibited by thiol reagents. Phenylmethylsulfonyl fluoride, aprotinin, leupeptin, antipain, and chymostatin partially inhibited the protease. The inhibition by phenylmethylsulfonyl fluoride was prevented by dithiothreitol, and alpha 1-antitrypsin and soybean trypsin inhibitor did not inhibit. No inhibition was observed with metalloprotease inhibitors. The alkaline protease is active over a broad range of pH with optimum activity for the degradation of oxidized glutamine synthetase around pH 9.0. Its activity is not stimulated by MgATP. A study of the products of insulin B chain degradation demonstrated major cleavage sites at Gln13-Ala14, Leu15-Tyr16, Cys(SO3H)19-Gly20, Gln4-His5, and Leu17-Val18. Based on its endopeptidase activity and its inhibitor specificity, the alkaline protease should be classified as a cysteine proteinase. It appears to be distinct from previously described proteinases and is likely involved in nonlysosomal mechanisms of intracellular protein turnover.
Rivett
A J
AJ
D000818:Animals; D002845:Chromatography; D003546:Cysteine Endopeptidases; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005260:Female; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011480:Protease Inhibitors; D051381:Rats; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D007328:Insulin; D011480:Protease Inhibitors; D013439:Sulfhydryl Reagents; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D005974:Glutamate-Ammonia Ligase