Purification and characterization of a high molecular weight proteinase (macropain) from human erythrocytes.
An alkaline proteinase, previously identified in rat liver and heart, has been purified from the soluble fraction of human erythrocytes. The proteinase has an apparent molecular weight of 600 000 and is composed of eight subunits with molecular weights ranging from 32 000 to 21 000. The proteinase degrades both protein and synthetic peptide substrates with a broad pH optimum of 7.5-11.0. Among the synthetic peptides tested, tripeptides with arginine at the P1 position (e.g. Z-Val-Leu-Arg-4-methoxy-2-napthylamine and Boc-Leu-Gly-Arg-4-methylcoumarin-7-amide) are particularly good substrates. The proteinase appears to be sulfhydryl-dependent and is inhibited completely by mersalyl acid and by hemin; inhibitors of serine and metallo-type proteinases have no effect on proteinase activity. Interestingly, a variety of other proteinase inhibitors such as leupeptin, chymostatin and N-ethylmaleimide failed to completely inhibit protein-hydrolyzing activities of the enzyme. These results indicate that these activities may be accounted for by at least two different catalytic sites. Proteinase activity is stable in the presence of 1 M urea, 0.5% Triton X-100 or 0.03% SDS and is not affected by ATP. Based on the high molecular weight and sulfhydryl-dependence, we have named this proteinase macropain.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D010447:Peptide Hydrolases / Q000097:blood*; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D046911:Macromolecular Substances; D009842:Oligopeptides; D011480:Protease Inhibitors; D013439:Sulfhydryl Reagents; D000255:Adenosine Triphosphate; D010447:Peptide Hydrolases