Isolation, and catalytic and immunochemical properties of cathepsin D-like acid proteinase from rat erythrocytes.
An erythrocyte membrane-associated cathepsin D-like acid proteinase, termed "EMAP," was purified to homogeneity from freshly collected rat blood in a yield of 60-65%. The molecular weight of the enzyme was determined to be 80,000-82,000 by Sephadex G-100 gel filtration. The enzyme was inhibited strongly by pepstatin and partially by HgCl2, Pb(NO3)2, and iodoacetic acid. The preferred substrate for the enzyme was hemoglobin. The enzyme also hydrolyzed serum albumin and casein, but to lesser extents, with an optimum pH of 3.5-4.0. However, it could not hydrolyze leucyl-2-naphthylamide, benzyloxycarbonyl-Phe-Arg-4-methyl-7-coumarylamide or other synthetic substrates at pH values ranging from 3.5 to 9.5. The enzyme was very similar to human EMAP in a number of enzymatic properties, whereas it differed from rat cathepsin D in several respects, such as pH stability, molecular weight, isoelectric point, and chromatographic properties. Immunologically, the enzyme cross-reacted with the rabbit antibody prepared against human EMAP. The patterns of immunoelectrophoresis, immunoblotting, and immunoprecipitation of the enzyme were remarkably similar, if not identical, to those of human EMAP. In contrast, rat EMAP showed no reaction with the rabbit antibody raised to rat spleen cathepsin D. These results indicate that EMAP is a unique cathepsin D-like acid proteinase different from ordinary cathepsin D.
D000818:Animals; D002402:Cathepsin D / Q000378:metabolism; D020059:Cathepsin E; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000097:blood*; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007159:Immunologic Tests; D008970:Molecular Weight; D011480:Protease Inhibitors; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D011480:Protease Inhibitors; D010450:Endopeptidases; D020059:Cathepsin E; D002402:Cathepsin D