Studies on cathepsins of rat liver lysosomes. II. Comparative studies on multiple forms of cathepsin A.
The multiple forms of cathepsin A (AI, AII, and AIII) purified from the lysosome fraction of rat liver by Sephadex G-200 and DEAE-Sephadex chromatographies were studied comparatively. Forms AI, AII and AIII were stable between pH 3.0 and 5.5, and had pH optima for CBZ-Glu-Phe at 5.6, 5.8, and 5.9, respectively. These activities were rapidly lost on heating above 60 degrees. Their isoelectric points were at 4.7, 4.8, and 4.9, and the Michaelis constants for CBZ-Glu-Phe were calculated as 10, 6.6, and 4.2 X 10(-4)M, respectively. Activity was inhibited by Ag+, Au3+, Hg2+, iodine, and p-chloromercuribenzoate (PCMB). Diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), toluenesuffonyl fluoride (TSF), and sodium dodecyl sulfate (SDS) were inhibitory at a concentration of 10(-3)M. Soybean trypsin inhibitor, pepstatin, leupeptins, and antipain were not inhibitory, while chymostatin caused slight inhibition. No distinct difference was observed in the effects of these compounds on the multiple forms of cathepsin A despite differences in the molecular weights of these forms (100,000, 200,000, and 420,000, respectively). In immuno-diffusion analysis, cathepsin AI, AII, and AIII which had been treated with EDTA, dithiothreitol, PCMB, and a high concentration of NaCl, gave the same precipitin patterns as the untreated enzymes, but treatment with 6 M urea caused a slight alteration of the pattern. After SDS-treatment (50 mM or more), the precipitin lines of these multiple forms fused and gave a single, identical line. This suggests that the different forms of the cathepsin A are all composed of subunits which are immunologically identical or closely related, and that the subunits are mainly bound by hydrophobic forces. This conclusion is supported by results obtained by poliacrylamide gel electrophoresis in the presence of SDS.
D000818:Animals; D002403:Cathepsins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002729:Chloromercuribenzoates / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007527:Isoenzymes / Q000032:analysis; D007531:Isoflurophate / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D051381:Rats; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014361:Trypsin Inhibitors / Q000494:pharmacology
D002729:Chloromercuribenzoates; D007527:Isoenzymes; D008670:Metals; D013439:Sulfhydryl Reagents; D014361:Trypsin Inhibitors; D007531:Isoflurophate; D012967:Sodium Dodecyl Sulfate; D002403:Cathepsins