The structure and function of acid proteases. VIII. Purification and characterization of cathepsins D from Japanese monkey lung.
Two kinds of cathepsin D were found in Japanese monkey lung and were named cathepsins D-I and D-II. Cathepsin D-I was partially purified by ammonium sulfate fractionation and DEAE-cellulose column chromatography. It had properties common to other ordinary cathepsins D in terms of the elution position from a DEAE-cellulose column at pH 8.0, the pH-dependence of activity toward acid-denatured hemoglobin, and the molecular weight of 35,000 as determined by Sephadex G-100 gel filtration. On the other hand, cathepsin D-II was purified about 1,000-fold by a combination of ammonium sulfate fractionation and column chromatographies on DEAE-cellulose and Sephadex G-100. It was a very acidic protein as judged from its elution position from a DEAE-cellulose column at pH 8.0, and the high mobility toward the anode on disc gel electrophoresis at pH 8.6. Its molecular weight was determined to be 35,000 by Sephadex G-100 gel filtration and 39,000 by SDS-polyacrylamide gel electrophoresis. It was optimally active at pH 2.8 against acid-denatured hemoglobin as a substrate, showing 80% of the optimal activity at pH 1.0, and almost no activity above pH 4.0. This pH-profile of activity was similar to that of monkey pepsin C (gastricsin). It did not hydrolyze N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine, a synthetic substrate for pepsin, but was inhibited by a series of pepsin inhibitors such as pepstatin, 1,2-epoxy-3-(p-nitrophenoxy)propane, p-bromophenacyl bromide, and diazoacetyl-DL-norleucine methyl ester, although the diazo reagent was a rather weak inhibitor of the enzyme. The amino acid composition of cathepsin D-II was found to be fairly different from those of other cathepsins D. However, it showed a striking resemblance to that of Japanese monkey pepsinogen C, suggesting some evolutionary relationship between them.
D000098:Acetophenones / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001391:Azo Compounds / Q000494:pharmacology; D002403:Cathepsins / Q000302:isolation & purification / Q000378:metabolism*; D004852:Epoxy Compounds / Q000494:pharmacology; D000882:Haplorhini; D006863:Hydrogen-Ion Concentration; D008168:Lung / Q000201:enzymology*; D008251:Macaca; D008970:Molecular Weight; D009596:Nitrophenols / Q000494:pharmacology; D009646:Norleucine / Q000031:analogs & derivatives / Q000494:pharmacology; D010436:Pepstatins / Q000494:pharmacology
D000098:Acetophenones; D000596:Amino Acids; D001391:Azo Compounds; D004852:Epoxy Compounds; D009596:Nitrophenols; D010436:Pepstatins; D009646:Norleucine; D002403:Cathepsins