CATHEPSIN-H - ENDOAMINOPEPTIDASE FROM RAT-LIVER LYSOSOMES

Abstract

Cathepsin H is an endoaminopeptidase belonging to the group of thiol enzymes. It was purified from rat liver lysosomes by gel filtration on Sephadex G-75, chromatography on CM-Sephadex C-50, on DEAE-Cellulose DE-52 and subsequently on an organomercurical adsorbent. The MW of cathepsin H was found to be 28,000, and the isoelectric point was estimated to be at pH 7.1 by analytical isoelectric focusing. Cathepsin H was designated an endoaminopeptidase because it catalyzes the hydrolysis of proteins, N-terminal substituted proteins and amino acid derivatives, respectively, as well as of peptides of various chain length and N-terminal free amino acid derivatives. Cathepsin H shows amidase and esterase activity, but it does not show carboxypeptidase activity. The finding of the amino- and endopeptidase nature of cathepsin H was revealed mainly by results obtained with inhibitors and by the rather high temperature stability of the enzyme. The chloromethyl ketone of leucine proves to be the strongest inhibitor of the aminopeptidase and endopeptidase activity, whereas leupeptin has a less inhibitory action. Aminopeptidase substrates are competitive inhibitors of the endopeptidase activity, and endopeptidase substrates inhibit competitively the aminopeptidase activity. Cathepsin H shows highest activity at pH 6.0 in the presence of 1-5 mM GSH [glutathione] and EDTA. The enzyme is stable for several months at slightly acid pH values in a deep frozen state.