Characterization of Hydrolase H, a New Muscle Protease Possessing Aminoendopeptidase Activity

Abstract

A newly-identified protease from rabbit skeletal muscle, named hydrolase H [Okitani, A. et al. (1980) Agric. Biol. Chem. 44, 1705–1708] has been shown to hydrolyze l-leucine β-naphthylamide as well as α-N-benzoyl-dl-arginine β-naphthylamide. The enzyme hydrolyzes prolamine in the manner of an endopeptidase and hydrolyzes tripeptides and tetrapeptides in the manner of an aminopeptidase. Thus it has been concluded that the enzyme possesses the properties of both endopeptidase and aminopeptidase and that it should be classified as an aminoendopeptidase. Both activities of endopeptidase and aminopeptidase are maximal at pH 7.5–8.0 and enhanced remarkably by thiol compounds. Both activities are stable at pH 7–9 and protected by 2-mercaptoethanol. They are inhibited by monoiodoacetic acid, leupeptin, Zn²⁺ and Ni²⁺, but not affected by EDTA, trypsin inhibitor, pepstatin, antipain and phenylmethylsulfonyl fluoride. These results suggest that the enzyme is a thiol protease. The enzyme is composed of three kinds of subunits, the chain M_r of which are 51000, 72000 and 92000.