Purification and properties of renin and gamma-renin from the mouse submaxillary gland.

Abstract

The purification of renin from male mouse submaxillary glands on a 100-mg scale yielded substantial amounts of a closely related protease, tentatively designated y-renin.y-Renin and renin quantitatively produced angiotensin I from synthetic tetradecapeptide renin substrate.Renin would, and y-renin would not, produce angiotensin I from hog angiotensinogen (natural renin substrate).y-Renin was purified to homogeneity in polyacrylamide gel electrophoresis by a combination of ion exchange chromatography, molecular sieve chromatography, and affinity chromatography on pepstatin A-aminohexyl-Sepharose. y-Renin had a mobility on polyacrylamide gel electrophoresis in sodium dodecyl sulfate corresponding to a molecular weight of 26,000 when run without a reducing agent.y-Renin reduced with 2-mercaptoethanol showed two bands when electrophoresed on polyacrylamide in sodium dodecyl sulfate, with apparent molecular weights of 7,200 and 4,400.The amino acid composition and NHz-terminal sequences of y-renin were determined.Reduced, alkylated, and dialyzed y-renin had two NHzterminal sequences which were 75% homologous to the sequence from residues 1 to 40 and 137 to 176 of the ysubunit of mouse nerve growth factor.y-Renin was also homologous in sequence to a number of other serine proteases.y-Renin hydrolyzed the p-nitroanilide ester of benzoyl arginine at a maximal rate of 0.18 pmol ofpnitroaniline produced/min/mg of protein at pH 8.00 in 0.10 M Tris-HC1 at 25 "C, with a Michaelis constant of 38 p ~. Benzamidine and pepstatin were competitive inhibitors of thep-nitroanilide ester of benzoyl arginine hydrolysis by y-renin, and diisopropylfluorophosphate was an irreversible inhibitor; all three inhibitors also inhibited the cleavage of synthetic tetradecapeptide renin substrate by y-renin.It is proposed that renin and y-renin may be in mouse saliva to cleave peptides which are homologous to angiotensinogen and which have potent vasoactivity and central nervous system action.Renin (EC 3.4.99.19) is an important component of the renin-angiotensin system (for reviews, see Refs.1-3).Interest in the structure of renin has been stimulated by the recent success in the treatment of hypertension by Captopril(4) and subsequently by MK-421 ( 5 ) , both of which are inhibitors of the angiotensin-converting enzyme (EC 3.4.15.1), and which were developed as the result, in part, of analysis of the crystal