Abstract
The results reported in the present paper and N‐terminal sequence homologies established by other authors strongly support the assumption that the gastric protease previously called bovine pepsin I or bovine pepsin B belongs to the aspartate protease group and corresponds to gastricsin (or pepsin C) (EC 3.4.23.3) from other species.Bovine gastricsin was prepared from commercial extracts of adult bovine vells by a procedure involving DEAE‐cellulose chromatography, gel filtration on Sephacryl S‐200 and a further DEAE‐cellulose chromatography. The preparation thus obtained was shown to be free of chymosin and bovine pepsin A by immunodiffusion, selective inactivation in urea and isoelectric focusing. Its molecular weight was estimated by gel filtration to be 32800.Bovine gastricsin displayed microheterogeneity on isoelectric focusing with pl values of the components ranging from 3.5 to 4.0. Chromatography of bovine gastricsin on hydroxyapatite separated three fractions, none of them being homogeneous by isoelectric focusing. Concanavalin‐A‐Sepharose 4B bound bovine gastricsin to some extent, but without any significant fractionation. Proteolytic activity could be detected directly on the isoelectric focusing gel for all the components of gastricsin and its fractions from hydroxyapatite and concanavalin‐A‐ Sepharose 4B.Bovine gastricsin and its fractions from hydroxyapatite have similar amino acid compositions, different from those of bovine chymosin and pepsin A but obviously related to those of human, simian and porcine gastricsins.Bovine gastricsin which is inactivated by reaction with diazoacetyl‐DL‐norleucine methyl ester and with 1,2‐epoxy‐(p‐nitrophenoxy)propane in a 1:1 and 1:2 stoichiometry, respectively, is able to hydrolyse a synthetic hexapeptide, Leu‐Ser‐Phe(NO2)‐Nle‐Ala‐Leu‐OMe, used as reference substrate for aspartate proteases, and exhibits a low activity towards N‐acetyl‐l‐phenylalanyl‐l‐diiodotyrosine. Its specific clotting activity with x‐casein as substrate is only half of that of chymosin and pepsin A.
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