Germinating barley grains contain five acid carboxypeptidases with complementary substrate specificities

Abstract

The high carboxypeptidase activity possessed by extracts of germinating barley grains around pH 5 was fractionated by sequential chromatography on DEAE-cellulose, hydroxypatite and Sephacryl S-200, and the fractions were assayed for hydrolytic activity against eight benzyloxycarbonyl-dipeptides (Z-dipeptides) and one Z-tripeptide. The results indicated that the overall activity of the extracts was due to five carboxypeptidases, each with a characteristic activity spectrum against the nine substrates used. Three of the enzymes liberated carboxy-terminal amino acids, including proline, provided that there was not a proline residue in the penultimate position. The other two enzymes were prolylcarboxypeptidases acting only on substrates with a proline residue in the penultimate position. The enzymes were designated barley carboxypeptidases I to V, and their best or most characteristic substrates and approximate molecular weights were: I, Z-Phe-Ala, 100 000; II, Z-Ala-Arg, 110 000; III, Z-Phe-Phe, 43 000; IV, Z-Pro-Ala, 170 000; V, Z-Gly-Pro-Ala, 95 000. All five enzymes were inhibited both by diisopropylfluorophosphate and by p-hydroxymercuribenzoate. There were differences in their sensitivities, however, carboxypeptidases II and IV requiring high concentrations of the inhibitors for extensive inhibition. The five barley enzymes resembled in several respects the three lysosomal carboxypeptidases of mammalian tissues.