Involvement of carboxypeptidase in the degradation of the mung bean (Vigna radiata) trypsin inhibitor during germination and early seedling growth

Abstract

Examination of the substrate specifity of the carboxypeptidase activity of ungerminated and germinated mung beans (Vigna radiata (L.) Wilczek) reveals the presence of two distinct enzymes. The first of these, carboxypeptidase I, is maximally active against carbobenzyloxy-Ala-Phe. It is present in large amounts in the cotyledons of ungerminated seeds, and declines rapidly during germination. The second, carboxypeptidase II, is most active against carbobenzyloxy-Phe-Ala. This enzyme increases in the cotyledons during germination and seedling growth. The possible involvement of one or both of these enzymes in the degradation of the native mung bean trypsin inhibitor (MBTI - F) and its proteolytically modified forms (MBTI - E, - C, and - E') during germination was also examined. The enzyme catalyzing one step in the conversion of MBTI - E to MBTI - C has been isolated with 557-fold purification from germinated mung beans. The inhibitor-degrading enzyme has a molecular weight of approximately 60,000. It is optimally active against MBTI - E at between pH 4.0 to 4.5. No activity was found with MBTI - F, - E', or - C as substrates. The enzyme has been identified as the carboxypeptidase II on the basis of (1) coelution during chromatography, (2) action on synthetic and natural substrates, (3) sensitivity to enzyme inhibitors, and (4) temporal variation during germination. The MBTI degrading carboxypeptidase removes the two carboxyl-terminal residues from MBTI - E to produce an inhibitor species that co-migrates with MBTI - C on polyacrylamide gel electrophoresis, but has not been subjected to the internal cleavage and proteolysis at the amino-terminus found in MBTI - C.