PURIFICATION, CDNA CLONING AND CHARACTERIZATION OF PROTEINASE-B, AN ASPARAGINE-SPECIFIC ENDOPEPTIDASE FROM GERMINATING VETCH (VICIA-SATIVA L) SEEDS

Proteinase B, an asparagine specific endopeptidase, has been purified from germinating vetch (Vicia sativa L.) seeds. The final preparation consists of two enzymically active proteins with molecular masses of a pproximately 39 kDa and 37 kDa. Synthetic substrates were used to conf irm cleavage specificity of the proteinase B preparation. As expected, the enzyme cleaves the substrates at the C-terminal side of Asn resid ues. The octapaptide ETRNGVEE was digested most efficiently. When Gly was replaced by Ile or Glu, cleavage took place with lower efficiency. Polyclonal antibodies displayed both proteins in cotyledon extracts o f germinated vetch seeds. In addition, a strong cross-reacting protein band was found in cotyledon extracts of developing seeds, indicating the presence of a very similar enzyme during seed development. cDNA cl ones encoding proteinase B precursor have been obtained on the basis o f the N-terminal amino acid sequence DDDFEGTRWAILLAGS, by means of the polymerase chain reaction. The cDNA clones contain an open reading fr ame of 1479 bp encoding a polypeptide of 493 amino acids. The precurso r displayed 59% sequence identity to the cDNA-derived amino acid seque nce of a vacuolar Asn-specific enzyme from the developing castor bean endosperm which is thought to catalyze the post-translational processi ng of pro-proteins into the mature forms. Proteinase B is synthesized de novo during seed germination. The results of Southern-blot analyses suggested that there are at least two genes for proteinase B.