PROTEINASE-A, A STORAGE-GLOBULIN-DEGRADING ENDOPEPTIDASE OF VETCH (VICIA-SATIVA, L.) SEEDS, IS NOT INVOLVED IN EARLY STEPS OF STORAGE-PROTEIN MOBILIZATION

Proteinase A is a papain-like cysteine endopeptidase of vetch (Vicia s ativa L.) which was assumed to initiate storage-globulin breakdown jus t after the onset of seed germination. This enzyme was purified from c otyledons of vetch seedlings. On gelatin-containg SDS gels, active pro teinase A migrated with an apparent molecular mass of 21 kDa, whereas after heat denaturation its molecular size on SDS/PAGE was 29 kDa. Alt hough proteinase A is capable of hydrolyzing storage globulins in vitr o it could not be localized in the protein-body fraction of cotyledons from germinating seeds. cDNA clones encoding proteinase A precursor h ave been obtained by PCR. The precursor is composed of an N-terminal s ignal sequence followed by a propeptide. the region encoding mature pr oteinase A, and a C-terminal KDEL sequence. Mature proteinase A with a derived molecular mass of 25244 Da doss not have the KDEL sequence. T he derived amino acid sequence of the proteinase A precursor is 78.2% identical to sulfhydryl-endopeptidase (SH-EP) a cysteine endopeptidase from germinating Vigna mungo seedlings. Northern blot analysis indica ted that proteinase A mRNA appears de novo in cotyledons of 1-day-germ inated vetch seeds, where its amount increases up to day 6. No protein ase A mRNA uas detected in other vetch organs, not even in the embryo axis, which contains stored globulins. By means of antibodies raised a gainst the purified and against recombinantly produced proteinase A, t he 29-kDa bands of mature proteinase A were detected in cotyledon extr acts of 6-day-germinated seeds when globulin degradation has already f ar proceeded. The reported data do not agree with the proposed trigger ing role of proteinase A in storage globulin breakdown during germinat ion.