Soybean subtilisin-like protease involved in initiating storage protein degradation

A partial cDNA fragment (1.1 kb) of protease C1, an enzyme that initiates the proteolytic degradation of the beta -conglycinin storage proteins in the soybean (Glycine max [L.] Merrill. cv. Amsoy 71) through limited action at Glurich regions, has been cloned by reverse transcription-polymerase chain reaction (RT-PCR). The sequence was extended toward the 5' end by another 314 bases. The nucleotide sequence shows that protease C1 belongs to the subtilisin family of serine proteases. The sequence encompasses the critical Asp, His and Ser residues of the catalytic triad, as well as the Asn at the binding site. Northern analysis shows the presence of 2.5 kb mRNA not only in seedling also in developing seeds. The developing seeds, and even dry seeds. phenylmethylsulfonyl fluoride (PMSF) and EDTA-sensitive protease activity that only cleaves the alpha- and alpha'-, but not the beta -subunits, of soybean beta -conglycinin. The discrete proteolytic intermediates produced are of the same sizes as those produced by pure enzyme, as are the final 50 kDa and 48 kDa products. The activity is also sensitive to inhibition by synthetic poly-L-Glu, all characteristics of purified protease C1. These data suggest that protease C1, or an enzyme very similar to it. is synthesized in a form that is active in vitro. Because seeds do accumulate beta -conglycinin and because there is very little evidence of the proteolytic products of protease C1 action in extracts of dry seeds, one can surmise that the protease C1 is not particularly active in vivo during seed maturation. GenBank lists the sequence of the 1.1 kb fragment with accession number AAD02075.