A high molecular weight glutamyl endopeptidase and its endogenous inhibitors from cucumber leaves

We purified a glutamyl endopeptidase that is a major foliar endopeptidase i n cucumber. The endopeptidase had a molecular mass of 400 kDa, consisted of four subunits of 97 kDa, and was inactivated by SH-modifying reagents. Its optimum pH and optimum temperature were 8.0 and 30-37 degreesC, respective ly. An internal amino acid sequence of the endopeptidase was highly homolog ous to a partial sequence of unidentified proteins deduced from genetic inf ormation for Arabidopsis thaliana, soybean and rice, but not to the sequenc es of bacterial glutamyl endopeptidases or animal proteases. Therefore, the unidentified proteins might be glutamyl endopeptidases and be widely distr ibuted only among plant species. The activity of the cucumber glutamyl endo peptidase was inhibited by at least three inhibitors existing in cucumber l eaves. One of the inhibitors was a competitive inhibitor of 25 kDa, which d id not significantly inhibit commercial endopeptidases derived from animals and microorganisms. This suggests that the cucumber glutamyl endopeptidase might be controlled by endogenous inhibitors in vivo.