A New Endopeptidase Activity Induced in the Last Stage of Tracheary Element Differentiation of Zinnia Cells

Abstract

Endopeptidase activities during in vitro tracheary element (TE) differentiation of Zinnia cells were investigated using peptidyl 4-methylcoumaryl-7-amide (MCA) compounds as substrate. Endopeptidase activities against Boc-Val-Leu-Lys-MCA, with an acidic pH optimum and against Boc-Phe-Ser-Arg-MCA, with an alkaline pH optimum were found to be induced preferentially in cells cultured in TE differentiation-inductive medium, although the former activity appeared earlier than the latter one during the culture. Each activity was eluted as a single peak in DEAE-Sepharose column chromatography. The nature of Boc-Val-Leu-Lys-MCA hydrolyzing activity was similar to that of a papain-like cysteine protease as reported previously by our group. On the other hand, the partially purified Boc-Phe-Ser-Arg-MCA hydrolyzing activity was completely inhibited by leupeptin and weakly by E-64, but not by pepstatin A, 1, 10-phenanthroline, or PMSF, suggesting that this activity results from a cysteine protease(s). These results reveal the Boc-Phe-Ser-Arg-MCA hydrolyzing activity is due to a novel cysteine protease with an alkaline pH optimum which may function during TE differentiation.