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Endopeptidase activities during the differentiation of Zinnia cells into tracheary elements (TEs) were examined with several peptidyl 4-methyl-7-coumarylamido (MCA) as substrates. The activity that hydrolysed carbobenzoxy-Phe-Arg-MCA (Z-Phe-Arg-MCA) at pH 5 increased in a differentiation-related manner: this activity, which was not observed in freshly isolated mesophyll cells was induced by the combination of alpha-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) that is necessary for differentiation of TEs, but not by NAA or BA alone. The activity in cells cultured in TE-inductive medium that contained both NAA and BA increased very rapidly between the 48th and 60th hours of culture, when the number of TE increased rapidly. A protease responsible for this activity with a molecular mass of 30 kDa was partially purified from cells which had been cultured in the TE-inductive medium and included many immature TEs. Strong inhibition by L-3-trans-carboxyoxiran-2-carbonyl-L-Leu- -agmatin (E-64), and activation by dithiothreitol (DTT) indicated that this protease belongs to a family of cysteine endopeptidases (EC 3.4.22).
Minami et al. (Fri,) studied this question.