Purification and Properties of Two Enzymes Hydrolyzing Synthetic Substrates, N-α-Benzoyl-D,L-argininep-Nitroanilide and Leucinep-Nitroanilide, from Pea Seeds

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Abstract
Two hydrolases hydrolyzing separate synthetic substrates, N-α-benzoyI-d,l-arginine p-nitroanilide (BAPA) and leucine p-nitroanilide (LPA), were purified 230-fold from dry pea seeds by ammonium sulfate fractionation, heat treatment, DEAE-cellulose and Sephadex G-100 column chromatographies, and isoelectric focusing; one of them (BAPAase) hydrolyzed BAPA, and the other (LPAase), LPA. BAPAase and LPAase were separated from each other by DEAE-cellulose column chromatography. Both enzymes were none of metallo-, serine- and thiol-type enzymes, and BAPAase, but not LPAase, was activated by divalent cations. Both enzymes had no ability to hydrolyze globulins prepared from the seeds. Neither BAPAase nor LPAase activity in the cotyledons changed during 5 days of germination. The levels of both hydrolases were not influenced by the removal of embryo at 4~hr imbibition stage, or by supply of cycloheximide to the seeds. It is concluded that neither BAPAase nor LPAase participates in the initial breakdown of reserve proteins.