Fusahiro Ogata

Evidence for and properties of a tripeptidyl peptidase in soybean (Glycine max Merr.) extract

Abstract A tripeptidyl peptidase which sequentially releases tripeptide units from free amino-terminal ends of peptides was detected in soybean (Glycine max Merr.) extract. The partially purified preparation of the peptidase possesses activity which releases p-nitroaniline from Ala-Pro-Phe-Np, but not from Phe-Np, Pro-Phe-Np and Boc-Ala-Pro-Phe-Np. Tripeptidyl p-nitroanilides with hydrophobic amino acids at the P1 position were better substrates for the peptidase than were those with charged amino acids. The peptidase also released tripeptides from natural peptides; two tripeptides are released sequentially from cholecystokinin-octapeptide, only the first tripeptide from angiotensin I, but none from bradykinin. The pH optimum of the peptidase for the hydrolysis of Ala-Pro-Phe-Np was around 7. The activity was completely inactivated by diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride, and was sensitive to N-ethylmaleimide, thus this peptidase is classified as a serine protease; however, the sulfhydryl group is also required for activity. The apparent native molecular weight of the enzyme was estimated to be about 1 000 000 as determined by gel filtration. This seems to be the first documented evidence for the presence of tripeptidyl peptidase in plant cells.