ISOLATION AND PARTIAL CHARACTERIZATION OF AN ENDOPEPTIDASE FROM ENTEROLOBIUM-CONTORTISILIQUUM SEEDS

1. Aqueous extracts of Enterolobium contortisiliquum seeds contain an endopeptidase of Mr 60,000 with specificity for basic amino acid resid ues. The enzyme was purified by chromatography on DEAE Sephadex, follo wed by gel filtration on Sephadex G-75 and affinity chromatography on Zinc-Sepharose. The overall purification was 300-fold and the yield ab out 46%. 2. The endopeptidase hydrolyzes benzoyl-arginine-p-nitroanili de (Bz-Arg-pNan) and acetyl-phenylalanine-arginine-p-nitroanilide (Ac- Phe-Arg-pNan) with Km 14.4 mM and 0.062 mM, respectively. Succinyl-phe nylalanine-p-nitroanilide (Suc-Phe-pNan) and tosyl-arginine methyl est er (TAME) were not hydrolyzed. E. contortisiliquum endopeptidase also cleaves a seed protein of low molecular weight from the same E. contor tisiliquum seeds, and converts Met-Lys-bradykinin into bradykinin (Arg -Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg). 3. Metals (1.0 mM) such as Cr3+, Fe 3+ and Zn2+ ions inactivate the enzyme when Bz-Arg-pNan was the substr ate. Enzyme activity is abolished by EDTA but is partially restored by Cu2+, Al3+, Ba2+, Mn2+, Mg2+, Fe2+, Ca2+ and Co2+ ions. The endopepti dase is not inhibited by the previously purified E. contortisiliquum i nhibitors of trypsin and cysteine proteinases, or by soybean trypsin i nhibitor (Oliva et al. (1987). Brazilian Journal of Medical and Biolog ical Research, 20: 767-770).