title: Isolation and partial characterization of an endopeptidase from Enterolobium contortisiliquum seeds.
abstract: 1. Aqueous extracts of Enterolobium contortisiliquum seeds contain an endopeptidase of M(r) 60,000 with specificity for basic amino acid residues. The enzyme was purified by chromatography on DEAE Sephadex, followed by gel filtration on Sephadex G-75 and affinity chromatography on Zinc-Sepharose. The overall purification was 300-fold and the yield about 46%. 2. The endopeptidase hydrolyzes benzoyl-arginine-p-nitroanilide (Bz-Arg-pNan) and acetyl-phenylalanine-arginine-p-nitroanilide (Ac-Phe-Arg-pNan) with Km 14.4 mM and 0.062 mM, respectively. Succinyl-phenylalanine-p-nitroanilide (Suc-Phe-pNan) and tosyl-arginine methyl ester (TAME) were not hydrolyzed. E. contortisiliquum endopeptidase also cleaves a seed protein of low molecular weight from the same E. contortisiliquum 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+, Fe3+ and Zn2+ ions inactivate the enzyme when Bz-Arg-pNan was the substrate. Enzyme activity is abolished by EDTA but is partially restored by Cu2+, Al3+, Ba2+, Mn2+, Mg2+, Fe2+, Ca2+ and Co2+ ions. The endopeptidase is not inhibited by the previously purified E. contortisiliquum inhibitors of trypsin and cysteine proteinases, or by soybean trypsin inhibitor (Oliva et al. (1987). Brazilian Journal of Medical and Biological Research, 20:767-770).
authors.affiliation: Departamento de Tecnologia de Alimentos, Universidade Federal Rural do Rio de Janeiro, Brasil.
authors.forename: G A
authors.initials: GA
authors.lastname: Silva
authors.forename: M L
authors.initials: ML
authors.lastname: Oliva
authors.forename: M U
authors.initials: MU
authors.lastname: Sampaio
authors.forename: M S
authors.initials: MS
authors.lastname: Araujo
authors.forename: C A
authors.initials: CA
authors.lastname: Sampaio
mesh_terms: D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D003546:Cysteine Endopeptidases / Q000302:isolation & purification*; D007887:Fabaceae / Q000201:enzymology*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010946:Plants, Medicinal; D011480:Protease Inhibitors / Q000378:metabolism; D012639:Seeds / Q000201:enzymology*
chemical_list: D011480:Protease Inhibitors; D003546:Cysteine Endopeptidases