Serine proteinase from Cucurbita ficifolia seed; purification, properties, substrate specificity and action on native squash trypsin inhibitor (CMTI I)

• Published in: Biological chemistry Hoppe-Seyler Vol. 371; no. 9; pp. 889 - 895
• Main Authors: Dryjanski, M, Otlewski, J, Polanowski, A, Wilusz, T
• Format: Journal Article
• Language: English
• Published: Germany 01.09.1990
• Subjects: Amino Acid Sequence; Animals; casein; Cattle; Cucurbita ficifolia; enzyme activity; Insulin - metabolism; Kinetics; Molecular Sequence Data; Peptides - metabolism; Plant Proteins - metabolism; purification; seeds; Seeds - enzymology; Serine Endopeptidases - isolation & purification; Serine Endopeptidases - metabolism; serine proteinases; Substrate Specificity; substrates; trypsin inhibitors; Trypsin Inhibitors - metabolism
• ISSN: 0177-3593
• DOI: 10.1515/bchm3.1990.371.2.889

A proteinase was purified from resting seeds of Cucurbita ficifolia by ammonium sulfate fractionation and successive chromatography on CM-cellulose, (Sephacryl) S-300 and TSK DEAE-2SW (HPLC) columns. Inhibition by DFP and PMSF suggests that the enzyme is a serine proteinase. The apparent molecular mass of this enzyme is ca. 77 kDa. The optimum activity for hydrolysis of casein and Suc-Ala-Ala-Pro-Phe-pNA is around pH 10.5. The following peptide bonds in the oxidized insulin B-chain were hydrolysed by the proteinase: Phe1-Val2, Asn3-Gln4, Gln4-His5, Cya7-Gly8, Glu13-Ala14, Ala14-Leu15, Cya19-Gly20, Pro28-Lys29 and Lys29-Ala30. The proteinase is more selective towards the native squash seed trypsin inhibitor (CMTII) and primarily cuts off only its N-terminal arginine. The inhibitor devoided of the N-terminal arginine residue is still active against trypsin.