Isolation and primary structures of seven serine proteinase inhibitors from Cyclanthera pedata seeds.
Seven new trypsin inhibitors, CyPTI I-VII, were purified from ripe seeds of Cyclanthera pedata by affinity chromatography on immobilized chymotrypsin in the presence of 5 M NaCl followed by preparative native PAGE at pH 8.9. The CyPTIs (Cyclanthera pedata trypsin inhibitors) belong to a well-known squash inhibitor family. They contain 28-30 amino acids and have molecular weights from 3031 to 3367 Da. All the isolated inhibitors strongly inhibit bovine beta-trypsin (K(a)>10(11) M(-1)) and, more weakly, bovine alpha-chymotrypsin (K(a) approximately 10(4)-10(6) M(-1)). In the presence of 3 M NaCl the association constants of CyPTIs with alpha-chymotrypsin increased a few hundred fold. Taking advantage of this phenomenon, a high concentration of NaCl was used to isolate the inhibitors by affinity chromatography on immobilized chymotrypsin. It was found that although one of them, CyPTI IV, had split the Asn25-Gly26 peptide bond, its inhibitory activity remained unchanged. The hydrolyzed bond is located downstream of the reactive site. Presumably, the inhibitor is a naturally occurring, double-chain protein arising during posttranslational modifications.
Kowalska
Jolanta
J
Institute of Biochemistry and Molecular Biology, University of Wrocław, Tamka 2, 50-137 Wrocław, Poland. jolanta.kowalska@bf.uni.wroc.pl
Zabłocka
Agnieszka
A
Wilusz
Tadeusz
T
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002918:Chymotrypsin / Q000737:chemistry; D028464:Cucurbita / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D012639:Seeds / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry*; D015842:Serine Proteinase Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology*; D014357:Trypsin / Q000737:chemistry
D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D014357:Trypsin