title: Interactions of serine proteinases with pNiXa, a serpin of Xenopus oocytes and embryos.
abstract: In a previous study, kinetic assays showed that pNiXa, an Ni(II)-binding serpin of Xenopus oocytes and embryos, strongly inhibits bovine chymotrypsin, weakly inhibits porcine elastase, and does not inhibit bovine trypsin. In this study, analyses by SDS-PAGE and gelatin zymography showed that an SDS-resistant complex is formed upon the interaction of pNiXa with bovine chymotrypsin. No such pNiXa-enzyme complex was detected after pNiXa interactions with porcine elastase, bovine trypsin, or human cathepsin G. The major products of pNiXa cleavage by the four proteinases were partially sequenced by Edman degradation. The cleavage products were also tested by immunoblotting with an antibody to the His-cluster of pNiXa, and by radio-blotting with 63Ni(II). These assays showed that chymotrypsin and elastase cleave pNiXa at the P1-P1 (Thr-Lys) peptide bond near the C-terminus, while trypsin and cathepsin G cleave pNiXa at specific peptide bonds near the N-terminus, within an interesting 26-residue segment, rich in Lys and Gln, that separates the His-cluster of pNiXa from the rest of the molecule. The segment lacks homology to other serpins, but resembles a domain of Xenopus POU3 transcription factor. This study identifies the specific sites for interactions of four serine proteinases with pNiXa, indicates that pNiXa inhibition of chymotrypsin involves a serpin-like mechanism, and shows that 63Ni(II)-binds to the His-cluster of pNiXa.
authors.affiliation: Department of Laboratory Medicine, School of Medicine, University of Connecticut, Farmington 06030, USA.
authors.forename: J
authors.initials: J
authors.lastname: Kotyza
authors.forename: A H
authors.initials: AH
authors.lastname: Varghese
authors.forename: G
authors.initials: G
authors.lastname: Korza
authors.forename: F W
authors.initials: FW
authors.lastname: Sunderman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism* / Q000494:pharmacology; D056649:Cathepsin G; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002417:Cattle; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009532:Nickel / Q000378:metabolism; D009865:Oocytes / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D015843:Serpins / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000378:metabolism; D013552:Swine; D014357:Trypsin; D014981:Xenopus / Q000196:embryology; D029867:Xenopus Proteins
chemical_list: D002352:Carrier Proteins; D010446:Peptide Fragments; D015842:Serine Proteinase Inhibitors; D015843:Serpins; D029867:Xenopus Proteins; C077804:SERPINA6 protein, Xenopus; D012967:Sodium Dodecyl Sulfate; D009532:Nickel; D002403:Cathepsins; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C534094:CTSG protein, human; D056649:Cathepsin G; D010196:Pancreatic Elastase; D014357:Trypsin