INTERACTIONS OF SERINE PROTEINASES WITH PNIXA, A SERPIN OF XENOPUS OOCYTES AND EMBRYOS

In a previous study, kinetic assays showed that pNiXa, an Ni(II)-bindi ng serpin of Xenopus oocytes and embryos, strongly inhibits bovine chy motrypsin, weakly inhibits porcine elastase, and does not inhibit bovi ne trypsin. In this study, analyses by SDS-PAGE and gelatin zymography showed that an SDS-resistant complex is formed upon the interaction o f pNiXa with bovine chymotrypsin. No such pNiXa-enzyme complex was det ected 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 cleava ge products were also tested by immunoblotting with an antibody to the His-cluster of pNiXa, and by radio-blotting with Ni-63(II). These ass ays showed that chymotrypsin and elastase cleave pNiXa at the P-1-P-1( ') (Thr-Lys) peptide bond near the C-terminus, while trypsin and cathe psin G cleave pNiXa at specific peptide bonds near the N-terminus, wit hin an interesting 26-residue segment, rich in Lys and Gln, that separ ates the His-cluster of pNiXa from the rest of the molecule. The segme nt lacks homology to other serpins, but resembles a domain of Xenopus POU3 transcription factor. This study identifies the specific sites fo r interactions of four serine proteinases with pNiXa, indicates that p NiXa inhibition of chymotrypsin involves a serpin-like mechanism, and shows that Ni-63(II)-binds to the His-cluster of pNiXa. (C) 1998 Elsev ier Science B.V.