Opossum serum alpha 1-proteinase inhibitor: purification, linear sequence, and resistance to inactivation by rattlesnake venom metalloproteinases.

Opossum (Didelphis virginiana) serum was fractionated with (NH4)2SO4 and then chromatographed on DEAE-Sepharose and phenyl-Sepharose. Affinity chromatography on a protein A-Sepharose-antibody column removed traces of opossum serum metalloproteinase inhibitors, and resulted in a homogeneous preparation of opossum alpha 1-proteinase inhibitor (alpha 1-PI). The inhibitor is a single-chain glycoprotein (17.7% carbohydrate) with an estimated M(r) = 54,000. An opossum liver cDNA library was immunoscreened, and clones containing cDNA encoding for the open reading frame for opossum alpha 1-PI were isolated. The cDNA inserts contained nucleotide sequences corresponding to the amino-terminal and an internal peptide sequence of opossum alpha 1-PI which had been separately determined by protein sequence analysis. The entire inserts coded for a protein consisting of a 21-residue signal peptide and a 389-residue mature protein. Opossum alpha 1-PI shows 51-58% identity with other mammalian alpha 1-PI amino acid sequences, and the conserved residues expected for a member for the serpin family have been retained. The carbohydrate attachment sites and the reactive site residues (M-S) of opossum alpha 1-PI are identical to those of human alpha 1-PI. Opossum alpha 1-PI formed stable enzyme/inhibitor complexes with trypsin, chymotrypsin, and human neutrophil elastase, but did not react with thrombin or with snake venom serine proteinases. Opossum alpha 1-PI was inactivated by papain or Pseudomonas aeruginosa elastase, and electrophoretic analysis of the reaction products indicated limited proteolysis in the reactive site loop of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)