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)
Catanese
J J
JJ
Molecular and Cellular Biology Department, Roswell Park Cancer Institute, Buffalo, New York 14263.
Kress
L F
LF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002853:Chromatography, Liquid; D003435:Crotalid Venoms / Q000494:pharmacology*; D004247:DNA; D006801:Humans; D006868:Hydrolysis; D008666:Metalloendopeptidases / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009893:Opossums / Q000097:blood; D017386:Sequence Homology, Amino Acid; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism
D003435:Crotalid Venoms; D000515:alpha 1-Antitrypsin; D004247:DNA; D008666:Metalloendopeptidases