Studies on the turnover of methionine oxidized alpha-1-protease inhibitor in rats.
Alpha-1-protease inhibitor (alpha-1-PI) is the major regulator of extracellular leukocyte elastase activity and can be rendered impotent against elastase by oxidation of a critical methionine, residue 358. Alpha-1-PI was isolated from rat plasma by affinity chromatography on Sepharose-bound anhydrochymotrypsin, DEAE-cellulose anion-exchange, and Sephadex G-150 gel filtration. The product was radiolabeled using non-oxidative conditions with Bolton-Hunter reagent, and an aliquot subsequently oxidized with N-chlorosuccinimide. Turnover studies in rats indicated that both native and oxidized alpha-1-PI had half-lives of 170 min. Using partially purified human neutrophil methionine sulfoxide-peptide reductase (Met(O)PR), it was demonstrated that oxidized product could be converted back "in vitro" to an active inhibitor of elastase. To assess whether oxidized alpha-1-PI underwent reduction "in vivo," methionine-oxidized rat inhibitor was injected into the rats, aliquots of plasma samples were withdrawan and passed through a Sepharose-bound anhydrochymotrypsin affinity resin, and bound functional alpha-1-PI was eluted with 0.1 M chymostatin. Radioactive counting of bound and unbound fractions indicated that reduction does not occur in vivo and suggested that, at least under homeostatic conditions, the Met(O)PR is confined to intracellular sites where it does not have access to the circulating protein.
D000818:Animals; D001798:Blood Proteins / Q000302:isolation & purification; D006207:Half-Life; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D008297:Male; D008715:Methionine / Q000097:blood*; D051150:Methionine Sulfoxide Reductases; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D011480:Protease Inhibitors / Q000097:blood* / Q000302:isolation & purification; D011485:Protein Binding; D051381:Rats; D013997:Time Factors; D000515:alpha 1-Antitrypsin
D001798:Blood Proteins; D007457:Iodine Radioisotopes; D011480:Protease Inhibitors; D000515:alpha 1-Antitrypsin; D008715:Methionine; D010088:Oxidoreductases; D051150:Methionine Sulfoxide Reductases; C027219:methionine sulfoxide reductase; D010196:Pancreatic Elastase