Evidence for a thiol ester in duck ovostatin (ovomacroglobulin).
The structure and the mechanism for proteinase inhibition of the egg white protein ovostatin (ovomacroglobulin) are similar to those of plasma alpha 2-macroglobulin, but previous studies have shown that chicken ovostatin lacks a reactive thiol ester (Nagase, H., and Harris, E. D., Jr. (1983) J. Biol. Chem. 258, 7490-7498). Here we show that duck ovostatin has conserved such a thiol ester and is capable of inhibiting both metallo- and serine proteinases stoichiometrically. Evidence for thiol esters was established by the following results with duck ovostatin: 1) autolysis into fragments of Mr = 123,000 and 60,000 occurred by heating in sodium dodecyl sulfate, but was prevented by treatment with CH3NH2; 2) covalent linkages were formed with proteinases on complex formation; 3) reaction with CH3NH2 generated 3.6 SH groups/mol, and 3.9 mol of [14C]CH3NH2 were incorporated per mol of protein; and 4) saturation with a proteinase liberated 3.8 SH groups/mol of the inhibitor. Conformational rearrangement of duck ovostatin upon reacting with CH3NH2 or proteinases was demonstrated by an increased mobility of the protein in polyacrylamide gel electrophoresis. CH3NH2-treated duck ovostatin was able to bind and inhibit proteinases without forming covalent bonds, but, unlike unmodified ovostatin, its inhibitory activity was destroyed by freezing and thawing. Complexes formed between CH3NH2-treated duck ovostatin and a proteinase were not dissociable except under denaturing conditions. These results and other evidence indicate that covalent bond formation through reaction with a thiol ester is a separate process from the trapping and inhibition of proteinases by this family of proteins.
D000595:Amino Acid Sequence; D000818:Animals; D001329:Autolysis; D002645:Chickens; D004372:Ducks; D004527:Egg Proteins / Q000032:analysis* / Q000494:pharmacology; D008259:Macroglobulins; D008666:Metalloendopeptidases; D008970:Molecular Weight; D010206:Papain / Q000378:metabolism; D011480:Protease Inhibitors / Q000032:analysis / Q000494:pharmacology; D011487:Protein Conformation; D012697:Serine Endopeptidases; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000032:analysis*; D013820:Thermolysin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D000511:alpha-Macroglobulins
D004527:Egg Proteins; D008259:Macroglobulins; D011480:Protease Inhibitors; D013438:Sulfhydryl Compounds; D000511:alpha-Macroglobulins; D012967:Sodium Dodecyl Sulfate; C036828:ovomacroglobulin; D012697:Serine Endopeptidases; D014357:Trypsin; D010206:Papain; D008666:Metalloendopeptidases; D013820:Thermolysin