Studies on the proteinase-A inhibitor I3A from yeast.
The purification and some properties of the two inhibitors I2A and I3A of proteinase A from yeast have previously been described [Saheki et al, (1974) Eur. J. Biochem. 47, 325]. An improved method for the preparation of I3A which is less time-consuming and leads to higher yields is presented. Based on amino acid analysis, I3A contains 68 amino acids per molecule. The molecular weight was 7676. The inhibitor contained no proline, no arginine, no cysteine and no tryptophan, but did contain a large number of the polar amino acids glutamate + glutamine, aspartate + asparagine and lysine. Neither by dansylation nor by Edman degradation could an N-terminal amino acid be detected. Changes in the circular dichroism upon transition from pH 6.9 to 3.0 suggest different tertiary structures at these pH values. Experiments on the kinetics of inhibition of proteinase A revealed an apparent Ki value of 5.5 X 10(-8) M for I3A and 1.6 X 10(-8) M for pepstatin. A "non-stoichiometric inhibition" of a "pseudo-irreversible" type is concluded from the kinetic data. A hydrophobic type of binding of I3A to yeast proteinase A is suggested from experiments demonstrating a large decrease in the percentage of inhibition caused by addition of 2 M urea, 2 M guanidine hydrochloride, 0.125% Triton or 0.125% cholic acid.
D000596:Amino Acids / Q000032:analysis; D002793:Cholic Acids / Q000494:pharmacology; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000032:analysis / Q000302:isolation & purification; D006146:Guanidines / Q000494:pharmacology; D006454:Hemoglobins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010436:Pepstatins / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology; D011480:Protease Inhibitors; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014508:Urea / Q000494:pharmacology
D000596:Amino Acids; D002793:Cholic Acids; D004791:Enzyme Inhibitors; D006146:Guanidines; D006454:Hemoglobins; D010436:Pepstatins; D011480:Protease Inhibitors; D011092:Polyethylene Glycols; D014508:Urea