Purification of the Ca2+-dependent proteinase inhibitor from bovine cardiac muscle and its interaction with the millimolar Ca2+-dependent proteinase.

A protein inhibitor of the Ca2+-dependent proteinase has been purified from bovine cardiac muscle by using the following steps in succession: salting out 17,600 X gmax supernatants from muscle homogenates in 50 mM Tris acetate, pH 7.5, 4 mM EDTA between 25 and 65% ammonium sulfate saturation; eluting between 25 and 120 mM KCl from a DEAE-cellulose column at pH 7.5; salting out between 30 and 60% ammonium sulfate saturation; Ultrogel-22 gel permeation chromatography at pH 7.5; heating to 80 degrees C followed by immediate cooling to 0 degree C; 6% agarose gel permeation chromatography in 4 M urea, pH 7.5; and elution from a phenyl-Sepharose hydrophobic column between 0.7 and 0.5 M ammonium sulfate. Approximately 1.16-1.69 mg of purified Ca2+-dependent proteinase inhibitor are obtained from 1 kg of bovine cardiac muscle, fresh weight. Bovine cardiac Ca2+-dependent proteinase inhibitor has an Mr of 115,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a pI of 4.85-4.95, very little alpha-helical structure, a very low specific absorbance of 1.647 (A1% 280), and very low contents of histidine, tryptophan, phenylalanine, and tyrosine. Bovine cardiac Ca2+-dependent proteinase inhibitor probably contains a single polypeptide chain in nondenaturing solvents. One 115-kDa inhibitor polypeptide inactivates 10 110-kDa millimolar Ca2+-requiring proteinase (millimolar Ca2+-dependent proteinase) molecules in assays of purified proteins. Inhibition of millimolar proteinase by the proteinase inhibitor did not change in the pH range 6.2-8.6. The inhibitor requires Ca2+ to bind to millimolar Ca2+-dependent proteinase. The Ca2+ concentration required for one-half-maximum binding of millimolar Ca2+-dependent proteinase to the inhibitor was 0.53 mM, compared with a Ca2+ concentration of 0.92 mM required for one-half maximum activity of millimolar Ca2+-dependent proteinase in the absence of the proteinase inhibitor. Unless millimolar Ca2+-dependent proteinase is located subcellularly in a different place than the proteinase inhibitor or unless the proteinase/inhibitor interaction is regulated, millimolar proteinase could never be active in situ.