Evidence for non-competitive inhibition between two calcium-dependent activated neutral proteinases and their specific inhibitor.

ABSTRACT

Two muscle thiol proteinases causing partial degradation of myofibrillar constituents were isolated and purified from skeletal muscle. The two proteinases that differ significantly in calcium requirements were designated respectively high- and low-Ca2+-requiring proteinase. Both are inhibited, in vitro, by a specific inhibitor which is a protein also isolated from skeletal muscle. Experiments using carboxymethylated monomeric proteinases and inhibitor-conjugated Sepharose were carried out in order to understand the mechanism of control of the proteinases by the inhibitor. The results using increasing inhibitor concentrations show a non-competitive inhibition for both enzymes. The Ki value for the low-Ca2+-requiring form was 0.3 microM, while the Ki value for the high-Ca2+-requiring form was 0.9 microM. Likewise, the low-Ca2+-requiring form needs about 3-fold more inhibitor than the high-Ca2+-requiring form for the same per cent inhibition.