Kinetic Characterization of the Inhibition of Purified Cynomolgus Monkey Lactate Dehydrogenase Isozymes by Gossypol

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Abstract
This report describes the results of the first step in a sequence of experiments designed to test the hypothesis that the sperm-specific isozyme of lactate dehydrogenase (LDH-C4), is a site of action of the potential male contraceptive agent gossypol. Cynomolgus monkey LDH-A4, LDH-B4, and LDH-C4 were purified and kinetically characterized. LDH-A4 and LDH-B4 exhibited “linear mixed-type” inhibition by gossypol with both lactate and pyruvate as variable substrates. LDH-C4 also exhibited “linear mixed-type” inhibition with lactate as substrate. However, the C4 isozyme exhibited “parabolic mixed-type” inhibition by gossypol and substrate inhibition with pyruvate as substrate, the latter due to abortive complex formation. Of the three isozymes, LDH-C4 exhibited the lowest apparent Km for pyruvate and the highest apparent Km for lactate. The LDH-C4 form was found to be the most sensitive isozyme to gossypol inhibition, since it had the lowest apparent K1 values for gossypol inhibition. The effect of gossypol on coenzyme binding to LDH-C4 was examined and gossypol binding was found to inhibit binding and release of NADH but not NAD+, an effect possibly due to its interaction with the more hydrophobic loop region of LDH-C4.