Effect of Tubulin on the Activity of the Muscle Isoenzyme of Lactate-Dehydrogenase

Abstract

The interaction between muscle-type lactate dehydrogenase (LDH m) and tubulin was investigated by monitoring the combined effect of NADH and tubulin on steady-state kinetics and the combined effect of NADH and pH on complex formation between tubulin and the enzyme. Steady-state kinetics showed that LDH m is inhibited by tubulin. Experiments with heart-type lactate dehydrogenase (LDH h) showed that the inhibition is unique to the muscle-type enzyme. The magnitude of the inhibition is dependent upon the concentration of NADH as well as the pH of the buffer medium. The enzyme was less sensitive to inhibition at 50μM NADH than at 10μM NADH. Since this effect of NADH is not due to an ionic strength contribution, it is deemed to be specific. In contrast to the absence of tubulin, its presence induced a modification of the kinetic behavior of LDH m; i.e., the velocity dependence on NADH concentration displayed a marked sigmoid response. The inhibition of LDH m by tubulin is more pronounced at lower pH values than at higher pH values. The pH-dependent inhibitory profile is shifted to the left (i.e., p K a is decreased) with increasing concentrations of NADH. This pattern is remarkably similar to that observed for the binding of the enzyme on Sepharose immobilized tubulin and is consistent with the premise that inhibition is a result of interaction between these proteins. NAD + was much less effective than NADH in dissociating LDH m from immobilized tubulin. Results from these in vitro studies are consistent with similar observations dealing with other glycolytic enzymes and cytoskeleton proteins, which show that enzyme catalytic properties are modified upon binding.