Effect of several anions on the activity of mitochondrial malate dehydrogenase from pig heart

Mitochondrial malate dehydrogenase (mMDH) shows a complex dependence upon i onic environment that includes kinetic and structural effects. We measured mMDH activity in several buffers (phosphate, MOPS, and MES) at pH 6.5 and 7 .5, and in the presence of a number of anions, at highly diluted enzyme con centrations where mMDH showed significant loss of activity. Under these con ditions, mMDH activity shows a non-linear dependence on enzyme concentratio n, in agreement with the existence of a dimer-monomer equilibrium, where on ly the dimeric form is active. According to this hypothesis, the dissociati on constant of mMDH dimer has been determined to be 5.4 nM in the MES buffe r at pH 6.5. Either the presence of a small anion like phosphate, or an inc rease of the pH from 6.5 to 7.5 shifts the equilibrium in favor of the dime ric form with the two effects appearing to be additive. To extend the study , we analysed the effect of a number of anions on the mMDH activity in 50 m M MOPS buffer at pH 7.5. All the anions had a dual effect: at low concentra tions, they increased the activity of mMDH, while at high concentrations, t hey inhibited it. A more accurate analysis of the data revealed that the ac tivation capacity of all the anions tested was similar, although they diffe red in their inhibitory influence. To show these differences more clearly, the experiment was repeated in 50 mM phosphate buffer at pH 7.5, under cond itions where almost all activations were due to the buffer. The analysis of the results obtained under these conditions revealed the following sequenc e of inhibition potency: phosphate < acetate < sulfate < chloride < bromide < nitrate < perchlorate < thiocyanate, that was in good agreement with the ir chaotropicity. The behaviour of mMDH in the presence of different ions a nd/or ionic strength indicates that at low ionic concentrations, increased activity is likely due to a stabilization of the dimeric form of the enzyme . Further increases in the ionic concentration, especially if chaotropic io ns are used, lead to a loss of enzyme activity that appears to be a consequ ence of structural changes in the enzyme rather than kinetic effects. (C) 2 001 Elsevier Science B.V, All rights reserved.