Inhibition of aconitase by alloxan and the differential modes of protection of glucose, 3-O-methylglucose, and mannoheptulose

Abstract

Alloxan inhibited aconitase with a half maximal inhibitory concentration of 0.5 mM in sonically disrupted and 2.3 mM in intact isolated liver mitochondria. For dialuric acid the half maximal inhibitory concentrations were 1.1 mM and 2.5 mM, respectively. Ninhydrin and N-ethylmaleimide (NEM) also inhibited aconitase with half maximal inhibitory concentrations in the submillimolar range and t-butylhydroperoxide (BuOOH) in the millimolar range, which, however, were not different for disrupted and intact mitochondria. Only the aconitase substrate citrate, but not glucose provided protection of the enzyme against inhibition. In intact liver cells the half maximal inhibitory concentration for alloxan was 6.8 mM. Again, dialuric acid and BuOOH were less potent inhibitors while ninhydrin and NEM were more potent inhibitors of aconitase in intact liver cells. In intact liver cells, glucose and 3-O-methylglucose, but not mannoheptulose and citrate provided protection against alloxan inhibition. The results show that aconitase is not an enzyme particularly sensitive towards alloxan inhibition and thus apparently not a primary site for mediation of alloxan toxicity as it is the glucokinase. This makes a primary site of alloxan action in the mitochondria extremely unlikely. On the other hand the results demonstrate that both the intact mitochondrial and plasma membrane as uptake barriers provide protection against alloxan toxicity. In addition the results clearly show, that 3-O-methylglucose provides protection against alloxan action only at the level of the plasma membrane through inhibition of alloxan uptake into the cell, while the site of protection of mannoheptulose is only the sugar binding site of the glucokinase. In contrast, glucose is shown here to be the only sugar with a dual protective effect both through inhibition of alloxan uptake through the plasma membrane like 3-O-methylglucose and through protection of the glucokinase sugar binding site against alloxan inhibition of the enzyme like mannoheptulose. In the light of these results the unique protective potency of glucose as compared to that of other sugars is not surprising.