Abstract
Long-chain fatty acid esters of CoA activate (Na+ + K+)-ATPase (the sodium pump) when ATP is suboptimal. To explore the nature of the interactions of these CoA derivatives with the pump, reversible effects of palmitoyl-CoA on the purified membrane-bound kidney enzyme were studied under conditions where interference from the irreversible membrane-damaging effect of the compound was ruled out. With 50 microM ATP, while saturating palmitoyl-CoA increased (Na+ + K+)-ATPase activity, it caused partial inhibition of Na+-ATPase activity without affecting the steady-state level of the phosphoenzyme. Palmitoyl-CoA did not change the K0.5 of ATP for Na+-ATPase, but it altered the complex Na+ activation curve to suggest the antagonism of the low-affinity, but not the high-affinity, Na+ sites. At a low ATP concentration (0.5 microM), K+ inhibited Na+-ATPase as expected. In the presence of palmitoyl-CoA and 0.5 microM ATP, however, K+ became an activator, as it is at high ATP concentrations. The activating effect of palmitoyl-CoA on (Na+ + K+)-ATPase activity was reduced with increasing pH (6.5-8.5), but its inhibitory effect on Na+-ATPase was not altered in this pH range. The data show two distinct actions of palmitoyl-CoA: 1) blockade of the extracellular "allosteric" Na+ sites whose exact role in the control of the pump is yet to be determined, and 2) activation of the pump through increased rate of K+ deocclusion. Since in their latter action the fatty acid esters of CoA are far more effective than ATP at a low-affinity regulatory site, we suggest that these CoA derivatives may be the physiological ligands of this regulatory site of the pump.
Highlights
CoA derivatives with the pump, reversible effects of purified enzyme to learn more about the mechanism of this palmitoyl-CoA on the purified membrane-bound kid- control
The findings indicate that theCoA derivatives actiney enzyme were studied under conditions where in- vate the pump by increasing the rate of release of K’ from terference from the irreversible membrane-damaging the K+-occluded state of the enzyme
When the effect of 30 PM palmitoyl-CoA on the steady-state level of acidstable phosphoenzyme obtainedfrom 50 PM ATP in the presence of 100 mM Na' and 5 mM M P was determined at 24 "C in four separate experiments, the controllevel was 2.1 f 0.2 nmol of Pi/mg of protein and that in the presence of palmitoyl-CoA was 2.3 f 0.2 nmol of Pi/mg of protein. These data, in conjunctionwith the partial natureof the inhibition of Na+-dependent ATPase caused by palmitoyl-CoA (Fig. 2), clearly show that palmitoyl-CoA and ATP(at the high-affinity catalytic site) may bind simultaneously and without significant interactions
Summary
In expericell membranes [3].In the latter system, witas demonstrated ments of Fig. l a , the effect of the palmitoyl-CoA:enzyme ratio that the activating effects of these compounds on the pump on time-dependent irreversible inhibition of the enzyme was were exerted from the intracellular, but not the extracellular,studied. Since there have been disagreements on whether or not the highand low-affinity ATP sites arethe same [6, 7] and since only a high-affinity ATP site is apparent in the expression of the Na+-dependent ATPaseactivity of the enzyme, it became of interest to examine the acyl-CoA effects on this activity.
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