Binding of malonyl - CoA to isolated mitochondria

1. [14C]Malonyl-CoA bound to intact mitochondria isolated from rat liver and heart in a manner consistent with the presence of two independent classes of binding sites in each tissue. 2. The binding characteristics for mitochondria obtained from fed male rats were: for heart, KD(1) = 1 -l8 nM, KD(2) = 30uM, N1 = 7pmol/mg of protein, N2 = approx. 660pmol/mg of protein; for liver, KD(1) = 0.1 M, KD(2) = 5.6 UM, N1 = 11 pmol/mg of protein, N2 = 165 pmol/mg of protein. In the presence of 40MUMpalmitoyl-CoA the characteristics of binding at the high-affinity sites were changed, so that for heart KD(1) = 0.26gM, with no change in N1 and for liver KD(1) = approx. 2Mm, with N1 increased to approx. 40pmol/mg of protein. Differences between the two tissues in tightness of malonyl-CoA binding at the high-affinity sites explains the considerably greater sensitivity of heart CPT1 (overt form of carnitine palmitoyltransferase) to inhibition by malonyl-CoA [Saggerson & Carpenter, (1981) FEBS Lett. 129, 229-232; McGarry, Mills, Long & Foster (1983) Biochem. J. 214, 21-28]. 3. Starvation (24h) did not change the characteristics of [14C]malonyl-CoA binding to liver mitochondria and did not alter the I50 (concentration giving 50% inhibition) for displacement of [14C]malonyl-CoA by palmitoyl-CoA. Therefore the decreased sensitivity of liver CPT1 to inhibition by malonyl-CoA in starvation [Saggerson & Carpenter (1981) FEBSLett. 129,225-228; Bremer (1981) Biochim. Biophys. Acta 665, 628-631] is not explained by differences in malonyl-CoA binding. 4. Percentage occupancy of the high-affinity sites in heart mitochondria by malonyl-CoA correlated closely with percentage inhibition of CPT1 measured under similar conditions. This finding supports the proposal that the high-affinity binding sites are the functional sites mediating inhibition of CPT1 by malonyl-CoA. Similar experiments with liver mitochondria also suggested that the occupancy of high-affinity sites by malonyl-CoA regulates CPT1 activity. 5. 5,5'-Dithiobis-(2-nitrobenzoic acid), which decreased the sensitivity of heart or liver CPT1 to inhibition by malonyl-CoA [Saggerson & Carpenter (1982) FEBS Lett. 137, 124-128], also decreased [14C]malonyl-CoA binding to the high-affinity sites of heart mitochondria. 6. N1 values for [t4C]malonylCoA binding to high-affinity sites in liver mitochondria were determined in various physiological states which encompassed a 7-fold range ofCPT1 maximal activity (fed, starved, pregnant, hypothyroid, foetal). The N1 value did not change in these states. This finding supports the proposal that the high-affinity site is unlikely to be the catalytic unit of CPT1. 7. The physiological importance of the low-affinity binding sites for malonyl-CoA is unknown.