Is carnitine palmitoyltransferase inhibited by a malonyl-CoA-binding unit in the mitochondria?

Malonyl-CoA inhibits the carnitine-dependent transport of activated fatty acids into mitochondria by inhibiting the outer carnitine palmitoyltransferase (McGarry et al., 1978). Recent studies in several laboratories have shown that malonyl-CoA is not an ordinary competitive inhibitor of the enzyme. Liver mitochondria from fasted or diabetic rats are less inhibited by malonyl-CoA than are mitochondria from normal fed rats (Cook et af., 1980; Bremer, 1981; Saggerson & Carpenter, 1982). When mitochondria from fasted rats are preincubated with malonyl-CoA its inhibitory effect is increased in a subsequent assay (Zammit, 1983), and when liver mitochondria are preincubated in the absence of malonyl-CoA its inhibitory effect is decreased. The rate of this 'desensitization' is accelerated by palmitoyl-CoA and by salts (Bremer rt ul., 1985). The desensitization is also more rapid in liver mitochondria from fasted or thyrotoxic rats than in mitochondria from fed or hypothyroid rats (Bergseth et al., 1986). Isolated mitochondria have been found to contain highaffinity binding sites for malonyl-CoA (Mills rt al., 1983; Bird & Saggerson, 1984; Zammit & Corstophine, 1985). However, the extracted purified carnitine palmitoyltransferase is completely insensitive to malonyl-CoA (Fiol & Bieber, 1984). Extraction and inhibitor studies also indicate that malonyl-CoA binds to a regulatory component in the membrane different from the enzyme (Bremer et al., 1985; Declercq et al., 1985). We have now found that rat liver mitochondria desensitized in a KCIcontaining medium can be 're-sensitized' by washing the mitochondria in a sucrose medium in the absence of malonyl-CoA. We have also found that the component(s) binding malonyl-CoA can be completely solubilked by extraction with Triton X-100 and KCI, and completely separated from the carnitine palmitoyltransferase. Rat liver mitochondria were prepared as previously described (Bremer et al., 1985). A complete extraction of carnitine palmitoyltransferase and malonyl-CoA binding component(s) was obtained with 5% Triton X-lOO/O.S MKCI/IOrnM-Hepes (pH 7.4)/1 mM-EGTA. Carnitine palmitoyltransferase was assayed as previously described (Bremer et al., 1985). Protein was assayed by the Lowry et al. (1951) method. Malonyl-CoA was analysed in freshly isolated rat liver mitochondria as described by Singh et al. (1984). These analyses showed that the mitochondria contained 5-10 pmol of malonyl-CoA/mg of protein. This malonylCoA must be relatively firmly bound since it survived the mitochondria isolation procedure. We have also found that added [2-'4C]malonyl-CoA is relatively rapidly degraded by the isolated mitochondria. When 3-15 nmol of labelled malonyl-CoA was incubated with mitochondria (8 mg of protein) under the preincubation conditions given in the legend to Fig. 1, about 50% of the malonyl-CoA had disap-