Cholate separates the catalytic and malonyl-CoA-binding components of carnitine palmitoyltransferase from liver outer mitochondrial membranes.
Sodium cholate was used as an anionic detergent to discriminate the two components of liver overt carnitine palmitoyltransferase (CPT1); namely a catalytic entity and a regulatory component that bound malonyl-CoA. Cholate solubilized approx. 40% of the malonyl-CoA binding entity from mitochondrial outer membranes without appreciable solubilization of CPT1 activity. Cholate did not interfere with binding of [14C]malonyl-CoA to outer membranes or to crude total mitochondrial membrane fractions. By contrast, the non-ionic detergent Tween-20 was ineffective in solubilizing the malonyl-CoA binding entity and also substantially interfered with the binding of [14C]malonyl-CoA. Both detergents appeared to cause total disengagement of the malonyl-CoA binding entity from the catalytic entity of CPT1 only when some inner membrane material was present. 'Reconstitution' experiments were performed in which a malonyl-CoA sensitivity conferring factor in cholate extracts from outer membranes was associated with CPT derived from inner membranes (CPT2). The IC50 for inhibition of CPT2 by malonyl-CoA in this artificial system was similar to that observed with CPT1 in situ in outer membranes. Extracts containing malonyl-CoA sensitivity conferring factor derived from outer membranes of fed or 48 h fasted rats were associated with CPT2 derived from fed rats. The outer membrane extracts from fasted animals conferred a lower maximum responsiveness to malonyl-CoA, but appeared to have a higher affinity for CPT2 than the extracts from fed rats. These results suggest that physiological state can alter the intrinsic properties of the malonyl-CoA sensitivity confering factor.
Ghadiminejad
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I
Department of Biochemistry and Molecular Biology, University College London, U.K.
Saggerson
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D
D000818:Animals; D002334:Carnitine O-Palmitoyltransferase / Q000187:drug effects* / Q000378:metabolism; D002384:Catalysis; D019826:Cholic Acid; D002793:Cholic Acids / Q000494:pharmacology*; D003902:Detergents / Q000494:pharmacology*; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000201:enzymology*; D008297:Male; D008316:Malonyl Coenzyme A / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000201:enzymology*; D011136:Polysorbates / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility
D002793:Cholic Acids; D003902:Detergents; D011136:Polysorbates; D008316:Malonyl Coenzyme A; D002334:Carnitine O-Palmitoyltransferase; D019826:Cholic Acid