Solubilization and separation of two distinct carnitine acyltransferases from hepatic microsomes: characterization of the malonyl-CoA-sensitive enzyme

Abstract

Conditions have been developed for the solubilization of hepatic microsomal carnitine acyltransferase activity in good yield, with excellent long-term stability and with retention of malonyl-CoA sensitivity. Solubilized microsomal carnitine acyltransferase activity can be separated into malonyl-CoA-sensitive and -insensitive activities either by gel filtration on Superdex 200 or by anion-exchange chromatography on Resource Q. On gel filtration the apparent molecular masses of the malonyl-CoA-sensitive and -insensitive activities are approx. 300 kDa and 60 kDa respectively. The malonyl-CoA-sensitive and -insensitive activities have different fatty-acyl-chain-length specificities and different stabilities in the detergent octyl glucoside. Together these findings indicate that the malonyl-CoA-sensitive and -insensitive activities are due to different enzymes. The malonyl-CoA sensitivity of the inhibitable enzyme is markedly increased on reconstitution into soybean L-alpha-lecithin liposomes, demonstrating that phospholipids play a crucial role in the inhibition by this metabolite. Evidence is also provided that the malonyl-CoA-sensitive microsomal carnitine acyltransferase is a different enzyme from the malonyl-CoA-sensitive carnitine palmitoyltransferase found in the mitochondrial outer membrane. The possible physiological role of the two microsomal acyltransferases is discussed.

Inhibition of liver microsomal carnitine acyltransferases by sulphonylurea drugs

“…Studies with primary cultures of rat hepatocytes [7 9] have led to the conclusion that fatty acids incorporated into secreted VLDL triacylglycerols are largely derived from lipolysis of a hepatic cytoplasmic pool of triacylglycerol. As a corollary to this it would seem likely that the synthesis of these discrete pools of VLDL and cytoplasmic store triacylglycerols should be spatially separated and we have speculated [5] that provision of fatty acyl units to the lumen of the secretory compartment may be part of this separation. *Corresponding author.…”

“…Dimethylsulphoxide was from Fisons. Animals and all other reagents were as described previously [5],…”

“…Membrane residues depleted of malonyl-CoA-insensitive CAT were obtained by freeze/thawing and recentrifugation of membranes as described in [5].…”

“…In liver carnitine acyltransferase (CAT) activities have been reported in mitochondria, in peroxisomes, in microsomes and in nuclei [14]. Recently we [5] and others [6] have provided evidence that rat liver microsomes contain two distinct CATs. One of these activities is membrane-bound, appears to be oriented towards the cytosolic compartment and can be inhibited by physiological concentrations of malonylCoA.…”

“…The physiological function of these microsomal CATs is not yet clearly established. It has been suggested [5] that they might be part of a system that enables transport of fatty acyl units between the cytosol and the lumen of the endoplasmic reticulum (this presupposes the existence also of a microsomal carnitine:acylcarnitine translocase). Studies with primary cultures of rat hepatocytes [7 9] have led to the conclusion that fatty acids incorporated into secreted VLDL triacylglycerols are largely derived from lipolysis of a hepatic cytoplasmic pool of triacylglycerol.…”