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Quantitative carbon-by-carbon analysis would be useful in determining the origin and fate of carbons involved in fatty acid metabolism. Incorporation of 13 C from 2-[ 13 C]acetate into specific carbons of liver fatty acids was lowest at the n—2 carbon of saturates and monoenes but was 47% greater at acyl Cl than at C2, suggesting substantial redistribution of the 13 C from C2 to Cl of acetyl CoA or malonyl CoA prior to 13 C incorporation into fatty acids during de novo synthesis or during elongation. Thus, 13 C derived from exogenous acetate can be quantitatively measured and is differentially incorporated into individual carbons depending on position in the fatty acid molecule.

A soluble extract was obtained on treatment of rat liver mitochondrial outer membranes with cholate which bound (14C]malonyl-CoA but was essentially free of carnitine palmitoyltransferase (CPT) activity. Extraction of mitochondrial inner membranes with cholate readily solubilized a CPT activity which was insensitive to malonyl-CoA. Combination of these two extracts caused the CPT derived from inner membranes to become inhibitable by malonyl-CoA.

The formation of malonyl-CoA in rat heart is catalyzed by cytosolic acetyl-CoA carboxylase. The existence of this enzyme in heart is difficult to prove by the abundant occurrence of mitochondrial propionyl-CoA carboxylase, which is also able to catalyze the carboxylation of acetyl-CoA. We used the calcium paradox as a tool to separate cytosolic components from the remaining heart, and found that acetyl-CoA carboxylase activity was preferentially released, like lactate dehydrogenase and carnitine, while propionyl-CoA carboxylase was almost fully retained. Acetyl-CoA carboxylase activity was determined after activation by citrate ion and Mg2+. The activity decreased to 64% by 48 h of fasting.

Carnitine octanoyltransferase (COT), an enzyme that facilitates the transport of medium chain fatty acids through peroxisomal membranes, is inhibited by malonyl-CoA. cDNAs encoding full-length wild-type COT and one double mutant variant from rat peroxisomal COT were expressed in Saccharomyces cerevisiae. Both expressed forms were expressed similarly in quantitative terms and exhibited full enzyme activity. The wild-type-expressed COT was inhibited by malonyl-CoA like the liver enzyme. The activity of the enzyme encoded by the double mutant H131A/H340A was completely insensitive to malonyl-CoA in the range assayed (2–200 μM). These results indicate that the two histidine residues, H131 and H340, are the sites responsible for inhibition by malonyl-CoA. Another mutant variant, H327A, abolishes the enzyme activity, from which it is concluded that it plays an important role in catalysis.

We report the cloning and nucleotide sequence of the gene encoding malonyl coenzyme A-acyl carrier protein transacylase of Escherichia coli, Malonyl transacylase has been overexpressed 155-fold compared to a wild-type strain, Overexpression of this enzyme alters the fatty acid composition of a wild-type E. coli strain; increased amounts of cis-vaccenate are incorporated into the membrane phospholipids.

An inbred strain of rat (gsd/gsd) with a glycogen storage disease has been described in [1]. The defect in these animals appears to result from a deficiency of liver phosphorylase kinase [2]. Besides exhibiting mild hypoglycaemia in the fed state and inability to mobilise glycogen in the fasted state, these animals show abnormalities in lipid metabolism in that they have diminished rates of hepatic fatty acid and cholesterol synthesis and considerably lower concentrations of plasma triacylglycerols [3]. It is apparent that these animals may be a useful model for studies of equivalent forms of human glycogen storage diseases and it was therefore of interest to investigate some enzymatic aspects of lipid metabolism in this condition. Accordingly measurements have been made of hepatic glycerolphosphate acyltransferase (GPAT) activities (mitochondrial and microsomal) and dihydroxyacetonephosphate acyltransferase (DHAPAT) activity. These activities have been found to change in a number of physiological states and change in parallel with changes in hepatic triacylglycerol synthesis and secretion [4-7] . The activity of the overt form of carnitine pahnitoyltransferase (CPT1) has also been measured in liver mitochondria and the sensitivity of this enzyme to inhibition by malonyl CoA determined. This is thought to be an important factor in the regulation of hepatic fatty acid oxidation [8] and the sensitivity to this effector has been shown to change in at least one physiological state, namely fasting [9].

Carnitine palmitoyltransferase I was isolated from octylglucoside extracts of rat liver mitochondrial outer membranes. This native enzyme was digested proteolytically with V8 protease. Five major peptides were obtained all of which were found in the amino acid sequence predicted from the full-length cDNA sequence of the protein. One peptide was found to correspond to the extreme N-terminus of the deduced amino acid sequence. Therefore, the mature protein retains the N-terminus of the nascent protein after import into the mitochondrial membrane. Knowledge of the identity of the N-terminus of the mature protein allows a reappraisal of the role of the two main, N-terminal hydrophobic domains of the protein and of the possible topology of the protein within the membrane.

The effects ofmalonyl CoA on carnitine palmityltransferase I (CPT-I), fatty acid-supported state 3 respiration, and carnitine reversal of palmityl CoA inhibition of state 3 respiration and of the adenine nucleotide translocator, were studied in isolated rat heart mitochondria. Malonyl CoA was a potent competitive inhibitor of CPT-I with an I50 of 0.8 μM. Fasting did not affect CPT-I activity or the I50 value of malonyl CoA. Malonyl CoA inhibited fatty acid-supported respiration and prevented carnitine from reversing the inhibition of the adenine nucleotide translocator by palmityl CoA. These findings suggest that malonyl CoA may affect fatty acid oxidation in the heart.

Carnitine acyltransferase of liver mitochondria prepared from obese Zucker rats has a higher sensitivity to inhibition by malonyl-CoA compared with carnitine acyltransferase of mitochondria prepared from lean Zucker rats.