The activities of carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) in rat liver were markedly increased by administration of di(2-ethyl-hexyl)phthalate. COT and CPT were purified from the enzyme-induced rat liver. COT was a 66,000-dalton polypeptide. The molecular weight of native CPT was 280,000--320,000 daltons, and the enzyme consisted of 69,200-dalton polypeptides. CAT, COT, and CPT were immunologically different. COT exhibited activity with all of the substrates tested (acyl-CoA's and acylcarnitines of saturated fatty acids having carbon chain lengths of C2--C20), though maximum activity was observed with hexanoyl derivatives. CPT exhibited catalytic activity with medium- and long-chain acyl derivatives. 2-Bromo-palmitoyl-CoA inactivated COT but not CPT. Malonyl-CoA inhibited CPT but not COT. CPT was confined to mitochondria, whereas COT was found in peroxisomes and the soluble compartment but not in mitochondria.

The identity of long-chain acyl-CoA synthetase in microsomes, mitochondria, and peroxisomes of rat liver was examined by using the antibody raised against a purified preparation of the microsomal enzyme. The enzyme activities of these three organelles and the purified microsomal enzyme were titrated by the antibody in a very similar fashion when the activity was measured in terms of palmitoyl-CoA synthetase activity. It was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitates and by Western blot analysis that the enzymes of all three organelles consisted of a polypeptide with the same molecular weight as that of the purified enzyme, and that the specific enzyme activity of the antigenic protein in all three subcellular compartments was nearly the same. The presence of other palmitoyl-CoA synthetase activity in these organelles could not be confirmed. Immunocytochemical study to locate the antigenic site with protein A-gold complex showed that the gold particles were closely associated with the membranes of these organelles. The cell-free translation product in a rabbit reticulocyte lysate protein-synthesizing system and the subunit of the mature enzyme labeled with [35S]methionine in the liver slices exhibited the same mobility as the subunit of the purified enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme in microsomes, mitochondria, and peroxisomes was labeled at nearly the same rate when the liver slices were incubated with [35S]methionine.

1. Rats were fed on a diet containing 0, 1, 2, or 4% di-(2-ethylhexyl)phthalate (DEHP), a widely used plasticizer, for four weeks. 2. The level of plasma triglyceride was decreased in both the 1% DEHP and the 2% DEHP groups maintained for 1 to 2 weeks, but that of cholesterol was not changed. Plasma free fatty acid and ketone bodies increased in all DEHP groups. 3. The level of blood glucose was depressed to about 80% in the 2% and the 4% DEHP groups. Liver glycogen decreased markedly. Isotopic incorporations of 14C from [2-14C]pyruvate into blood glucose and liver glycogen were decreased. The changes in the contents of intermediates in the liver indicate that gluconeogenesis is inhibited at the reaction(s) between 3-phosphoglycerate and fructose 1,6-diphosphate. 4. Hepatic fatty acid synthesis increased about 2-fold on DEHP administration. The content of phospholipid in the liver was increased, whereas that of triglyceride was decreased. The rate of phospholipid synthesis was increased, but that of triglyceride was not changed.

1. Rats were fed on a diet containing ethyl p-chlorophenoxyisobutyrate (0.3%, w/w) for 14 days. 2. The alterations of contents of intermediates in the liver indicate that gluconeogenesis is inhibited at the reaction(s) between 3-phosphoglycerate and fructose 1,6-diphosphate. The [nad+]/([nadh] ratios in cytoplasm and mitochondria were increased about 3- and 4-fold, respectively. Marked increases in the contents of CoA and its thioesters were found. 3. Hepatic fatty acid synthesis increased about 3-fold. There was no evidence of inhibition of the acetyl-CoA carboxylase [EC 6.4.1.2] reaction by the drug.