Microsomal long-chain acyl-CoA thioesterase (carboxylesterase ES-4) is regulated by thyroxine.

Long chain acyl-CoA thioesterase activity is mainly located in microsomes after subcellular fractionation of liver from untreated rats. The physiological function and regulation of expression of this activity is not known. In the present study we have investigated the effect of thyroxine on expression of carboxylesterase ES-4, the major acyl-CoA thioesterase of liver microsomes. Thyroidectomy of rats decreased the palmitoyl-CoA thioesterase activity to about 25% of normal activity. This decrease was accompanied by similar decreases at the protein and mRNA levels (31% and 57%, respectively, of controls). Treatment with thyroxine completely reversed the effect of thyroidectomy and resulted in elevated levels in both thyroidectomized and control rats. For reasons of comparison we also studied the possibility that ES-10 and ES-2, two other members of the same gene family, are affected by thyroxine. ES-10 was not changed at the protein or mRNA level by any of the treatments, while ES-2 expression in liver was decreased by thyroxine treatment. The data shows that changes in activity and expression of ES-4 correlate to thyroxine status in the rat suggesting a physiological regulatory role by this hormone. Since thyroxine regulates the expression of lipogenic enzymes, these results are consistent with a function for this microsomal acyl-CoA thioesterase in fatty acid synthesis and/or secretion, rather than in oxidative degradation of fatty acids.

Isolation and characterization of novel long-chain acyl-CoA thioesterase/carboxylesterase isoenzymes from Candida rugosa.

Long-chain acyl-CoA thioesterases, which catalyze the cleavage of acyl-CoA's to free fatty acids and CoASH, are abundant in animal cells. However, in yeast little is known about presence and function of acyl-CoA thioesterase activity. Therefore a commercial lipase preparation from the yeast Candida rugosa was investigated and found to contain high myristoyl-CoA thioesterase activity. Hydrophobic interaction chromatography separated the activity into three peaks, of which two enzymes (YTE-1 and YTE-2) were purified to apparent homogeneity with molecular masses of about 40 kDa as determined by size-exclusion chromatography and SDS-PAGE. The employed purification protocol resulted in final preparations with specific activities of about 90 micromol/mg/min with myristoyl-CoA as substrate. YTE-1 and YTE-2 showed similar kinetic properties and YTE-1 was characterized in detail. Acyl-CoA chain-length specificity showed that YTE-1 was not active on acyl-CoAs shorter than decanoyl-CoA, at the substrate concentrations tested. The best substrates were C14-C18 acyl-CoAs with Vmax values of about 150 micromol/mg/min and Km values of 15-46 microM. The enzyme was very active with lauroyl-CoA (Vmax about 400 micromol/mg/min) although the Km was high (about 325 microM). The purified enzyme was also active on short-chain nitrophenyl esters but inactive with tributyrin. Treatment of the protein with N-glycosidase F decreased the molecular mass about 1-2 kDa, indicating the presence of carbohydrate of the high mannose type. Diisopropyl fluorophosphate (DFP) inhibited the enzyme activity efficiently and the protein was covalently labeled with [3H]DFP. p-Chloromercuribenzoic acid inhibited the thioesterase activity but did not affect carboxylesterase activity. N-terminal sequence analysis and labeling by DFP suggest that these long-chain acyl-CoA thioesterases belong to a novel group of yeast serine esterases.

Isolation of carboxylester lipase (CEL) isoenzymes from Candida rugosa and identification of the corresponding genes.

The yeast Candida rugosa produces extracellular lipases which are widely used for industrial purposes. A commercial lipase preparation from this yeast can be separated into several isoenzymes which differ in carbohydrate content, isolelectric point, substrate specificity, and primary sequence. We have here purified and characterized three lipases, which also hydrolyze p-nitrophenyl esters, from a commercial preparation of this yeast. These three carboxylester lipases (CELs) elute differently on hydrophobic interaction chromatography, and have different carbohydrate contents and substrate specificities. Sequence analysis of their amino termini and peptides generated by LysC treatment showed that CEL-1 and CEL-3 probably have identical primary structure while CEL-2 was proven to be a different enzyme. Sequence comparison showed that both CEL-1 and CEL-3 are products of the LIP1 gene and that CEL-2 is the gene product of LIP2, cloned by Longhi et al. (Biochim. Biophys. Acta 1131, 227-232, 1992).

Formation of fatty acid ethyl esters in rat liver microsomes. Evidence for a key role for acyl-CoA: ethanol O-acyltransferase.

Fatty acid ethyl esters have been detected in high concentrations in organs commonly damaged by alcohol abuse and are regarded as being important non-oxidative metabolites of ethanol. The formation of fatty acid ethyl esters (FAEEs) has been ascribed to two enzymic activities, acyl-CoA : ethanol O-acyltransferase (AEAT) and FAEE synthase. In the present study we determined AEAT and FAEE synthase activities in isolated rat liver microsomes and further characterized the microsomal AEAT activity in more detail. The determined AEAT and FAEE synthase activities were found to be similar (about 1.7 nmol.min-1.mg-1). However, the AEAT activity was increased about sixfold by the addition of 250 microm bis-(4-nitrophenyl) phosphate (a serine esterase inhibitor) to the incubation whereas FAEE synthase activity was completely inhibited. p-Hydroxymercuribenzoic acid (a cysteine-reacting compound) also stimulated AEAT activity (about fourfold) but had no effect on FAEE synthase activity. The effects of the inhibitors suggest that the formation of FAEEs by AEAT was severely counteracted by enzymic hydrolysis of the substrate (acyl-CoA) and to a lesser extent the product by serine esterases. dl-Melinamide, a hypocholesterolaemic drug, was found to be a very potent inhibitor of AEAT activity with an IC50 value of about 2.5 microm. Furthermore, we compared the activities of two purified microsomal carboxylesterases, ES-4 and ES-10, and identified ES-4 as the enzyme responsible for hydrolysis of FAEEs. The two carboxyesterases were also tested for FAEE synthase activity, but neither had any detectable activity. Esterase ES-4 was found to have some AEAT activity, but it was low. When measured under optimal conditions without competing hydrolysis the capacity of AEAT is thus considerably higher than FAEE synthase and the results are consistent with an important role for AEAT in the formation of ethyl esters. As the ratio acyl-CoA/non-esterified fatty acids is high under normal conditions, AEAT is probably the most important enzyme in fatty acid ethyl ester formation.

Characterization of enzymes involved in formation of ethyl esters of long-chain fatty acids in humans.

Elevated fatty acid ethyl ester (FAEE) concentrations have been detected in postmortem organs from alcoholics and patients acutely intoxicated by alcohol, and FAEE have been implicated as mediators of ethanol-induced organ damage. The formation of FAEE is catalyzed by acyl-coenzyme A:ethanol O-acyltransferase (AEAT) and by FAEE synthase, which utilize acyl-CoA and free fatty acids, respectively, as substrates. Because little is known about the capacity of various human tissues to synthesize and hydrolyze FAEE, we investigated formation of FAEE by AEAT and FAEE synthase in tissue homogenates from human gastric ventricular and duodenal mucosa, pancreas, liver, heart, lung, and adipose tissue, gallbladder mucosa, and in serum. Liver, duodenal mucosa, and pancreas were found to have the highest capacities to synthesize FAEE, mainly due to AEAT. FAEE hydrolyzing activity was highest in liver and pancreas, but hardly detectable in adipose tissue or heart. Because fatty acids and alcohol are absorbed by the intestinal mucosa, intestine may be a major site of FAEE synthesis, and FAEE may be delivered via the circulation to other organs and taken up by lipoprotein receptor-mediated uptake. A very low rate of FAEE hydrolysis was detected in heart and adipose tissue, which probably accounts for the previously observed accumulation of FAEE in these organs.

Acyl-coenzyme A:cholesterol O-acyltransferase is not identical to liver microsomal carboxylesterase.

Acyl-coenzyme A (CoA):cholesterol O-acyltransferase (ACAT) is responsible for esterification of cholesterol in the cell. The enzyme has never been purified, but two cDNA sequences coding for this enzyme were recently reported. One of the sequences was identical to human liver carboxylesterase. We have used inhibitors to elucidate the relation between microsomal carboxylesterase, acyl-CoA hydrolase (ACH), and ACAT activities in rat liver. Low concentrations of serine esterase inhibitors strongly inhibited carboxylesterase and acyl-CoA hydrolase activities but stimulated ACAT activity. At higher concentrations, ACAT activity was also inhibited. A sulfhydryl-modifying agent was found to be a potent inhibitor of ACAT without affecting carboxylesterase activity. Similarly, two specific ACAT inhibitors, DL-melinamide and PD 138142-15, inhibited ACAT activity but did not affect carboxylesterase or ACH activities. Our data thus exclude ACAT as a liver microsomal carboxylesterase. The complex inhibition patterns observed with serine esterase inhibitors indicate that carboxylesterases and ACHs may interfere with ACAT activity by competing for the substrate. It is obvious that final identification of ACAT requires demonstration of an active homogenous protein.