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Factors affecting the activity and stability of the palmitoyl-coenzyme A hydrolase of rat brain.

ABSTRACT: Palmitoyl-CoA hydrolase (EC 3.1.2.2) catalyses the irreversible hydrolysis of long-chain acyl-CoA thioesters. This enzyme is found primarily in the postmicrosomal supernatant fraction prepared from homogenates of rat brain. Either of two forms of the hydrolase, a lower-molecular-weight species of approx. 70000 or a higher-molecular-weight species of approx. 130000 can be isolated by gel filtration. The higher-molecular-weight form is obtained from columns of Sephadex G-200 eluted with buffer containing 10mum-palmitoyl-CoA or 20% (v/v) glycerol, whereas the lower-molecular-weight form is obtained when the eluting buffer does not contain palmitoyl-CoA or glycerol. The two forms of the hydrolase have the same pH optimum of 7.5, are equally sensitive to the thiol-blocking reagents p-hydroxymercuribenzoate, HgCl(2), and 5,5'-dithiobis-(2-nitrobenzoic acid), and exhibit the same K(m) (1.8mum) with palmitoyl-CoA as substrate. The two forms differ in the availability or reactivity of certain external thiol groups, as determined by covalent chromatography with activated thiol Sepharose. Dilute solutions of the lower-molecular-weight form of the hydrolase rapidly lose activity (50% in 60min at 0 degrees C), but there is no change in the K(m) with palmitoyl-CoA as substrate during this progressive inactivation. Dilutions of the hydrolase in buffer containing 10mum-palmitoyl-CoA retain full activity. However, addition of palmitoyl-CoA to solutions of the lower-molecular-weight form will not restore previously lost hydrolase activity. The evidence supports the conclusion that the substrate palmitoyl-CoA promotes the formation of a relatively stable dimer from two unstable subunits. This process may not be reversible, since the removal of palmitoyl-CoA or glycerol from solutions of the higher-molecular-weight form does not result in the appearance of the lower-molecular-weight form of the hydrolase.

SUBMITTER: Knauer TE

PROVIDER: S-EPMC1186659 | biostudies-other | 1979 Jun

REPOSITORIES: biostudies-other

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Project description:Activities of phosphatidate phosphohydrolase and palmitoyl-CoA hydrolase were determined in cardiac subcellular fractions prepared from rabbits which has received tri-iodothyronine and from hamsters with hereditary cardiomyopathy (strain BIO 14.6). 1. Both mitochondrial and microsomal fractions of hyperthyroid rabbit hearts produced 4-5 times as much diacylglycerol 3-phosphate from glycerol 3-phosphate and palmitate as did those of euthyroid hearts. 2. Phosphatidate phosphohydrolase, measured with phosphatidate emulsion, was activated by 1mm-Mg(2+) in all but the mitochondrial fraction of euthyroid rabbit hearts. The activation was more pronounced in subcellular fractions isolated from hyperthyroid hearts, so that the measured activities were significantly increased above those of the controls. The highest activity was found in the microsomal and lysosomal fractions. 3. In the absence of Mg(2+) during incubation, the difference in phosphohydrolase activities between eu- and hyper-thyroid states was not significant. 4. The phosphohydrolase of subcellular fractions of control hamsters did not respond to addition of 0.5-8.0mm-Mg(2+). The enzyme from cardiomyopathic hearts was slightly inhibited by this bivalent cation and therefore significant increases in activity were observed only in the absence of Mg(2+) from the assay system. 5. The rate of reaction by soluble phosphatidate phosphohydrolase was similar regardless of the nature of the substrate. Both when microsomal-bound phosphatidate was used as the substrate and when phosphatidate suspension was used, the activity of soluble enzyme was lower than that of the microsomal and lysosomal enzymes measured with phosphatidate suspension; this was especially so when the assay was carried out in the absence of Mg(2+). Neither tri-iodothyronine nor cardiomyopathy influenced the soluble phosphohydrolase activity in the two species. 6. Neither tri-iodothyronine nor cardiomyopathy significantly changed palmitoyl-CoA hydrolase activities in subcellular fractions. 7. Microsomal diacylglycerol acyltransferase and myocardial triacylglycerol content were also unchanged in the hyperthyroid state.

Project description:1. The specific activities for palmitoyl-CoA synthetase and for sn-glycerol 3-phosphate esterification, with palmitoyl-CoA generated either by the endogenous synthetase or from palmitoyl-(-)-carnitine, CoA and excess of carnitine palmitoyltransferase, were measured with rat liver mitochondria. 2. The mean specific activity of palmitoyl-CoA synthetase was approximately five- and seven-fold the rates of sn-glycerol 3-phosphate esterification from palmitate and palmitoyl-(-)-carnitine respectively. No significant correlation was found in different rats between the activities of palmitoyl-CoA synthetase and sn-glycerol 3-phosphate esterification from either acyl precursor. However, there was a significant correlation (r=0.83, P<0.001) between the rates of glycerolipid synthesis from palmitate and palmitoyl-(-)-carnitine. 3. The mean molar composition of the glycerolipid synthesized from palmitate was 58% lysophosphatidate, 31% phosphatidate and 11% neutral lipid. With palmitoyl-(-)-carnitine the equivalent values were 70, 23 and 7%, which were significantly different. 4. When palmitoyl-CoA synthetase had been inactivated by 60-70% after preincubation of mitochondria at 37 degrees C, it became rate-limiting in glycerolipid biosynthesis. Additions of 1-5mm-ATP prevented inactivation of palmitoyl-CoA synthetase. 5. Preincubation also inhibited the oxidation of palmitate, palmitoyl-CoA, palmitoyl-(-)-carnitine and malate plus glutamate. These inhibitions could not be prevented by addition of ATP. 6. Diversion of palmitoyl-CoA to form palmitoyl-(-)-carnitine did not inhibit sn-glycerol 3-phosphate esterification. 7. The palmitoyl-CoA pool synthesized by the palmitoyl-CoA synthetase was augmented by adding partially purified synthetase or carnitine palmitoyltransferase and palmitoyl-(-)-carnitine. No stimulation of palmitate incorporation into glycerolipids occurred. 8. At low concentrations of Mg(2+), palmitate, ATP and CoA the velocity with palmitoyl-CoA synthetase decreased more than that of glycerolipid synthesis from palmitate. 9. It is concluded that in the presence of optimum substrate concentrations the activity of sn-glycerol 3-phosphate acyltransferase and not of palmitoyl-CoA synthetase is rate-limiting in the synthesis of phosphatidate and lysophosphatidate in isolated rat liver mitochondria.

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Factors affecting the activity and stability of the palmitoyl-coenzyme A hydrolase of rat brain.

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