Rat liver microsomal palmitoyl-coenzyme A synthetase. Structural properties.
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ABSTRACT:
SUBMITTER: Bar-Tana J
PROVIDER: S-EPMC1177492 | biostudies-other | 1973 Mar
REPOSITORIES: biostudies-other
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Project description:The partial and exchange reactions of long-chain fatty acid activation were determined by using purified microsomal long-chain fatty acyl-CoA synthetase (EC 6.2.1.3). No significant ATP formation from palmitoyl-AMP and PP(i), nor palmitoyl-AMP-dependent CoA disappearance could be demonstrated. Similarly, no palmitate-dependent [(32)P(2)]PP(i)-ATP exchange was catalysed by the pure enzyme. The above partial and exchange reactions were, however, catalysed by the parent microsomal fraction at a rate similar to that of the overall reaction. The implications of these results are discussed.
| S-EPMC1174268 | biostudies-other
Project description:The isolation and purification of palmitoyl-CoA synthetase from rat liver microsomes is described. Several methods suitable for enzyme assay are described. The general properties and kinetic parameters of the purified enzyme were determined and are discussed in relationship to microsomal fatty acid activation.
| S-EPMC1176785 | biostudies-other
Project description:Palmitoyl-CoA hydrolase (EC 3.1.2.2) and palmitoyl-L-carnitine hydrolase (EC 3.1.1.28) activities from rat liver were investigated. 1. Microsomal and mitochondrial-matrix palmitoyl-CoA hydrolase activities had similar pH and temperature optima, although the activities showed different temperature stability. They were inhibited by Pb2+ and Zn2+. The palmitoyl-CoA hydrolase activities in microsomal fraction and mitochondrial matrix were differently affected by the addition of Mg2+, Ca2+, Co2+, K+ and Na+ to the reaction mixture. ATP, ADP and NAD+ stimulated the microsomal activity and inhibited the mitochondrial-matrix enzyme. The activity of both the microsomal and mitochondrial-matrix hydrolase enzymes was specific for long-chain fatty acyl-CoA esters (C12-C18), with the highest activity for palmitoyl-CoA. The apparent Km for palmitoyl-CoA was 47 microM for the microsomal enzyme and 17 microM for the mitochondrial-matrix enzyme. 2. The palmitoyl-CoA hydrolase and palmitoyl-L-carnitine hydrolase activities of microsomal fraction had similar pH optima and were stimulated by dithiothreitol, but were affected differently by the addition of Pb2+, Mg2+, Ca2+, Mn2+ and cysteine. The two enzymes had different temperature-sensitivities. 3. The data strongly suggest that palmitoyl-CoA hydrolase and palmitoyl-L-carnitine hydrolase are separate microsomal enzymes, and that the hydrolysis of palmitoyl-CoA in the microsomal fraction and mitochondria matrix was catalysed by two different enzymes.
| S-EPMC1161132 | biostudies-other
Project description:1. Rat liver microsomal stearoyl-CoA desaturase activity was shown to be stimulated by both bovine serum albumin and a basic cytoplasmic protein from rat liver. 2. Partially purified desaturase is unaffected by either of these two proteins. 3. Bovine serum albumin appears to exert its effect on the crude system by protecting the desaturase substrate, stearoly-CoA, from the action of endogenous thiolesterases. 4. By using partially purified enzyme preparations, it was possible to establish the substate specificity of the delta9-fatty acyl-CoA desaturase with the C14, C15, C16, C17, C18 and C19 fatty acyl-CoA substrates. Maximum enzyme activity was shown with stearoyl-CoA decreasing with both palmitoyl-CoA and nonadecanoyl-CoA, as reported previously for free fatty acids. 5. Both cytochrome b5 and NADH-cytochrome b5 reductase (EC 1.6.2.2) are required for these studies and a method is described for the purification of homogeneous preparations of detergent-isolated cytochrome b5 from rat liver. 6. From amino acid analyses, a comparison was made of the hydrophobicity of the membrane portion of cytochrome b5 with the hydrophobicity reported for stearoyl-CoA desaturase. The close resemblance of the two values suggested that unlike cytochrome b5 and its reductase, the stearoyl-CoA desaturase may be largely buried in the endoplasmic reticulum.
| S-EPMC1164521 | biostudies-other
Project description:The mechanism of long-chain fatty acid activation catalysed by highly purified microsomal palmitoyl-CoA synthetase was investigated. The kinetics of the overall reaction were found to conform to the Bi Uni Uni Bi Ping Pong mechanism. (18)O was transferred from [(18)O]palmitate to AMP and palmitoyl-CoA exclusively. The enzyme intermediate formed appeared to consist of enzyme-bound palmitate; this formation occurred only in the presence of ATP. However, the involvement of palmitoyl-AMP in the reaction catalysed by the purified enzyme has proved difficult to establish.
| S-EPMC1177459 | biostudies-other
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.
| S-EPMC1177645 | biostudies-other
Project description:An enzyme-bound intermediate of the overall reaction catalysed by rat liver microsomal long-chain fatty acyl-CoA synthetase is described. It was found to contain equimolar amounts of adenylate and fatty acid moieties bound to protein, and was stabilized by ATP. The intermediate reacted with CoA to give palmitoyl-CoA.
| S-EPMC1165843 | biostudies-other
Project description:Choloyl-CoA synthetase (EC 6.2.1.7) was characterized for the first time under appropriated assay conditions. The p/ optimum for the reaction is pH 7.2.-7.3. The reaction has an absolute requirement for bivalent cation. Several different metal ions fulfil this requirement, but Mn2+ and Mg2+ were the most effective. The KAppm (apparent Km) for CoA, extrapolated from kinetic data, is 50 micronM, but in fact the rate of reaction is increased little by concentrations of CoA above 25 micronM. The KAppm for ATP is 600 micronM. High concentrations of ATP appear to cause substrate inhibition. The KAppm for cholate was 6 micronM. The enzyme was inhibited by treating the microsomal fraction with N-ethylmaleimide. The inclusion of various conjugated and unconjugated bile salts in the assay also inhibited the enzyme. Unconjugated bile salts were more potent inhibitors than the conjugated bile salts. High concentrations of oleic acid inhibited the enzyme. The properties of choloyl-CoA synthetase were not modified by alterations of the properties of the lipid phase of the microsomal membrane. Treatment with phospholipase A did not alter activity directly. Triton N-101 and Triton X-100 also were without effect on activity, and the enzyme was insensitive to temperature-induced phase transitions within the lipid portion of the membrane. The enzyme can be solubilized from the microsomal membrane in an active form by treatment with Triton N-101.
| S-EPMC1164704 | biostudies-other
Project description:A cDNA which encodes a carboxylesterase of 561 amino acid residues including a cleavable signal peptide is described. The enzyme expressed in COS cells migrates during PAGE (SDS-, and non-denaturing) as a single prominent band in the region of liver ES-4. It ends in the C-terminal cell-retention signal -HNEL, which, in COS cells overexpressing the enzyme, appears to be slightly less efficient than the signals -HTEL and -HVEL of ES-3 and ES-10 respectively. Glycosylation is not essential for intracellular retention, but leads to a higher activity. As do many carboxylesterases, the enzyme expressed in COS cells hydrolyses omicron-nitrophenyl acetate and alpha-naphthyl acetate. It also hydrolyses acetanilide, although less efficiently than ES-3, and, distinctively, palmitoyl-CoA. In addition to the four canonical Cys residues of the carboxylesterases, it contains a fifth, unpaired Cys336, which apparently is not essential for the catalytic properties. Indeed, treatment with iodoacetamide or substitution of Cys336 by Phe does not markedly alter the activity of the enzyme on the various substrates. The predicted structure of ES-4 is highly homologous to that of two other recently cloned esterases which also end in -HNEL [Yan, Yang, Brady and Parkinson (1994) J. Biol. Chem. 269, 29688-29696; Yan, Yang, and Parkinson (1995) Arch. Biochem. Biophys. 317, 222-234]. Together, these isoenzymes probably account for the closely spaced bands observed in the region of ES-4 in non-denaturing PAGE.
| S-EPMC1216984 | biostudies-other
Project description:Acetyl coenzyme A synthetase-1 (AceCS1) catalyzes the synthesis of acetyl coenzyme A from acetate and coenzyme A and is thought to play diverse roles ranging from fatty acid synthesis to gene regulation. By using an affinity-purified antibody generated against an 18-mer peptide sequence of AceCS1 and a polyclonal antibody directed against recombinant AceCS1 protein, we examined the expression of AceCS1 in the rat brain. AceCS1 immunoreactivity in the adult rat brain was present predominantly in cell nuclei, with only light to moderate cytoplasmic staining in some neurons, axons, and oligodendrocytes. Some nonneuronal cell nuclei were very strongly immunoreactive, including those of some oligodendrocytes, whereas neuronal nuclei ranged from unstained to moderately stained. Both antibodies stained some neuronal cell bodies and axons, especially in the hindbrain. AceCS1 immunoreactivity was stronger and more widespread in the brains of 18-day-old rats than in adults, with increased expression in oligodendrocytes and neurons, including cortical pyramidal cells. Expression of AceCS1 was substantially up-regulated in neurons throughout the brain after controlled cortical impact injury. The strong AceCS1 expression observed in the nuclei of CNS cells during brain development and after injury is consistent with a role in nuclear histone acetylation and therefore the regulation of chromatin structure and gene expression. The cytoplasmic staining observed in some oligodendrocytes, especially during postnatal brain development, suggests an additional role in CNS lipid synthesis and myelination. Neuronal and axonal localization implicates AceCS1 in cytoplasmic acetylation reactions in some neurons.
| S-EPMC3047483 | biostudies-literature