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Computer-based approaches identified PTE2 as a candidate human peroxisomal acyl-CoA thioesterase gene. The PTE2 gene product is highly similar to the rat cytosolic and mitochondrial thioesterases, CTE1 and MTE1, respectively, and terminates in a tripeptide sequence, serine-lysine-valine(COOH), that resembles the consensus sequence for type-1 peroxisomal targeting signals. PTE2 was targeted to peroxisomes and recombinant PTE2 showed intrinsic acyl-CoA thioesterase activity with a pH optimum of 8.5. A comparison of PTE2 and PTE1 thioesterase activities across multiple acyl-CoA substrates indicated that while PTE1 was most active on medium-chain acyl-CoAs, with little activity on long-chain acyl-CoAs, PTE2 displayed high activity on medium- and long-chain acyl-CoAs. The identification of PTE2 therefore offers an explanation for the observed long-chain acyl-CoA thioesterase activity of mammalian peroxisomes.  相似文献   

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Shahi P  Kumar I  Sharma R  Sanger S  Jolly RS 《The FEBS journal》2006,273(11):2374-2387
A novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis has been isolated and characterized. The protein was extracted from the cells with 1 m NaCl, which required 1.5-fold, single-step purification to yield near-homogeneous preparations. In solution, the protein exists as homomeric aggregates, of mean diameter 21.6 nm, consisting of 22-kDa subunits. MS/MS data for peptides obtained by trypsin digestion of the thiosterase did not match any peptide from Escherichia coli thioesterases or any other thioesterases in the database. The thioesterase was associated exclusively with the surface of cells as revealed by ultrastructural studies using electron microscopy and immunogold labeling. It hydrolyzed saturated and unsaturated fatty acyl-CoAs of C12 to C18 chain length with Vmax and Km of 3.58-9.73 micromol x min(-1) x (mg protein)(-1) and 2.66-4.11 microm, respectively. A catalytically important histidine residue is implicated in the active site of the enzyme. The thioesterase was active and stable over a wide range of temperature and pH. Maximum activity was observed at 65 degrees C and pH 10.5, and varied between 60% and 80% at temperatures of 25-70 degrees C and pH 6.5-10. The thioesterase also hydrolyzed p-nitrophenyl esters of C2 to C12 chain length, but substrate competition experiments demonstrated that the long-chain acyl-CoAs are better substrates for thioesterase than p-nitrophenyl esters. When assayed at 37 and 20 degrees C, the affinity and catalytic efficiency of the thioesterase for palmitoleoyl-CoA and cis-vaccenoyl-CoA were reduced approximately twofold at the lower temperature, but remained largely unaltered for palmitoyl-CoA.  相似文献   

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Peroxisomes function in beta-oxidation of very long and long-chain fatty acids, dicarboxylic fatty acids, bile acid intermediates, prostaglandins, leukotrienes, thromboxanes, pristanic acid, and xenobiotic carboxylic acids. These lipids are mainly chain-shortened for excretion as the carboxylic acids or transported to mitochondria for further metabolism. Several of these carboxylic acids are slowly oxidized and may therefore sequester coenzyme A (CoASH). To prevent CoASH sequestration and to facilitate excretion of chain-shortened carboxylic acids, acyl-CoA thioesterases, which catalyze the hydrolysis of acyl-CoAs to the free acid and CoASH, may play important roles. Here we have cloned and characterized a peroxisomal acyl-CoA thioesterase from mouse, named PTE-2 (peroxisomal acyl-CoA thioesterase 2). PTE-2 is ubiquitously expressed and induced at mRNA level by treatment with the peroxisome proliferator WY-14,643 and fasting. Induction seen by these treatments was dependent on the peroxisome proliferator-activated receptor alpha. Recombinant PTE-2 showed a broad chain length specificity with acyl-CoAs from short- and medium-, to long-chain acyl-CoAs, and other substrates including trihydroxycoprostanoyl-CoA, hydroxymethylglutaryl-CoA, and branched chain acyl-CoAs, all of which are present in peroxisomes. Highest activities were found with the CoA esters of primary bile acids choloyl-CoA and chenodeoxycholoyl-CoA as substrates. PTE-2 activity is inhibited by free CoASH, suggesting that intraperoxisomal free CoASH levels regulate the activity of this enzyme. The acyl-CoA specificity of recombinant PTE-2 closely resembles that of purified mouse liver peroxisomes, suggesting that PTE-2 is the major acyl-CoA thioesterase in peroxisomes. Addition of recombinant PTE-2 to incubations containing isolated mouse liver peroxisomes strongly inhibited bile acid-CoA:amino acid N-acyltransferase activity, suggesting that this thioesterase can interfere with CoASH-dependent pathways. We propose that PTE-2 functions as a key regulator of peroxisomal lipid metabolism.  相似文献   

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The thioesterase activity of porcine pancreatic phospholipase A2 has been investigated with non-phospholipid substrates. The acyl-CoA hydrolase activity towards acyl-CoA derivatives is specific for long chain fatty acids (14 C, 16 C) but is unable to hydrolyze short chain acyl-CoA compounds (below 8 C). The same enzyme also shows protein deacylase activity liberating [3H]palmitic acid from [3H]palmitoyl-acyl carrier protein.  相似文献   

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Membrane preparations from Saccharomyces cerevisiae OC-2 catalyzed the acylation of glycerophosphate, 1-acyl and 2-acyl isomers of monoacylglycerophosphate, and 1-acyl and 2-acyl isomers of monoacylglycerylphosphorylcholine. The acyl-CoA:glycerophosphate acyltransferase system (EC 2.3.1.15) showed a broad specificity for acyl-CoAs when the maximal velocities were compared under optimized conditions. The acyl-CoA:2-acylglycerophosphate acyltransferase activity was much lower than the 1-acyl-glycerophosphate acyltransferase activity. Although the 1-acylglycerophosphate acyltransferase system utilized saturated and unsaturated acyl-CoAs at comparable rates, the acylations at the 1- and 2-positions were relatively more selective for palmitate and oleate, respectively, when assayed in the presence of palmitoyl-CoA, oleoyl-CoA, 1-acylglycerophosphate, and 2-acylglycerophosphate. The acyl-CoA:1-acylglyceryl-phosphorylcholine acyltransferase system (EC 2.3.1.23) was relatively more specific for unsaturated acyl-CoAs, while the acyl-CoA:2-acylglycerylphosphorylcholine acyltransferase system (EC 2.3.1.23) utilized both palmitoyl-CoA and oleoyl-CoA at a comparable rate. Although various acyltransferase systems showed a different degree of specificity for acyl-CoAs, the positional distribution of fatty acids in the phospholipid molecules could not be explained simply by the observed specificities. Zymolyase, β-1,3-glucanase from Arthrobacter luteus, was used successfully for the protoplast formation. Subcellular fractionation of the protoplast revealed that these acyltransferase activities were localized mainly in the microsomal fraction. However, the glycerophosphate and 1-acylglycerophosphate acyltranferase activities in the mitochondrial fraction could not be explained by the contamination of microsomes in this fraction. These observations are apparently inconsistent with a current concept that the mitochondrial fraction is the major site of phospholipid synthesis in yeast.  相似文献   

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Thioesterase superfamily member 1 (Them1; synonyms acyl-CoA thioesterase 11 and StarD14) is highly expressed in brown adipose tissue and limits energy expenditure in mice. Them1 is a putative fatty acyl-CoA thioesterase that comprises tandem hot dog-fold thioesterase domains and a lipid-binding C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. To better define its role in metabolic regulation, this study examined the biochemical and enzymatic properties of Them1. Purified recombinant Them1 dimerized in solution to form an active fatty acyl-CoA thioesterase. Dimerization was induced by fatty acyl-CoAs, coenzyme A (CoASH), ATP, and ADP. Them1 hydrolyzed a range of fatty acyl-CoAs but exhibited a relative preference for long-chain molecular species. Thioesterase activity varied inversely with temperature, was stimulated by ATP, and was inhibited by ADP and CoASH. Whereas the thioesterase domains of Them1 alone were sufficient to yield active recombinant protein, the START domain was required for optimal enzyme activity. An analysis of subcellular fractions from mouse brown adipose tissue and liver revealed that Them1 contributes principally to the fatty acyl-CoA thioesterase activity of microsomes and nuclei. These findings suggest that under biological conditions, Them1 functions as a lipid-regulated fatty acyl-CoA thioesterase that could be targeted for the management of metabolic disorders.  相似文献   

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Long-chain acyl-CoA thioesterases hydrolyze long-chain acyl-CoAs to the corresponding free fatty acid and CoASH and may therefore play important roles in regulation of lipid metabolism. We have recently cloned four members of a highly conserved acyl-CoA thioesterase multigene family expressed in cytosol (CTE-I), mitochondria (MTE-I), and peroxisomes (PTE-Ia and -Ib), all of which are regulated via the peroxisome proliferator-activated receptor alpha (Hunt, M. C., Nousiainen, S. E. B., Huttunen, M. K., Orii, K. E., Svensson, L. T., and Alexson, S. E. H. (1999) J. Biol. Chem. 274, 34317-34326). Sequence comparison revealed the presence of putative active-site serine motifs (GXSXG) in all four acyl-CoA thioesterases. In the present study we have expressed CTE-I in Escherichia coli and characterized the recombinant protein with respect to sensitivity to various amino acid reactive compounds. The recombinant CTE-I was inhibited by phenylmethylsulfonyl fluoride and diethyl pyrocarbonate, suggesting the involvement of serine and histidine residues for the activity. Extensive sequence analysis pinpointed Ser(232), Asp(324), and His(358) as the likely components of a catalytic triad, and site-directed mutagenesis verified the importance of these residues for the catalytic activity. A S232C mutant retained about 2% of the wild type activity and incubation with (14)C-palmitoyl-CoA strongly labeled this mutant protein, in contrast to wild-type enzyme, indicating that deacylation of the acyl-enzyme intermediate becomes rate-limiting in this mutant protein. These data are discussed in relation to the structure/function of acyl-CoA thioesterases versus acyltransferases. Furthermore, kinetic characterization of recombinant CTE-I showed that this enzyme appears to be a true acyl-CoA thioesterase being highly specific for C(12)-C(20) acyl-CoAs.  相似文献   

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Calcium-independent phospholipase A(2) (iPLA(2)) is the major phospholipase A(2) activity in many cell types, and at least one isoform of this enzyme class is physically and functionally coupled to calmodulin (CaM) in a reversible calcium-dependent fashion. To identify the domain in recombinant iPLA(2)beta (riPLA(2)beta) underlying this interaction, multiple techniques were employed. First, we identified calcium-activated CaM induced alterations in the kinetics of proteolytic fragment generation during limited trypsinolysis (i.e. CaM footprinting). Tryptic digests of riPLA(2)beta (83 kDa) in the presence of EGTA alone, Ca(+2) alone, or EGTA and CaM together resulted in the production of a major 68-kDa protein whose kinetic rate of formation was specifically attenuated in incubations containing CaM and Ca(+2) together. Western blotting utilizing antibodies directed against either the N- or C-terminal regions of riPLA(2)beta indicated the specific protection of riPLA(2)beta by calcium-activated CaM at a cleavage site approximately 15 kDa from the C terminus. Moreover, calcium-activated calmodulin increased the kinetic rate of tryptic cleavage near the active site of riPLA(2)beta. Second, functional characterization of products from these partial tryptic digests demonstrated that approximately 90% of the 68-kDa riPLA(2)beta tryptic product (i.e. lacking the 15-kDa C-terminus) did not bind to a CaM affinity matrix in the presence of Ca(2+), although >95% of the noncleaved riPLA(2)beta as well as a 40-kDa C-terminal peptide bound tightly under these conditions. Third, when purified riPLA(2)beta was subjected to exhaustive trypsinolysis followed by ternary complex CaM affinity chromatography, a unique tryptic peptide ((694)AWSEMVGIQYFR(705)) within the 15-kDa C-terminal fragment was identified by RP-HPLC, which bound to CaM-agarose in the presence but not the absence of calcium ion. Fourth, fluorescence energy transfer experiments demonstrated that this peptide (694) bound to dansyl-calmodulin in a calcium-dependent fashion. Collectively, these results identify multiple contact points in the 15-kDa C terminus as being the major but not necessarily the only binding site responsible for the calcium-dependent regulation of iPLA(2)beta by CaM.  相似文献   

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We report that potassium leakage from cells leads to activation of the Ca2+-independent phospholipase A2 (iPLA2), and the latter plays a pivotal role in regulating the cleavage of pro-IL-1 beta by the IL-converting enzyme caspase-1 in human monocytes. K+ efflux led to increases of cellular levels of glycerophosphocholine, an unambiguous indicator of phospholipase A2 activation. Both maturation of IL-1 beta and formation of glycerophosphocholine were blocked by bromoenol lactone, the specific iPLA2 inhibitor. Bromoenol lactone-dependent inhibition of IL-1 beta processing was not due to perturbation of the export machinery for pro-IL-1 beta and IL-1 beta or to caspase-1 suppression. Conspicuously, activation of Ca2+-dependent phospholipase A2 did not support but rather suppressed IL-1 beta processing. Thus, our findings reveal a specific role for iPLA2 activation in the sequence of events underlying IL-1 beta maturation.  相似文献   

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Ramanadham S  Hsu FF  Zhang S  Jin C  Bohrer A  Song H  Bao S  Ma Z  Turk J 《Biochemistry》2004,43(4):918-930
The death of insulin-secreting beta-cells that causes type I diabetes mellitus (DM) occurs in part by apoptosis, and apoptosis also contributes to progressive beta-cell dysfunction in type II DM. Recent reports indicate that ER stress-induced apoptosis contributes to beta-cell loss in diabetes. Agents that deplete ER calcium levels induce beta-cell apoptosis by a process that is independent of increases in [Ca(2+)](i). Here we report that the SERCA inhibitor thapsigargin induces apoptosis in INS-1 insulinoma cells and that this is inhibited by a bromoenol lactone (BEL) inhibitor of group VIA calcium-independent phospholipase A(2) (iPLA(2)beta). Overexpression of iPLA(2)beta amplifies thapsigargin-induced apoptosis of INS-1 cells, and this is also suppressed by BEL. The magnitude of thapsigargin-induced INS-1 cell apoptosis correlates with the level of iPLA(2)beta expression in various cell lines, and apoptosis is associated with stimulation of iPLA(2)beta activity, perinuclear accumulation of iPLA(2)beta protein and activity, and caspase-3-catalyzed cleavage of full-length 84 kDa iPLA(2)beta to a 62 kDa product that associates with nuclei. Thapsigargin also induces ceramide accumulation in INS-1 cells, and this response is amplified in cells that overexpress iPLA(2)beta. These findings indicate that iPLA(2)beta participates in ER stress-induced apoptosis, a pathway that promotes beta-cell death in diabetes.  相似文献   

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The agonist-stimulated release of arachidonic acid (AA) from cellular phospholipids in many cell types (e.g. myocytes, beta-cells, and neurons) has been demonstrated to be primarily mediated by calcium-independent phospholipases A(2) (iPLA(2)s) that are inhibited by the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL). Recently, the family of mammalian iPLA(2)s has been extended to include iPLA(2)gamma, which previously could not be pharmacologically distinguished from iPLA(2)beta. To determine whether iPLA(2)beta or iPLA(2)gamma (or both) were the enzymes responsible for arginine vasopressin (AVP)-induced AA release from A-10 cells, it became necessary to inhibit selectively iPLA(2)beta and iPLA(2)gamma in intact cells. We hypothesized that the R- and S-enantiomers of BEL would possess different inhibitory potencies for iPLA(2)beta and iPLA(2)gamma. Accordingly, racemic BEL was separated into its enantiomeric constituents by chiral high pressure liquid chromatography. Remarkably, (S)-BEL was approximately an order of magnitude more selective for iPLA(2)beta in comparison to iPLA(2)gamma. Conversely, (R)-BEL was approximately an order of magnitude more selective for iPLA(2)gamma than iPLA(2)beta. The AVP-induced liberation of AA from A-10 cells was selectively inhibited by (S)-BEL (IC(50) approximately 2 microm) but not (R)-BEL, demonstrating that the overwhelming majority of AA release is because of iPLA(2)beta and not iPLA(2)gamma activity. Furthermore, pretreatment of A-10 cells with (S)-BEL did not prevent AVP-induced MAPK phosphorylation or protein kinase C translocation. Finally, two different cell-permeable protein kinase C activators (phorbol-12-myristate-13-acetate and 1,2-dioctanoyl-sn-glycerol) could not restore the ability of A-10 cells to release AA after exposure to (S)-BEL, thus supporting the downstream role of iPLA(2)beta in AVP-induced AA release.  相似文献   

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In a previous paper we showed that bradykinin (BK), interacting with its B2 receptor, inhibits proximal tubule Na+ -ATPase activity but does not change (Na+ +K+)ATPase activity. The aim of this paper was to investigate the molecular mechanisms involved in B2-mediated modulation of proximal tubule Na+ -ATPase by BK. To abolish B1 receptor-mediated effects, all experiments were carried out in the presence of (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu), des-Arg9-[Leu8]-BK (DALBK), a specific antagonist of B1 receptor. A dual effect on the Na+ -ATPase activity through the B2 receptor was found: short incubation times (1-10 min) stimulate the enzyme activity; long incubation times (10-60 min) inhibit it. The stimulatory effect of BK is mediated by activation of phosphoinositide-specific phospholipase C beta (PI-PLCbeta)/protein kinase C (PKC); its inhibitory action is mediated by Ca2+ -independent phospholipase A2 (iPLA2). Prior activation of the PI-PLCbeta/PKC pathway is required to activate the iPLA2-mediated inhibitory phase. These results reveal a new mechanism by which BK can modulate renal sodium excretion: coupling between B2 receptor and activation of membrane-associated iPLA2.  相似文献   

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Studies involving pharmacologic or molecular biologic manipulation of Group VIA phospholipase A(2) (iPLA(2)beta) activity in pancreatic islets and insulinoma cells suggest that iPLA(2)beta participates in insulin secretion. It has also been suggested that iPLA(2)beta is a housekeeping enzyme that regulates cell 2-lysophosphatidylcholine (LPC) levels and arachidonate incorporation into phosphatidylcholine (PC). We have generated iPLA(2)beta-null mice by homologous recombination and have reported that they exhibit reduced male fertility and defective motility of spermatozoa. Here we report that pancreatic islets from iPLA(2)beta-null mice have impaired insulin secretory responses to D-glucose and forskolin. Electrospray ionization mass spectrometric analyses indicate that the abundance of arachidonate-containing PC species of islets, brain, and other tissues from iPLA(2)beta-null mice is virtually identical to that of wild-type mice, and no iPLA(2)beta mRNA was observed in any tissue from iPLA(2)beta-null mice at any age. Despite the insulin secretory abnormalities of isolated islets, fasting and fed blood glucose concentrations of iPLA(2)beta-null and wild-type mice are essentially identical under normal circumstances, but iPLA(2)beta-null mice develop more severe hyperglycemia than wild-type mice after administration of multiple low doses of the beta-cell toxin streptozotocin, suggesting an impaired islet secretory reserve. A high fat diet also induces more severe glucose intolerance in iPLA(2)beta-null mice than in wild-type mice, but PLA(2)beta-null mice have greater responsiveness to exogenous insulin than do wild-type mice fed a high fat diet. These and previous findings thus indicate that iPLA(2)beta-null mice exhibit phenotypic abnormalities in pancreatic islets in addition to testes and macrophages.  相似文献   

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