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1.
The effect of fatty acids, palmitoyl-CoA, and N',N-dicyclohexylcarbodiimide on the ion conductance of the reconstituted brown adipose tissue mitochondria uncoupling protein was investigated. 1, 5, and 10 microM palmitic acid induced a specific, GDP inhibited, increase in proton conductance in proteoliposomes containing the uncoupling protein but not in proteoliposomes prepared with purified protein extracts of liver mitochondria. 10 microM oleic acid, like palmitic acid, increased proton conductance in proteoliposomes prepared with the uncoupling protein. Palmitoyl-CoA and caprylic acid had no effect on increasing proton conductance. Similar to the observation in mitochondria, there was no effect of palmitic acid on Cl-conductance, but unlike mitochondria its activation by palmitoyl-CoA or inhibition by N',N-dicyclohexylcarbodiimide was lost. The results, obtained in an isolated system, provide support for the contention that long chain fatty acids act as an acute physiological activator of the uncoupling protein.  相似文献   

2.
Microsomal acyl CoA:cholesterol acyltransferase (ACAT) is stimulated in vitro and/or in intact cells by proteins that bind and transfer both substrates, cholesterol, and fatty acyl CoA. To resolve the role of fatty acyl CoA binding independent of cholesterol binding/transfer, a protein that exclusively binds fatty acyl CoA (acyl CoA binding protein, ACBP) was compared. ACBP contains an endoplasmic reticulum retention motif and significantly colocalized with acyl-CoA cholesteryl acyltransferase 2 (ACAT2) and endoplasmic reticulum markers in L-cell fibroblasts and hepatoma cells, respectively. In the presence of exogenous cholesterol, ACAT was stimulated in the order: ACBP > sterol carrier protein-2 (SCP-2) > liver fatty acid binding protein (L-FABP). Stimulation was in the same order as the relative affinities of the proteins for fatty acyl CoA. In contrast, in the absence of exogenous cholesterol, these proteins inhibited microsomal ACAT, but in the same order: ACBP > SCP-2 > L-FABP. The extracellular protein BSA stimulated microsomal ACAT regardless of the presence or absence of exogenous cholesterol. Thus, ACBP was the most potent intracellular fatty acyl CoA binding protein in differentially modulating the activity of microsomal ACAT to form cholesteryl esters independent of cholesterol binding/transfer ability.  相似文献   

3.
Long-chain unsaturated fatty acids and fatty acyl CoA derivatives activated (Na++K+)-ATPase at suboptimal, but not optimal, ATP concentrations. Activation was obtained within a narrow range of fatty acid concentrations; higher acid levels inhibited the enzyme. The various CoA esters, however, activated with K0.5 values in the range of 0.15-10 microM; and with no inhibitory effects at concentrations up to 100 microM. Palmitoyl CoA, binding reversibly to a regulatory site, reduced K0.5 of ATP from 0.37 mM to 0.17 mM; and changed the Hill coefficient of the substrate-velocity curve from 0.86 to 0.63. These compounds may be physiological regulators that desensitize the function of this enzyme to diminishing ATP levels.  相似文献   

4.
The ability of purified rat liver and heart fatty acid binding proteins to bind oleoyl-CoA and modulate acyl-CoA synthesis by microsomal membranes was investigated. Using binding assays employing either Lipidex 1000 or multilamellar liposomes to sequester unbound ligand, rat liver but not rat heart fatty acid binding protein was shown to bind radiolabeled acyl CoA. Binding studies suggest that liver fatty acid binding protein has a single binding site acyl-CoA which is separate from the two binding sites for fatty acids. Experiments were then performed to determine how binding may influence acyl-CoA metabolism by liver microsomes or heart sarcoplasmic reticulum. Using liposomes as fatty acid donors, liver fatty acid binding protein stimulated acyl-CoA production, whereas that from heart did not stimulate production over control values. 14C-labeled fatty acid-fatty acid binding protein complexes were prepared, incubated with membranes, and acyl-CoA synthetase activity was determined. Up to 70% of the fatty acid could be converted to acyl-CoA in the presence of liver fatty acid binding protein but in the presence of heart fatty acid binding protein, only 45% of the fatty acid was converted. Liver but not heart fatty acid binding protein bound the acyl-CoA formed and removed it from the membranes. The amount of product formed was not changed by additional membrane, enzyme cofactors, or incubation time. Additional liver fatty acid binding protein was the only factor found that stimulated product formation. Acyl-CoA hydrolase activity was also shown in the absence of ATP and CoA. These studies suggest that liver fatty acid binding protein can increase the amount of acyl-CoA by binding this ligand, thereby removing it from the membrane and possibly aiding transport within the cell.  相似文献   

5.
The time-course for the induction of the uncoupling pathway in the inner membrane of brown-fat mitochondria from cold-adapting guinea pigs was studied. The amount of the protein was quantified from the capacity for high-affinity binding of GDP to the intact mitochondria, and was compared with the functional parameters diagnostic of the protein, namely the nucleotide-sensitive proton conductance and the sensitivity to uncoupling by low concentrations of fatty acids. A monophasic increase in nucleotide titre was observed, with no evidence of an early 'unmasking' of preexisting nucleotide-binding sites. The nucleotide-sensitive conductance increased in precise synchrony with the nucleotide-binding capacity. Mitochondria from newborn animals, and those from acutely cold-adapted animals, showed anomalously low sensitivities to uncoupling by fatty acids.  相似文献   

6.
This paper reviews characteristics of microsomal membrane structure; long chain fatty acids, acyl CoA derivatives, retinoids and the microsomal formation of acyl CoA derivatives and retinyl esters. It is analyzed how the movement of these molecules at the intracellular level is affected by their respective binding proteins (Fatty acid binding protein, acyl CoA binding protein and cellular retinol binding protein). Studies with model systems using these hydrophobic ligands and the lipid-binding or transfer proteins are also described. This topic is of interest especially because in the esterification of retinol the three substrates and the three binding proteins may interact. (Mol Cell Biochem20: 89–94, 1993)Abbreviations FABP(s) Fatty Acid Binding Protein(s) - CRBP Cellular Retinol Binding Protein - ACBP Acyl-CoA-Binding Protein  相似文献   

7.
Ramsey AJ  Chinkers M 《Biochemistry》2002,41(17):5625-5632
The protein serine/threonine phosphatase designated PP5 has little basal activity, and physiological activators of the enzyme have never been identified. Purified PP5 can, however, be activated by partial proteolysis or by the binding of supraphysiological concentrations of polyunsaturated long-chain fatty acids to its tetratricopeptide repeat (TPR) domain. To test whether activation of PP5 by polyunsaturated but not saturated fatty acids was an artifact of the lower solubility of saturated fatty acids, the effects of fatty acyl-CoA esters were examined. Saturated and unsaturated long-chain fatty acids are both freely water-soluble when esterified to CoA. Long-chain fatty acyl-CoA esters activated PP5 at physiological concentrations, with the saturated compounds being more effective. We investigated the effects of chain length and of the CoA moiety on PP5 activation. Chains of 16 carbons or more were required for optimal activation, with no activation observed below 10 carbons. On the basis of competition studies using acetyl-CoA, the function of the CoA moiety appeared to be to increase solubility of the fatty acyl moiety rather than to interact with a specific binding site. These data suggested that long-chain fatty acid-CoA esters might be physiological activators of PP5 and point to a potential link between fatty acid metabolism and signal transduction via this enzyme. Because heat shock protein 90 is also known to bind to the TPR domain of PP5 via its C-terminal domain (C90), we investigated its effect on PP5 activity. C90 activated the enzyme approximately 10-fold. Thus, we have identified two potential physiological activators of PP5.  相似文献   

8.
Abstract. H14CO3 was not incorporated into fatty acids by isolated pea leaf chloroplasts, which, therefore, do not possess a self-contained pathway for the synthesis of fatty acids from early intermediates of the Calvin cycle. Citrate, pyruvate, acetate and L-acetylcarnitine were all shown to act as sources of acetyl groups for fatty acid synthesis by pea leaf chloroplasts. L-acetylcarnitine was the best substrate, being incorporated into fatty acids at rates that were at least five-fold higher than those achieved with the other substrates. Citrate was incorporated into fatty acids at the lowest rate, followed by pyruvate, with acetate being incorporated at the second highest rate of all. When the isolated chloroplasts were ruptured, an inhibition of L-acetylcarnitine incorporation into fatty acids was noted, whilst acetate incorporation remained unaffected. L-acetylcarnitine also increased the ratio of monoenoic: saturated fatty acids synthesized, compared with a 1:1 ratio observed when citrate, pyruvate and acetate were supplied as substrates. It is suggested that L-carnitine and carnitine acyltransferases play a central role in plant acyl CoA metabolism by facilitating the transfer of activated acyl groups across membranes (acyl CoA barriers).  相似文献   

9.
We characterized the uncoupling activity of the plant uncoupling protein from Solanum tuberosum (StUCP) using mitochondria from intact potato tubers or from yeast (Saccharomyces cerevisiae) expressing the StUCP gene. Compared with mitochondria from transfected yeast, StUCP is present at very low levels in intact potato mitochondrial membranes (at least thirty times lower) as shown by immunodetection with anti-UCP1 antibodies. Under conditions that ruled out undesirable effects of nucleotides and free fatty acids on uncoupling activity measurement in plant mitochondria, the linoleic acid-induced depolarization in potato mitochondria was insensitive to the nucleotides ATP, GTP, or GDP. In addition, sensitivity to linoleic acid was similar in potato and in control yeast mitochondria, suggesting that uncoupling occurring in potato mitochondria was because of a UCP-independent proton diffusion process. By contrast, yeast mitochondria expressing StUCP exhibited a higher sensitivity to free fatty acids than those from the control yeast and especially a marked proton conductance in the presence of low amounts of linoleic acid. However, this fatty acid-induced uncoupling was also insensitive to nucleotides. Altogether, these results suggest that uncoupling of oxidative phosphorylation and heat production cannot be the dominant feature of StUCP expressed in native potato tissues. However, it could play a role in preventing reactive oxygen species production as proposed for mammalian UCP2 and UCP3.  相似文献   

10.
Martin GG  Huang H  Atshaves BP  Binas B  Schroeder F 《Biochemistry》2003,42(39):11520-11532
Although liver fatty acid binding protein (L-FABP) is known to bind not only long chain fatty acid (LCFA) but also long chain fatty acyl CoA (LCFA-CoA), the physiological significance of LCFA-CoA binding has been questioned and remains to be resolved. To address this issue, the effect of L-FABP gene ablation on liver cytosolic LCFA-CoA binding, LCFA-CoA pool size, LCFA-CoA esterification, and potential compensation by other intracellular LCFA-CoA binding proteins was examined. L-FABP gene ablation resulted not only in loss of L-FABP but also in concomitant upregulation of two other intracellular LCFA-CoA binding proteins, acyl CoA binding protein (ACBP) and sterol carrier protein-2 (SCP-2), by 45 and 80%, respectively. Nevertheless, the soluble fraction from livers of L-FABP (-/-) mice bound 95% less radioactive oleoyl-CoA than wild-type L-FABP (+/+) mice. The intracellular LCFA-CoA binding protein fraction (Fraction III) from wild-type L-FABP (+/+) mice, isolated by gel permeation chromatography of liver soluble proteins, exhibited one high-affinity binding and several low-affinity binding sites for cis-parinaroyl-CoA, a naturally occurring fluorescent LCFA-CoA. In contrast, high-affinity LCFA-CoA binding was absent from Fraction III of L-FABP (-/-) mice. While L-FABP gene ablation did not alter liver LCFA-CoA pool size, LCFA-CoA acyl chains of L-FABP (-/-) mouse livers were enriched 2.1-fold in C16:1 and decreased 1.9-fold in C20:0 fatty acids. Finally, L-FABP gene ablation selectively increased the amount of LCFAs esterified into liver phospholipid > cholesteryl ester, while concomitantly decreasing the amount of fatty acids esterified into triglycerides by 40%. In summary, these data with L-FABP (-/-) mice demonstrated for the first time that L-FABP is a physiologically significant contributor to determining liver cytosolic LCFA-CoA binding capacity, LCFA-CoA acyl chain distribution, and esterified fatty acid distribution.  相似文献   

11.
GDP binding, proton conductance and the specific concentration of uncoupling protein were measured in brown-adipose-tissue mitochondria of rats treated acutely with the novel beta-agonist, BRL 26830A. At 1 h after dosing with BRL 26830A, mitochondrial GDP binding was increased more than 2-fold. The increase in binding resulted from an increase in the number of binding sites. An iterative analysis of Scatchard binding data suggested that there is only one high-affinity GDP-binding site (Kd 0.3 microM) in brown-adipose-tissue mitochondria. The acute increase in GDP binding produced by treatment with BRL 26830A occurred without any alteration in the specific mitochondrial concentration of uncoupling protein, as determined by radioimmunoassay. Treatment with the beta-agonist did, however, lead to a small increase in the GDP-sensitive component of mitochondrial proton conductance. These results indicate that GDP-binding sites on uncoupling protein can be rapidly unmasked after treatment with a brown-fat-specific beta-agonist, and that the increase in binding reflects an increase in the activity of the mitochondrial proton-conductance pathway.  相似文献   

12.
Fatty acids behave as activators of the aP2 gene expression in committed, lipid-free, non-terminally differentiated Ob1771 cells. Like fatty acids, dexamethasone provokes a dose-dependent accumulation of aP2 mRNA. However, fatty acids and dexamethasone act through different mechanisms to activate the aP2 gene expression since i) fatty acids and dexamethasone act in a synergistic manner; ii) the effect of dexamethasone is rapid and transient (maximal effect after 8 h), whereas that of fatty acids is slower, and maintained as long as the inducer is present and is fully reversible upon fatty acid removal; iii) the induction of the aP2 gene expression by dexamethasone does not require ongoing protein synthesis, while the response to fatty acids is completely prevented by cycloheximide; and iv) the induction of the aP2 gene expression by fatty acids but not by dexamethasone is confined to preadipocyte cell lines. This suggests that the process of activation by fatty acids, rather than the expression of the aP2 gene, is unique to adipose cells. Besides their effects on the aP2 gene, fatty acids activate the expression of the acyl CoA synthetase gene which encodes another protein involved in fatty acid metabolism. Activation of both genes by fatty acids appears not to be mediated by the CCAAT enhancer binding protein, a nuclear factor reported as transactivator of the aP2 promoter activity, since the enhancer binding protein mRNA is not expressed under these conditions.  相似文献   

13.
Protein-mediated transport of exogenous long-chain fatty acids across the membrane has been defined in a number of different systems. Central to understanding the mechanism underlying this process is the development of the appropriate experimental systems which can be manipulated using the tools of molecular genetics. Escherichia coli and Saccharomyces cerevisiae are ideally suited as model systems to study this process in that both [1] exhibit saturable long-chain fatty acid transport at low ligand concentration; [2] have specific membrane-bound and membrane-associated proteins that are components of the transport apparatus; and [3] can be easily manipulated using the tools of molecular genetics. In E. coli, this process requires the outer membrane-bound fatty acid transport protein FadL and the inner membrane associated fatty acyl CoA synthetase (FACS). FadL appears to represent a substrate specific channel for long-chain fatty acids while FACS activates these compounds to CoA thioesters thereby rendering this process unidirectional. This process requires both ATP generated from either substrate-level or oxidative phosphorylation and the proton electrochemical gradient across the inner membrane. In S. cerevisiae, the process of long-chain fatty acid transport requires at least the membrane-bound protein Fat1p. Exogenously supplied fatty acids are activated by the fatty acyl CoA synthetases Faa1p and Faa4p but unlike the case in E. coli, there is not a tight linkage between transport and activation. Studies evaluating the growth parameters in the presence of long-chain fatty acids and long-chain fatty acid transport profiles of a fat1 strain support the hypothesis that Fat1p is required for optimal levels of long-chain fatty acid transport.  相似文献   

14.
In this article we comment on the various in vitro biochemical measurements employed to assess the thermogenic activity and capacity of brown adipose tissue. The meaning and significance of changes in tissue weight, protein content, cell number, and mitochondrial mass are each summarized. In addition, various indices of the proton conductance pathway-mitochondrial swelling, proton conductance, uncoupling protein concentration, and GDP binding studies--are discussed. The issue of unmasking and masking of GDP binding sites is reviewed; recent reports have clearly demonstrated unmasking and masking, and it is concluded that GDP binding studies are an index of the activity of uncoupling protein, rather than a measure of its concentration. It is suggested that tissue mass, mitochondrial content, mitochondrial GDP binding, and uncoupling protein concentration represent core measurements for the biochemical assessment of the thermogenic activity and capacity of brown adipose tissue. Auxiliary measurements include Scatchard analysis of GDP binding data to distinguish changes in the number of binding sites from potential changes in Kd, and mitochondrial swelling studies, as an additional index of proton permeability. The distinction between thermogenic activity (GDP binding, proton permeability) and capacity (uncoupling protein content), both on a per unit of mitochondrial protein and per tissue basis, is emphasized.  相似文献   

15.
The uncoupling protein 1 (UCP1) is a H(+) carrier which plays a key role in heat generation in brown adipose tissue. The H(+) transport activity of UCP1 is activated by long-chain fatty acids and inhibited by purine nucleotides. While nucleotide binding has been well characterized, the interaction of fatty acid with UCP1 remains unknown. Here I demonstrate the binding of fatty acids by competition with a fluorescent nucleotide probe 2(')-O-dansyl guanosine 5(')-triphosphate (GTP), which has been shown previously to bind at the nucleotide binding site in UCP1. Fatty acids but not their esters competitively inhibit the binding of 2(')-O-dansyl GTP to UCP1. The fatty acid effect was enhanced at higher pH, suggesting the binding of fatty acid anion to UCP1. The inhibition constants K(i) were determined by fluorescence titrations for various fatty acids. Short-chain (C<8) fatty acids display no affinity, whereas medium-chain (C10-14) and unsaturated C18 fatty acids exhibit stronger affinity (K(i)=65 microM, for elaidic acid). This specificity profile agrees with previous functional data obtained in both proteoliposomes and mitochondria, suggesting a possible physiological role of this fatty acid binding site.  相似文献   

16.
Liver fatty acid-binding protein (FABP) binds a variety of non-polar anionic ligands including fatty acids, fatty acyl CoAs, and bile acids. Previously we prepared charge reversal mutants and demonstrated the importance of lysine residues within the portal region in ligand and membrane binding. We have now prepared several tryptophan-containing mutants within the portal region, and one tryptophan at position 28 (L28W) has proved remarkably effective as an intrinsic probe to further study ligand binding. The fluorescence of the L28W mutant was very sensitive to fatty acid and bile acid binding where a large (up to 4-fold) fluorescence enhancement was obtained. In contrast, the binding of oleoyl CoA reduced tryptophan fluorescence. Positive cooperativity for fatty acid binding was observed while detailed information on the orientation of binding of bile acid derivatives was obtained. The ability of bound oleoyl CoA to reduce the fluorescence of L28W provided an opportunity to demonstrate that fatty acyl CoAs can compete with fatty acids for binding to liver FABP under physiological conditions, further highlighting the role of fatty acyl CoAs in modulating FABP function in the cell.  相似文献   

17.
A fatty acid binding protein (FABP) was isolated from bovine mammary cytosol by gel filtration and ion exchange chromatography. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated a mol. wt. of 12,000. Isoelectric focusing showed two bands at pH 5.6 and 5.8. FABP bound long chain fatty acids and their CoA thioesters, but not medium or short chain fatty acids. Affinity constant (Ka) for 18:1 was about 2 micromolar. Endogenously bound fatty acids included 16:0, 18:0 and 18:1, in both covalent and noncovalent association with FABP. Activities of microsomal phosphatidic acid phosphatase, fatty acid:CoA ligase or diacylglycerol acyltransferase were not affected by purified FABP in vitro.  相似文献   

18.
Male Long-Evans rats (9 weeks of age) were exposed to cold (5 degrees C) for 10 days. Then, sympathetic de-activation of brown adipose tissue (BAT) was performed either by BAT surgical denervation (Sy) or by warm re-exposure at 28 degrees C (WE) for 4 days. The incidence of the two treatments on thermogenic activity of BAT mitochondrial membranes and their lipid composition was investigated. Sy and WE induced a large decrease in GDP binding on the uncoupling protein (UCP) (43% and 82%, respectively). Several parameters of mitochondrial energization were investigated. Sy and WE substantially decreased UCP-dependent proton conductance (CmH+) over the whole range of protonmotive force. CmH+ showed greater variation than GDP binding. The low basal UCP-independent CmH+ was the same in all groups. Comparison of GDP binding and CmH+ with UCP content which is not modified revealed a masking of both the nucleotide binding site and the proton channel. Sy and WE induced the same increase of phosphatidylcholine to phosphatidylethanolamine ratio (16%) but had opposite effects on fatty acid unsaturation. The results were discussed with reference to functional significance of these variations in BAT mitochondrial thermogenic activity and lipid composition.  相似文献   

19.
Fatty acid synthase was purified from Mycobacterium tuberculosis var. bovis BCG. The method developed gave a 23% yield of the synthase and also yielded purified mycocerosic acid synthase. The fatty acid synthase is of unusually large size and composed of two 500-kDa monomers. The amino acid composition of the two synthases was not identical; the N-terminus of the fatty acid synthase was blocked, whereas that of the mycocerosic acid synthase was not. Western blot analysis of crude mycobacterial extracts with polyclonal antibodies prepared against each synthase showed a single band in each case with no cross-reactivity with the other synthase. Fatty acid synthase required both NADH (Km, 11 microM) and NADPH (Km, 14 microM). The Km for acetyl-CoA and malonyl-CoA were 5 and 6 microM, respectively. Fatty acids were released from the synthase as CoA esters. A bimodal distribution of fatty acids was obtained at around C16 and C26. The primer utilization also reflects the de novo synthesis and elongation capabilities of the enzyme; acetyl-CoA was the preferred primer but CoA esters up to C8 but not C12 and C14 could serve as primers, whereas C16 was readily used as a primer for elongation. Addition of CoA and CoA ester-binding oligosaccharides caused enhanced release of C16. Since this mycobacterial fatty acid synthase is twice as large as other multifunctional fatty acid synthases, it is tempting to suggest that this synthase represents a head to tail fusion of two fatty acid synthase genes coding for a double size protein with one-half producing C16 acid and the other elongating the C16 acid to a C26 acid. The monomer of fatty acid synthase from M. smegmatis was immunologically similar and equal in size to the synthase from M. tuberculosis.  相似文献   

20.
Incubations of p-nitrophenyl fatty acyl esters and estradiol-17 beta fatty acyl 17-esters with porcine esterase, human mammary tumor cytosol and rat uterine cytosol leads to ester hydrolysis of compounds with short chain fatty acids. Esters with long chain fatty acids show no hydrolysis except in the presence of Tween 80. Short chain fatty acid esters have a higher binding potency to the estrogen receptor than long chain fatty acid esters. Extraction of the nuclear receptor peak sedimenting at 4.6S and identification of the steroid showed that about 90% of the radioactivity was associated with estradiol and only 10% with estradiol esters. These studies show that estradiol fatty acyl esters act as a storage form from which estradiol is released by enzymatic hydrolysis.  相似文献   

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