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1.
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
2.
Fatty acid export from the chloroplast. Molecular characterization of a major plastidial acyl-coenzyme A synthetase from Arabidopsis
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Acyl-coenzyme A (CoA) synthetases (ACSs, EC 6.2.1.3) catalyze the formation of fatty acyl-CoAs from free fatty acid, ATP, and CoA. Essentially all de novo fatty acid synthesis occurs in the plastid. Fatty acids destined for membrane glycerolipid and triacylglycerol synthesis in the endoplasmic reticulum must be first activated to acyl-CoAs via an ACS. Within a family of nine ACS genes from Arabidopsis, we identified a chloroplast isoform, LACS9. LACS9 is highly expressed in developing seeds and young rosette leaves. Both in vitro chloroplast import assays and transient expression of a green fluorescent protein fusion indicated that the LACS9 protein is localized in the plastid envelope. A T-DNA knockout mutant (lacs9-1) was identified by reverse genetics and these mutant plants were indistinguishable from wild type in growth and appearance. Analysis of leaf lipids provided no evidence for compromised export of acyl groups from chloroplasts. However, direct assays demonstrated that lacs9-1 plants contained only 10% of the chloroplast long-chain ACS activity found for wild type. The residual long-chain ACS activity in mutant chloroplasts was comparable with calculated rates of fatty acid synthesis. Although another isozyme contributes to the activation of fatty acids during their export from the chloroplast, LACS9 is a major chloroplast ACS. 相似文献
3.
Characterization and solubilization of an acyl chain elongation system in microsomes of leek epidermal cells 总被引:5,自引:0,他引:5
Microsomes prepared from leek epidermal tissue readily elongate stearoyl-CoA to very long chain fatty acid with malonyl-CoA as the C2 unit. In the absence of stearoyl-CoA, but in the presence of ATP, microsomes elongate endogenous free fatty acids. Endogenous CoA is the source of CoA. Palmitoyl, stearoyl, and higher saturated acyl-CoAs are readily elongated by the microsomal system but oleoyl-CoA is ineffective; however, the higher monounsaturated acyl-CoAs can be elongated. Since the very long chain fatty acids of the leek epidermis are all saturated, it would appear that the reaction controlling the nature of the final acyl product is the inactivity of oleoyl-CoA as a substrate. There is no evidence that acyl carrier protein participates in the elongation reactions. Evidence is also presented suggesting that (a) there may be two elongation systems, one responsible for the conversion of stearoyl-CoA to arachidonyl-CoA and the second involved in the conversion of arachidonyl-CoA to very long chain fatty acids, and that (b) the elongation activities may be associated with a large polypeptide. 相似文献
4.
Christopher A. Jolly David C. Wilton Friedhelm Schroeder 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2000,1483(1):185-197
Liver and intestinal cytosol contain abundant levels of long chain fatty acyl-CoA binding proteins such as liver fatty acid binding protein (L-FABP) and acyl-CoA binding protein (ACBP). However, the relative function and specificity of these proteins in microsomal utilization of long chain fatty acyl-CoAs (LCFA-CoAs) for sequential transacylation of glycerol-3-phosphate to form phosphatidic acid is not known. The results showed for the first time that L-FABP and ACBP both stimulated microsomal incorporation of the monounsaturated oleoyl-CoA and polyunsaturated arachidonoyl-CoA 8–10-fold and 2–3-fold, respectively. In contrast, these proteins inhibited microsomal utilization of the saturated palmitoyl-CoA by 69% and 62%, respectively. These similar effects of L-FABP and ACBP on microsomal phosphatidic acid biosynthesis were mediated primarily through the activity of glycerol-3-phosphate acyltransferase (GPAT), the rate limiting step, rather than by protecting the long chain acyl-CoAs from microsomal hydrolase activity. In fact, ACBP but not L-FABP protected long chain fatty acyl-CoAs from microsomal acyl-CoA hydrolase activity in the order: palmitoyl-CoA>oleoyl-CoA>arachidonoyl-CoA. In summary, the data established for the first time a role for both L-FABP and ACBP in microsomal phosphatidic acid biosynthesis. By preferentially stimulating microsomal transacylation of unsaturated long chain fatty acyl-CoAs while concomitantly exerting their differential protection from microsomal acyl-CoA hydrolase, L-FABP and ACBP can uniquely function in modulating the pattern of fatty acids esterified to phosphatidic acid, the de novo precursor of phospholipids and triacylglycerols. This may explain in part the simultaneous presence of these proteins in cell types involved in fatty acid absorption and lipoprotein secretion. 相似文献
5.
Dunphy JT Schroeder H Leventis R Greentree WK Knudsen JK Silvius JR Linder ME 《Biochimica et biophysica acta》2000,1485(2-3):185-198
Both enzymatic and autocatalytic mechanisms have been proposed to account for protein thioacylation (commonly known as palmitoylation). Acyl-CoA binding proteins (ACBP) strongly suppress non-enzymatic thioacylation of cysteinyl-containing peptides by long-chain acyl-CoAs. At physiological concentrations of ACBP, acyl-CoAs, and membrane lipids, the rate of spontaneous acylation is expected to be too slow to contribute significantly to thioacylation of signaling proteins in mammalian cells (Leventis et al., Biochemistry 36 (1997) 5546-5553). Here we characterized the effects of ACBP on enzymatic thioacylation. A protein S-acyltransferase activity previously characterized using G-protein alpha-subunits as a substrate (Dunphy et al., J. Biol. Chem., 271 (1996) 7154-7159), was capable of thioacylating short lipid-modified cysteinyl-containing peptides. The minimum requirements for substrate recognition were a free cysteine thiol adjacent to a hydrophobic lipid anchor, either myristate or farnesyl isoprenoid. PAT activity displayed specificity for the acyl donor, efficiently utilizing long-chain acyl-CoAs, but not free fatty acid or S-palmitoyl-N-acetylcysteamine. ACBP only modestly inhibited enzymatic thioacylation of a myristoylated peptide or G-protein alpha-subunits under conditions where non-enzymatic thioacylation was reduced to background. Thus, protein S-acyltransferase remains active in the presence of physiological concentrations of ACBP and acyl-CoA in vitro and is likely to represent the predominant mechanism of thioacylation in vivo. 相似文献
6.
Long chain fatty acid synthesis was studied using etiolated leek seedling microsomes. In the presence of ATP, [2-14C]malonyl-CoA was incorporated into fatty acids of C16C26. The omission of ATP, even in the presence of acetyl-CoA, led to a complete loss of activity, which was restored by addition of exogeneous acyl-CoAs. Comparison of acyl-CoA (C12C24) elongation showed that stearoyl-CoA, in the presence of [2-14C]malonyl-CoA, was the more efficient precursor leading to the formation of fatty acids having a chain length of C20C26. [1-14C]C16CoA and [1-14C]C18CoA were elongated in the presence of malonyl-CoA, without degradation of the acyl chain. The time-course and the malonyl-CoA concentration curves showed that [1-14C]C18CoA was a better primer than [1-14C]C16CoA. Acyl-CoA elongation was also studied over the concentration range 4.5–45 μM [1-14C]C18CoA. Comparison of the radioactivity incorporated into the fatty acids formed using [2-14C]malonyl-CoA in the presence of C18CoA, on the one hand, and [1-14C]C18CoA in the presence of malonyl-CoA, on the other, demonstrated clearly that the acyl chain of the acyl-CoA was elongated by malonyl-CoA. 相似文献
7.
Lipid biosynthesis in developing mustard seed: formation of triacylglycerols from endogenous and exogenous Fatty acids 总被引:1,自引:1,他引:0
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Mukherjee KD 《Plant physiology》1983,73(4):929-934
Cotyledons of developing mustard (Sinapis alba L.) seed have been found to synthesize lipids containing the common plant fatty acids and very long-chain monounsaturated (icosenoic, erucic, and tetracosenic) and saturated (icosanoic, docosanoic, and tetracosanoic) fatty acids from various radioactive precursors. The in vivo pattern of labeling of acyl lipids, either from fatty acids synthesized `endogenously' from radioactive acetate or malonate, or from radioactive fatty acids added `exogenously', indicates the involvement of the following pathways in the biosynthesis of triacylglycerols. Palmitic, stearic, and oleic acid, synthesized in the acyl carrier protein-track, are channeled to the Coenzyme A (CoA)-track and converted to triacylglycerols via the glycerol-3-phosphate pathway. Pools of stearoyl-CoA and oleoyl-CoA are elongated to very long-chain saturated and monounsaturated acyl-CoA, respectively. Most of the very long-chain saturated acyl-CoAs acylate preformed diacylglycerols. Very long-chain monounsaturated acyl-CoAs are converted to triacylglycerols, partly via phosphatidic acids and diacylglycerols, and partly by acylation of preformed diacylglycerols. 相似文献
8.
Pech-Canul Á Nogales J Miranda-Molina A Álvarez L Geiger O Soto MJ López-Lara IM 《Journal of bacteriology》2011,193(22):6295-6304
FadD is an acyl coenzyme A (CoA) synthetase responsible for the activation of exogenous long-chain fatty acids (LCFA) into acyl-CoAs. Mutation of fadD in the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti promotes swarming motility and leads to defects in nodulation of alfalfa plants. In this study, we found that S. meliloti fadD mutants accumulated a mixture of free fatty acids during the stationary phase of growth. The composition of the free fatty acid pool and the results obtained after specific labeling of esterified fatty acids with a Δ5-desaturase (Δ5-Des) were in agreement with membrane phospholipids being the origin of the released fatty acids. Escherichia coli fadD mutants also accumulated free fatty acids released from membrane lipids in the stationary phase. This phenomenon did not occur in a mutant of E. coli with a deficient FadL fatty acid transporter, suggesting that the accumulation of fatty acids in fadD mutants occurs inside the cell. Our results indicate that, besides the activation of exogenous LCFA, in bacteria FadD plays a major role in the activation of endogenous fatty acids released from membrane lipids. Furthermore, expression analysis performed with S. meliloti revealed that a functional FadD is required for the upregulation of genes involved in fatty acid degradation and suggested that in the wild-type strain, the fatty acids released from membrane lipids are degraded by β-oxidation in the stationary phase of growth. 相似文献
9.
The kinetics of incorporation of [2-14C] acetate into lipids and acyl-CoAs in relation to added CoA and ATP by isolated spinach chloroplasts have been examined. The effect of the concentration of these cofactors on lipid and acyl-CoA synthesis was also studied. In the absence of cofactors, or when only one was present, the incorporation was very low and went mainly into lipids. When both cofactors were present a strong stimulation of both activities occurred. After 25 min, acyl-CoAs were more strongly labeled than lipids and both activities continued linearly for at least 60 min.Abbreviations ACP
acyl carrier protein
- FFA
free fatty acids 相似文献
10.
Acyl-CoA esters inhibit the plastidial glucose 6-phosphate (Glc-6-P) transporter and the adenylate transporter; the IC(50) values for the inhibition by oleoyl-CoA (18:1-CoA) are 200-400 nM and 1-2 microM respectively. The inhibition of either of these processes significantly reduces the flux of carbon from Glc-6-P or from acetate into long-chain fatty acids. The effect is dependent on the acyl chain length, e.g. lauryl-CoA is less inhibitory than oleoyl-CoA, causing 34 and 68% inhibition respectively of Glc-6-P uptake after 30 s. The inhibition of Glc-6-P and ATP transport is alleviated by addition of an equivalent concentration of acyl-CoA-binding protein (ACBP) or BSA. Acyl-CoAs do not inhibit pyruvate or glucose transporters. The endogenous concentrations of acyl-CoAs and ACBP are similar during embryo maturation. 相似文献
11.
12.
Angel Catalá 《Molecular and cellular biochemistry》1993,120(2):89-94
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
13.
14.
arachidonoyl-CoA. In summary, the data established for the first time a role for both L-FABP and ACBP in microsomal phosphatidic acid biosynthesis. By preferentially stimulating microsomal transacylation of unsaturated long chain fatty acyl-CoAs while concomitantly exerting their differential protection from microsomal acyl-CoA hydrolase, L-FABP and ACBP can uniquely function in modulating the pattern of fatty acids esterified to phosphatidic acid, the de novo precursor of phospholipids and triacylglycerols. This may explain in part the simultaneous presence of these proteins in cell types involved in fatty acid absorption and lipoprotein secretion. 相似文献
15.
The incorporation of fatty acids into lipid fractions was studied using a high-speed particulate fraction from germinating peas (Pisum sativum cv Feltham First). The acids were incorporated principally into the acyl-CoA and polar lipid fractions, with unsaturated fatty acids labelling the latter fraction at higher rates than saturated substrates. α-Hydroxylation also occurred. Oleate from oleoyl-CoA or oleoyl-acyl carrier protein was incorporated into polar lipids faster than from ammonium oleate. The incorporation of fatty acids into polar lipids was dependent on the presence of CoA and ATP in the medium and on the consequent generation of acyl-CoA's. Time-course studies and experiments when two fatty acid substrates were added consecutively confirmed the role of acyl-CoA's in the transfer of acyl groups to phospholipids. Although CoA was necessary when acyl-CoA's had to be generated, high concentrations were found to inhibit the rate ofacyl transfer. The results are discussed in terms ofthe ‘witching mechanism’ for controlling the fate of fatty acids in the plant cell. 相似文献
16.
17.
Structure and substrate specificity of β‐ketoacyl‐acyl carrier protein synthase III from Acinetobacter baumannii
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Woo Cheol Lee Min‐Cheol Jeong Yeongjoon Lee Chulhee Kwak Jee‐Young Lee Yangmee Kim 《Molecular microbiology》2018,108(5):567-577
Originally annotated as the initiator of fatty acid synthesis (FAS), β‐ketoacyl‐acyl carrier protein synthase III (KAS III) is a unique component of the bacterial FAS system. Novel variants of KAS III have been identified that promote the de novo use of additional extracellular fatty acids by FAS. These KAS III variants prefer longer acyl‐groups, notably octanoyl‐CoA. Acinetobacter baumannii, a clinically important nosocomial pathogen, contains such a multifunctional KAS III (AbKAS III). To characterize the structural basis of its substrate specificity, we determined the crystal structures of AbKAS III in the presence of different substrates. The acyl‐group binding cavity of AbKAS III and co‐crystal structure of AbKAS III and octanoyl‐CoA confirmed that the cavity can accommodate acyl groups with longer alkyl chains. Interestingly, Cys264 formed a disulfide bond with residual CoA used in the crystallization, which distorted helices at the putative interface with acyl‐carrier proteins. The crystal structure of KAS III in the alternate conformation can also be utilized for designing novel antibiotics. 相似文献
18.
Microsomes isolated from the developing cotyledons of the seeds of the safflower varieties, very-high-linoleate, Gila and high-oleate, were capable of exchanging the acyl groups in acyl-CoA with the fatty acids in position 2 of phosphatidylcholine. The specificity of the 'acyl-exchange' towards the acyl moiety in acyl-CoA was selective in the order: oleate greater than linoleate greater than linolenate. Stearoyl-CoA was completely selected against when presented in a mixed substrate with unsaturated 18-carbon acyl-CoAs. Microsomes, of the very-high-linoleate safflower variety, rapidly desaturated in situ-labelled [14C]oleoylphosphatidylcholine in the presence of NADH. Little oleate desaturation, however, was observed in the microsomes of the high-oleate variety. Microsomes of the Gila and high-oleate varieties of safflower rapidly synthesised phosphatidic acid by the acylation of glycerol 3-phosphate with acyl-CoA. The phosphatidic acid was metabolised to diacylglycerol, which was further acylated to triacylglycerol. A strong selectivity for linoleoyl-CoA was found for the acylation of glycerol 3-phosphate in both the Gila and high-oleate microsomes. On the basis of these results, we propose that the pattern of 18-carbon unsaturated fatty acids in the triacylglycerols of all 'oil'-producing seeds is a direct reflection of the fatty acids in the acyl-CoA pool. This, in turn, is governed by: A, the rate and specificity of the acyl exchange between acyl-CoA and phosphatidylcholine; B, the rate of oleate (and linoleate) desaturation in phosphatidylcholine; and C, the rate and specificity of the glycerophosphate acyltransferase. 相似文献
19.
Microsomal membrane preparations from rat lung catalyse the incorporation of radioactive linolenic acid from [14C]linolenoyl-CoA into position 2 of sn-phosphatidylcholine. The incorporation was stimulated by bovine serum albumin and free CoA. Free fatty acids in the incubation mixtures were not utilised in the incorporation into complex lipids. Fatty acids were transferred to the acyl-CoA pool during the incorporation of linolenic acid into phosphatidylcholine. An increase in lysophosphatidylcholine occurred in incubations containing both bovine serum albumin and free CoA and in the absence of acyl-CoA. The results were consistent with an acyl-CoA: lysophosphatidylcholine acyltransferase operating in both a forwards and backwards direction and thus catalysing the acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine. In incubations with mixed species of acyl-CoAs, palmitic acid was the major fatty acid substrate transferred to phosphatidylcholine in acyl exchange, whereas this acid was completely selected against in the acylation of added lysophosphatidylcholine. The selectivity for palmitoyl-CoA was particularly enhanced when the mixed acyl-CoA substrate was presented to the microsomes in molar concentrations equivalent to the molar ratios of the fatty acids in position 2 of sn-phosphatidylcholine. During acyl exchange, the predominant fatty acid transferred to phosphatidylcholine from acyl-CoA was palmitic acid, whereas arachidonic acid was particularly selected for in the reverse reaction from phosphatidylcholine to acyl-CoA. A hypothesis is presented to explain the differential selectivity for acyl species between the forward and backward reactions of the acyltransferase that is based upon different affinities of the enzyme for substrates at high and low concentrations of acyl donor. Acyl exchange between acyl-CoA and phosphatidylcholine offers, therefore, a possible mechanism for the acyl-remodelling of phosphatidylcholine for the production of lung surfactant. 相似文献
20.
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