Altered membrane association of p60v-src and a murine 63-kDa N-myristoyl protein after incorporation of an oxygen-substituted analog of myristic acid

A number of viral and cellular proteins contain covalently bound lipid. In a subset of these acyl proteins, the 14-carbon saturated fatty acid myristic acid is attached through an amide linkage to an NH2-terminal glycine residue. Myristoyl-CoA:protein N-myristoyltransferase (NMT) transfers the myristoyl moiety from myristoyl-CoA to these nascent proteins and is highly selective for fatty acid chain length. We have found that 10-(propoxy)decanoyl-CoA (11-oxymyristoyl-CoA), an analog of myristic acid with reduced hydrophobicity, acts as a substrate for NMT both in vitro and in vivo. Comparison of the in vitro kinetic properties of a number of synthetic octapeptide substrates of NMT using myristoyl-CoA or 11-oxymyristoyl-CoA indicated that there is an interaction between the acyl-CoA and peptide binding sites of this acyltransferase. Peptide catalytic efficiency with 11-oxymyristoyl-CoA was reduced relative to that with myristoyl-CoA, but the extent of the reduction varied widely among the octapeptides tested. These in vitro data accurately predicted that only a subset of myristoyl proteins synthesized in Saccharomyces cerevisiae and a murine myocyte-like cell line (BC3H1) would incorporate 11-oxy[3H]myristate. Substitution of the myristoyl moiety by the 11-oxymyristoyl moiety does not significantly affect the membrane association of most N-myristoyl proteins. However, for the tyrosine kinase p60v-src and a 63-kDa N-myristoyl protein in BC3H1 cells, analog incorporation results in marked redistribution from the membrane to the cytosolic fraction. These studies demonstrate the utility of heteroatom-containing analogs for analysis of the role of myristate in acyl protein targeting. The sequence-specific nature of analog incorporation and the protein-specific effects on membrane association suggests that these compounds may represent a useful class of antiviral and antitumor agents.     

Protein N-myristoylation in Escherichia coli: reconstitution of a eukaryotic protein modification in bacteria.

Protein N-myristoylation refers to the covalent attachment of a myristoyl group (C14:0), via amide linkage, to the NH2-terminal glycine residue of certain cellular and viral proteins. Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes this cotranslational modification. We have developed a system for studying the substrate requirements and biological effects of protein N-myristoylation as well as NMT structure-activity relationships. Expression of the yeast NMT1 gene in Escherichia coli, a bacterium that has no endogenous NMT activity, results in production of the intact 53-kDa NMT polypeptide as well as a truncated polypeptide derived from proteolytic removal of its NH2-terminal 39 amino acids. Each E. coli-synthesized NMT species has fatty acid and peptide substrate specificities that are indistinguishable from those of NMT recovered from Saccharomyces cerevisiae, suggesting that the NH2-terminal domain of this enzyme is not required for its catalytic activity. By using a dual plasmid system, N-myristoylation of a mammalian protein was reconstituted in E. coli by simultaneous expression of the yeast NMT1 gene and a murine cDNA encoding the catalytic (C) subunit of cAMP-dependent protein kinase (PK-A). The fatty acid specificity of N-myristoylation was preserved in this system: [9,10(n)-3H]myristate but not [9,10(n)3H]palmitate was efficiently linked to Gly-1 of the C subunit. [13,14(n)-3H]10-Propoxydecanoic acid, a heteroatom-containing analog of myristic acid with reduced hydrophobicity but similar chain length, was an effective alternative substrate for NMT that also could be incorporated into the C subunit of PK-A. Such analogs have recently been shown to inhibit replication of certain retroviruses that depend upon linkage of a myristoyl group to their gag polyprotein precursors (e.g., the Pr55gag of human immunodeficiency virus type 1). A major advantage of the bacterial system over eukaryotic systems is the absence of endogenous NMT and substrates, providing a more straightforward way of preparing myristoylated, analog-substituted, and nonmyristoylated forms of a given protein for comparison of their structural and functional properties. The system should facilitate screening of enzyme inhibitors as well as alternative NMT fatty acid substrates for their ability to be incorporated into a specific target protein. Our experimental system may prove useful for recapitulating other eukaryotic protein modifications in E. coli so that structure-activity relationships of modifying enzymes and their substrates can be more readily assessed.     

Analogs of palmitoyl-CoA that are substrates for myristoyl-CoA:protein N-myristoyltransferase.

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p; EC 2.3.1.97) is an essential enzyme that is highly selective for myristoyl-CoA in vivo. It is unclear why myristate (C14:0), a rare cellular fatty acid, has been selected for this covalent protein modification over more abundant fatty acids such as palmitate (C16:0), nor is it obvious how the enzyme's acyl-CoA binding site is able to discriminate between these two fatty acids. Introduction of a cis double bond between C5 and C6 of palmitate [(Z)-5-hexadecenoic acid] or a triple bond between C4 and C5 or C6 and C7 (Y4- and Y6-hexadecenoic acids) yields compounds that, when converted to their CoA derivatives, approach the activity of myristoyl-CoA as Nmt1p substrates in vitro. Kinetic studies of 42 C12-C18 fatty acids containing triple bonds, para-phenylene, or a 2,5-furyl group, as well as cis and trans double bonds, suggest that the geometry of the enzyme's acyl-CoA binding site requires that the acyl chain of active substrates assume a bent conformation in the vicinity of C5. Moreover, the distance between C1 and the bend appears to be a critical determinant for optimal positioning of the acyl-CoA in this binding site so that peptide substrates can subsequently bind in the sequential ordered bi-bi reaction mechanism. Identification of active, conformationally restricted analogs of palmitate offers an opportunity to "convert" wild-type or mutant Nmts to palmitoyltransferases so that they can deliver these C16 fatty acids to critical N-myristoylproteins in vivo. nmt181p contains a Gly-451-->Asp mutation, which causes a marked reduction in the enzyme's affinity for myristoyl-CoA. Strains of S. cerevisiae containing nmt1-181 exhibit temperature-sensitive myristic acid auxotrophy: their complete growth arrest at 37 degrees C is relieved when the medium is supplemented with 500 microM C14:0 but not with C16:0. The CoA derivatives of (Z)-5-hexadecenoic and Y6-hexadecynoic acids are as active substrates for the mutant enzyme as myristoyl-CoA at 24 degrees C. However, unlike C16:0, they produce growth arrest of nmt181p-producing cells at this "permissive" temperature, suggesting that these C16 fatty acids do not allow expression of the biological functions of essential S. cerevisiae N-myristoylproteins.     

Purification and characterization of yeast myristoyl CoA:protein N-myristoyltransferase.

Myristoyl CoA:protein N-myristoyltransferase (NMT) catalyzes the addition of myristic acid to the amino-terminal glycine residues of a number of eukaryotic proteins. Recently, we developed a cell-free system for analyzing NMT activity and have begun to characterize the substrate specificity of this enzyme by using a series of synthetic peptides. We have now purified NMT from Saccharomyces cerevisiae to apparent homogeneity. The native enzyme is a 55-kDa protein, exhibits no requirement for divalent cation, and appears to contain a histidine residue critical for enzyme activity. A total of 42 synthetic peptides have been used to define structure/activity relationships in NMT substrates. An amino-terminal glycine is required for acylation; substitution with glycine analogues produces peptides that are inactive as substrates or inhibitors of NMT. A broad spectrum of amino acids is permitted at positions 3 and 4, while strict amino acid requirements are exhibited at position 5. Replacement of Ala5 in the peptide Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg with Asp ablates the peptide's myristoyl-accepting activity. A serine at this position results in a decrease by a factor of approximately equal to 500 in the apparent Km in the context of three different sequences. Penta- and hexa-peptides are substrates, but with decreased affinity. These studies establish that structural information important for NMT-ligand interaction exists beyond the first two amino acids in peptide substrates and that the side chains of residue 5 play a critical role in the binding of substrates to this enzyme.     

Presence and properties of acyl coenzyme A synthetase for medium-chain fatty acids in rat intestinal mucosa.

A system was developed for assay of acyl coenzyme A (acyl CoA) activity on medium-chain fatty acids in rat intestinal mucosa. Using this system, we compared the characteristics of octanoyl (C8:0) CoA synthetase activity and palmitoyl (C16:0) CoA synthetase activity. Palmitoyl CoA synthetase activity as a function of palmitate concentration followed Michaelis-Menten kinetics, but octanoyl CoA synthetase activity as a function of octanoate concentration showed a biphasic reaction curve. The distributions of octanoyl CoA synthetase activity and palmitoyl CoA synthetase activity along the gastrointestinal tract were similar, both activities being present mainly in the middle portion of the small intestine. Incubation of octanoate with a homogenate of intestinal mucosa revealed that octnoate, like palmitate, is incorporated into phospholipids and triglycerides after its CoA activation. Oral administration of medium chain triglycerides induced a nearly 2-fold increase in octanoyl CoA synthetase activity in rat intestinal mucosa, whereas oral administration of long chain triglycerides did not affect the palmitoyl CoA synthetase activity. These results indicate that acyl CoA synthetase for medium-chain fatty acids in rat intestinal mucosa plays a key role in the utilization of medium-chain fatty acids for lipid synthesis, and that it is regulated in a different manner from acyl CoA synthetase for long-chain fatty acids.     

Protein fatty acid acylation: enzymatic synthesis of an N-myristoylglycyl peptide.

Incubation of Saccharomyces cerevisiae strain JR153 with either [3H]myristate or [3H]palmitate demonstrates the synthesis of proteins that contain covalently bound fatty acids. A unique set of proteins is labeled by each fatty acid. Detailed analysis of a 20-kDa protein labeled with myristic acid demonstrates that myristate is linked to the amino-terminal glycine. We describe an enzymatic activity in yeast that will transfer myristic acid to the amino terminus of the octapeptide Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg, whose sequence was derived from a known N-myristoylated acyl protein, the catalytic subunit of cAMP-dependent protein kinase of bovine cardiac muscle. The acylation reaction is dependent on ATP and CoA, is enriched in a crude membrane fraction, and will use myristate but not palmitate as the acyl donor. Specificity of the glycyl peptide substrate is demonstrated by the observation that other glycyl peptides do not competitively inhibit myristoylation of Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg.     

Regulation of rat heart triacylglycerol ester hydrolases by free fatty acids,fatty acyl CoA and fatty acyl carnitine

The regulation of triacylglycerol ester hydrolase (TG lipase) activity was studied in a pH 5.2 precipitate fraction from rat hearts. Neutral TG lipase activity (assayed at pH 7.5) was measured with both ethanolic and glycerol-dispersed triolein substrate preparations; acid lipase activity was determined at pH 5 with a glycerol-dispersed triolein substrate preparation containing lecithin. Neutral TG lipase activity was inhibited by free fatty acids (oleic acid) and by fatty acyl CoA compounds (oleoyl CoA, palmitoyl CoA); concentrations required for half-maximal inhibition were 100 μm and 20 to 25 μm, respectively. Palmitoyl carnitine resulted in an increase in neutral TG lipase activity. The inhibition of neutral TG lipase activity by oleoyl CoA could be prevented by increasing the content of albumin in the assay, and was reduced when the triolein substrate concentration was increased. Fatty acyl CoA compounds also inhibited acid TG lipase activity, but palmitoyl carnitine was the most effective inhibitor (concentration for half-maximal inhibition of 30 μm). The inhibition of neutral TG lipase activity by fatty acyl CoA is consistent with the observation that metabolic interventions that increase tissue levels of fatty acyl CoA result in an inhibition of rates of cardiac lipolysis.     

Effects of the fatty acid blocking agents, oxfenicine and 4-bromocrotonic acid, on performance in aerobic and ischemic myocardium

Two fatty acid blocking agents, oxfenicine (33 mg/kg) and 4-bromocrotonic acid (0.34 mg/kg/min for 70 min), were used to selectively adjust levels of long-chain acyl CoA and carnitine in aerobic and ischemic myocardium. The purpose of the study was to test whether the shift in these amphiphiles was associated with alterations of mechanical function in intact myocardium. The extracorporeally perfused swine heart preparation was used. Hearts were perfused at aerobic levels for 40 min following which flow to the anterior descending (LAD) circulation was reduced by 50% for the final 30 min of perfusion. All hearts were perfused with excess fatty acids to raise serum levels to 1.37 +/- 0.16 mumol/mol throughout the studies. Oxfenicine and 4-bromocrotonic acid affected a 20% (P less than 0.05 and P less than 0.05, respectively) further decline in 14CO2 production from labelled palmitate as compared with placebo hearts during regional ischemia. Accompanying this were downward shifts in acyl carnitine (-27 delta %, NS in aerobic tissue; -70 delta %, P less than 0.001 in ischemic tissue) and acyl CoA (-13 delta %, NS in aerobic tissue; -33 delta %, P less than 0.01 in ischemic tissue) for oxfenicine and upward shifts of acyl carnitine (+212 delta %, P less than 0.001 in aerobic tissue; -9 delta %, NS in ischemic tissue) and acyl CoA (+78 delta %, P less than 0.001 in aerobic tissue; +29 delta %, P less than 0.025 in ischemic tissue) for 4-bromocrotonic acid. These adjustments in amphiphiles were further associated with improved function (+55 delta % increase in max LV dP/dt, P less than 0.05) in oxfenicine-treated hearts and depressed function (+87 delta % increase in LVEDP, P less than 0.05) in 4-bromocrotonic acid-treated hearts. Thus, at comparable conditions of coronary flow, left ventricular pressure, and fatty acid availability and oxidation between treatments, depletion or build-up of CoA and carnitine esters as affected by selective inhibitors of fatty acid metabolism were causally linked to improved or impaired cardiac performance in intact hearts.     

Long‐chain fatty acid uptake and oxidation in the perfused liver of Walker‐256 tumour‐bearing rats

Abstract: Aims/Background: The effect of the Walker‐256 tumour on uptake and oxidation of long‐chain fatty acids was investigated in perfused livers of rats. Methods: Isolated livers were perfused in a non‐recirculating system. Fatty acid uptake, ketogenesis, oxygen uptake and ¹⁴CO₂‐production were measured as well as the activities of the acyl carnitine transferases I and II (CAT I and CAT II). Results: Basal oxygen uptake of livers from tumour‐bearing rats was lower. Ketone bodies production derived from the long‐chain fatty acids in livers from starved tumour‐bearing rats was lower relative to the controls, but ¹⁴CO₂ production was similar in both groups. The oxygen uptake increment and the mitochondrial NADH/NAD⁺ redox ratio were also decreased in tumour‐bearing rats. The extent of these differences was dependent on the chain length and structure of the fatty acid, the following decreasing sequence of differences between control and tumour‐bearing animals being valid: palmitate > oleate > stearate. The CAT I activity of the enzyme isolated from livers of tumour‐bearing rats was half that from normal rats when palmitoyl‐CoA and oleoyl‐CoA were the substrates. Conclusions: Ketogenesis from exogenous fatty acids is decreased in the livers of Walker‐256 tumour‐bearing rats in consequence of the diminished activity of the mitochondrial CAT I. The lower rates of oxygen uptake indicate a decreased ATP synthesis, which is consistent with the in vivo lower phosphorylation potential.     

Isolation and Preliminary Characterization of the Medium-Chain Fatty Acid:CoA Ligase Responsible for Activation of Short- and Medium-Chain Fatty Acids in Colonic Mucosa from Swine

The ATP-dependent activation of short- and medium-chain fatty acids to their respective CoA thioester adducts was investigated in the colonic mucosa from swine. Subcellular fractionation of a homogenate of the mucosa from the entire length of the colon revealed a predominantly mitochondrial localization for activity toward fatty acids ranging from propionate through laurate. These activities could be released from mitochondria in soluble form by freeze–thaw lysis. Purification of these activities revealed that they all appeared to reside with a single enzyme. This suggests that the entire colon contains a single form of medium-chain fatty acid:CoA ligase (MCFA:CoA ligase). The ligase also had activity toward benzoate and salicylate, although this activity was significantly lower than activity toward medium-chain fatty acids. The enzyme had the highest activity at V_max with butyrate as substrate and had the lowest K_m for octanoate. Butyrate and octanoate were mutually inhibitory. Activity toward both substrates was also efficiently inhibited by cyclohexane carboxylate. The molecular weight of the enzyme was estimated by gel filtration chromatography to be ca. 46,500. These data indicate that the colonic MCFA:CoA ligase is significantly different from the hepatic and kidney MCFA:CoA ligases.     

Fatty acyl CoA synthetase activity in normal and atherosclerotic rabbit aortic tissue.

The activity of fatty acyl CoA synthetase and fatty acyl CoA:cholesterol acyltransferase was determined in microsomal fractions from normal and atherosclerotic rabbit aortic tissue. No change in fatty acyl CoA synthetase activity was observed as a result of cholesterol feeding in contrast to the several-fold increase in the activity of fatty acyl CoA:cholesterol acyltransferase seen in atherosclerotic tissue. Inhibition of both enzymes was observed when clofibrate, or the tetrahydronapthyl analog of this drug were added in vitro. The inhibitory effects were most pronounced on the fatty acyl CoA:cholesterol acyltransferase.     

Studies on long chain fatty acid:CoA ligase from human small intestine.

Long chain fatty acid:CoA ligase (EC 6.2.1.3.) was examined in human small intestinal mucosa using the hydroxamate-trapping method. With optimal assay requirements using palmitate as substrate a significant difference of specific activities could be detected in the total homogenate from duodenum, 40.9 +/- 11.6 nmol/min per mg protein versus upper jejunum, 51.9 +/- 13.7 (P less than 0.05). The enzyme activity of the microsomal fraction of upper jejunum was 101.8 +/- 44 nmol/min per mg protein. ATP, CoA, and Mg2+ were essential constituents of the reaction. A broad pH-optimum was observed between 6.75 and 7.75 with a maximum at a pH of 7.25. Whereas palmitate in the presence of albumin revealed a wide range of optimal concentration in supporting maximal enzyme activity, oleate was found to strongly inhibit the reaction. Where substrate specificity with both the total homogenate and the microsomal fraction was concerned, maximal reaction rates were obtained with palmitate for the long chain saturated fatty acids C12:0' C14:0' C16:0' and C18:0' and with oleate for the long chain unsaturated fatty acids C18:1 C18:2' and C18:3' respectively. The highest specific activity of the enzyme was localised in the microsomal fraction. The kinetic data and properties of the long chain fatty acid: CoA ligase from human intestine are discussed with respect to the intestinal enzyme from other species.     

Expression of a delta 9 14:0-acyl carrier protein fatty acid desaturase gene is necessary for the production of omega 5 anacardic acids found in pest-resistant geranium (Pelargonium xhortorum).

Anacardic acids, a class of secondary compounds derived from fatty acids, are found in a variety of dicotyledonous families. Pest resistance (e.g., spider mites and aphids) in Pelargonium xhortorum (geranium) is associated with high levels (approximately 81%) of unsaturated 22:1 omega 5 and 24:1 omega 5 anacardic acids in the glandular trichome exudate. A single dominant locus controls the production of these omega 5 anacardic acids, which arise from novel 16:1 delta 11 and 18:1 delta 13 fatty acids. We describe the isolation and characterization of a cDNA encoding a unique delta 9 14:0-acyl carrier protein fatty acid desaturase. Several lines of evidence indicated that expression of this desaturase leads to the production of the omega 5 anacardic acids involved in pest resistance. First, its expression was found in pest-resistant, but not suspectible, plants and its expression followed the production of the omega 5 anacardic acids in segregating populations. Second, its expression and the occurrence of the novel 16:1 delta 11 and 18:1 delta 13 fatty acids and the omega 5 anacardic acids were specific to tall glandular trichomes. Third, assays of the recombinant protein demonstrated that this desaturase produced the 14:1 delta 9 fatty acid precursor to the novel 16:1 delta 11 and 18:1 delta 13 fatty acids. Based on our genetic and biochemical studies, we conclude that expression of this delta 9 14:0-ACP desaturase gene is required for the production of omega 5 anacardic acids that have been shown to be necessary for pest resistance in geranium.     

Inhibiting long-chain fatty acyl CoA synthetase does not increase agonist-induced release of arachidonate metabolites from human endothelial cells

BACKGROUND: Triacsin C, a fatty acid analog, inhibits endothelial nitric oxide synthetase (eNOS) palmitoylation, increases nitric oxide synthesis and enhances methacholine-induced relaxation of vascular rings. The experiments presented here tested the hypothesis that triacsin C increases the synthesis of PGI(2) and/or endothelial-derived hyperpolarizing factor. METHODS: Long-chain fatty acyl CoA synthetase activity (LCFACoAS), agonist-induced prostacyclin synthesis and agonist-induced release of radioactivity in endothelial cells labeled with [(3)H]arachidonic acid were measured in the presence and absence of triacsin C. RESULTS: Inhibition by triacsin C of palmitoyl CoA formation was significantly greater than inhibition of arachidonoyl CoA formation in solubilized endothelial cell preparations. While 24-hour triacsin C treatment significantly reduced basal 6-keto synthesis, it had no effect on agonist-stimulated synthesis. The release of arachidonic acid metabolites was examined in [(3)H]arachidonate-labeled cells. Triacsin C treatment had no effect on basal or vasopressin-, angiotensin-II-, bradykinin- or ionomycin-induced release of radioactivity, but significantly reduced release in response to isoproterenol or phenylephrine. Expression of neither immunoreactive eNOS nor immunoreactive inducible nitric oxide synthetase (iNOS) was changed by triacsin C treatment, but the fraction of immunoreactive eNOS in the cytoplasm of treated cells was significantly greater as compared to vehicle control cells. Phorbol myristoyl acetate or fenofibrate significantly increased in vitro LCFACoAS activity, and significantly decreased the nitrite/eNOS ratio. CONCLUSIONS: These data indicate that, while triacsin C can inhibit arachidonoyl CoA synthetase in endothelial cells, it does not increase the availability of endogenous substrate for basal or agonist-induced PGI(2) synthesis, nor does it enhance release of arachidonic acid or its metabolites.     

Inhibition of carnitine palmitoyl-CoA transferase activity and fatty acid oxidation by lactate and oxfenicine in cardiac muscle

High concentrations of lactate and oxfenicine inhibit fatty acid oxidation in cardiac muscle. The site of this inhibition was investigated in isolated perfused rat hearts. In hearts perfused with glucose (11 mM) and [U-14 C]palmitate (1.0 mM), addition of 5 mM lactate caused a 38% reduction in 14CO2 production. Tissue levels of long-chain acyl carnitine decreased suggesting that inhibition occurred at either fatty acyl CoA synthetase or carnitine-acyl CoA transferase. Cytosolic levels of acyl-CoA are low compared with mitochondrial levels and changes in acyl-CoA within the cytosolic compartment cannot be estimated directly. Consequently, the rate of conversion of 14C-palmitate to neutral lipids was used as an indicator of cytosolic acyl CoA levels. Lactate caused a 100% increase in 14C-fatty acid conversion to triglycerides suggesting that cytosolic levels of acyl-CoA increased in association with decreased acyl-carnitine. This indicates that lactate inhibited FFA oxidation at the level of carnitine-acyl CoA transferase. Oxfenicine (2 mM) reduced fatty acid oxidation by 45%, decreased acyl-carnitine levels by 80%, and increased conversion of 14C-palmitate to neutral lipids by 44%, suggesting that oxfenicine also inhibits fatty acid oxidation at the level of carnitine-acyl CoA transferase. These data further indicate that carnitine-acyl CoA transferase is an important site of control in the pathway of fatty acid oxidation.     

Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils

We demonstrate that naturally occurring C(14) and C(16)-specific acyl-acyl carrier protein (ACP) desaturases from plants can complement the unsaturated fatty acid (UFA) auxotrophy of an Escherichia coli fabA/fadR mutant. Under the same growth conditions, C(18)-specific delta(9)-stearoyl (18:0)-ACP desaturases are unable to complement the UFA auxotrophy. This difference most likely results from the presence of sufficient substrate pools of C(14) and C(16) acyl-ACPs but a relative lack of C(18) acyl-ACP pools in E. coli to support the activities of the plant fatty acid desaturase. Based on this, a substrate-dependent selection system was devised with the use of the E. coli UFA auxotroph to isolate mutants of the castor delta(9)-18:0-ACP desaturase that display enhanced specificity for C(14) and C(16) acyl-ACPs. Using this selection system, a number of desaturase variants with altered substrate specificities were isolated from pools of randomized mutants. These included several G188L mutant isolates, which displayed a 15-fold increase in specific activity with 16:0-ACP relative to the wild-type castor delta(9)-18:0-ACP desaturase. Expression of this mutant in Arabidopsis thaliana resulted in the accumulation of unusual monounsaturated fatty acids to amounts of >25% of the seed oil. The bacterial selection system described here thus provides a rapid means of isolating variant fatty acid desaturase activities for modification of seed oil composition.     

Acyl coenzyme A esters differentially activate cardiac and beta-cell adenosine triphosphate-sensitive potassium channels in a side-chain length-specific manner

Recent evidence demonstrates that long-chain acyl coenzyme A esters (CoAs) activate cardiac and beta-cell plasma-membrane (pmK(ATP)) adenosine triphosphate (ATP)-sensitive potassium channels. In this study, we have investigated the differential effects of acyl CoAs of short and medium side-chain length on cardiac and beta-cell pmK(ATP) isoforms. At the single-channel level, the addition of acyl CoAs of differing side-chain length (2 to 16 carbons) to the inside face of membrane patches from ventricular myocytes caused varying increases in pmK(ATP) channel open probability proportional to increases in acyl side-chain length (20 mumol/L acetyl CoA: 310% +/- 90%, 20 mumol/L decanoyl CoA: 570% +/- 150%). A similar dependence of activation on side-chain length was observed in recombinant pmK(ATP) channels (SUR2A/Kir6.2) with full activation of current requiring both the acyl and CoA moieties in the esterified form. We found the recombinant beta-cell K(ATP) channel (SUR1/Kir6.2) to be much less sensitive to medium-chain acyl CoAs (decanoyl CoA: 124% +/- 15% v 231% +/- 25% in SUR2A/Kir6.2), suggesting a role for the cardiac sulfonylurea receptor, SUR2A, in the molecular mechanism of activation by these compounds. We propose that fatty acid metabolism, and the resultant generation of acyl CoAs of varying side-chain length, may be an important regulator of cellular excitability via interactions with the K(ATP) channel.     

Direct demonstration of lipid sequestration as a mechanism by which rosiglitazone prevents fatty-acid-induced insulin resistance in the rat: comparison with metformin

Aims/hypothesis Thiazolidinediones can enhance clearance of whole-body non-esterified fatty acids and protect against the insulin resistance that develops during an acute lipid load. The present study used [³H]-R-bromopalmitate to compare the effects of the thiazolidinedione, rosiglitazone, and the biguanide, metformin, on insulin action and the tissue-specific fate of non-esterified fatty acids in rats during lipid infusion.Methods Normal rats were treated with rosiglitazone or metformin for 7 days. Triglyceride/heparin (to elevate non-esterified fatty acids) or glycerol (control) were then infused for 5 h, with a hyperinsulinaemic clamp being performed between the 3rd and 5th hours.Results Rosiglitazone and metformin prevented fatty-acid-induced insulin resistance (reduced clamp glucose infusion rate). Both drugs improved insulin-mediated suppression of hepatic glucose output but only rosiglitazone enhanced systemic non-esterified fatty acid clearance (plateau plasma non-esterified fatty acids reduced by 40%). Despite this decrease in plateau plasma non-esterified fatty acids, rosiglitazone increased fatty acid uptake (two-fold) into adipose tissue and reduced fatty acid uptake into liver (by 40%) and muscle (by 30%), as well as reducing liver long-chain fatty acyl CoA accumulation (by 30%). Both rosiglitazone and metformin increased liver AMP-activated protein kinase activity, a possible mediator of the protective effects on insulin action, but in contrast to rosiglitazone, metformin had no significant effect on non-esterified fatty acid kinetics or relative tissue fatty acid uptake.Conclusions/interpretation These results directly demonstrate the lipid steal mechanism, by which thiazolidinediones help prevent fatty-acid-induced insulin resistance. The contrasting mechanisms of action of rosiglitazone and metformin could be beneficial when both drugs are used in combination to treat insulin resistance.Abbreviations AMPK AMP-activated protein kinase - ¹⁴C-2DG [¹⁴C]-2-deoxyglucose - GIR glucose infusion rate - HGO hepatic glucose output - LCACoA long-chain fatty acyl CoA - PPAR peroxisome proliferator-activated receptor - TZDs thiazolidinediones     

Incorporation of 12-methoxydodecanoate into the human immunodeficiency virus 1 gag polyprotein precursor inhibits its proteolytic processing and virus production in a chronically infected human lymphoid cell line.

Covalent linkage of myristate (tetradecanoate; 14:0) to the NH2-terminal glycine residue of the human immunodeficiency virus 1 (HIV-1) 55-kDa gag polyprotein precursor (Pr55gag) is necessary for its proteolytic processing and viral assembly. We have shown recently that several analogs of myristate in which a methylene group is replaced by a single oxygen or sulfur atom are substrates for Saccharomyces cerevisiae and mammalian myristoyl-CoA:protein N-myristoyltransferase (EC 2.3.1.97; NMT) despite their reduced hydrophobicity. Some inhibit HIV-1 replication in acutely infected CD4+H9 cells without accompanying cellular toxicity. To examine the mechanism of their antiviral effects, we performed labeling studies with two analogs, 12-methoxydodecanoate (13-oxamyristate; 13-OxaMyr) and 5-octyloxypentanoate (6-oxamyristate; 6-OxaMyr), the former being much more effective than the latter in blocking virus production. [3H]Myristate and [3H]13-OxaMyr were incorporated into Pr55gag with comparable efficiency when it was coexpressed with S. cerevisiae NMT in Escherichia coli. [3H]6-OxaMyr was not incorporated, even though its substrate properties in vitro were similar to those of 13-OxaMyr and myristate. [3H]13-OxaMyr, but not [3H]6-OxaMyr, was also efficiently incorporated into HIV-1 Pr55gag and nef (negative factor) in chronically infected H9 cells. Analog incorporation produced a redistribution of Pr55gag from membrane to cytosolic fractions and markedly decreased its proteolytic processing by viral protease. 13-OxaMyr and 3'-azido-3'-deoxythymidine (AZT) act synergistically to reduce virus production in acutely infected H9 cells. Unlike AZT, the analog is able to inhibit virus production (up to 70%) in chronically infected H9 cells. Moreover, the inhibitory effect lasts 6-8 days. These results suggest that (i) its mechanism of action is distinct from that of AZT and involves a late step in virus assembly; (ii) the analog may allow reduction in the dose of AZT required to affect viral replication; and (iii) combinations of analog and HIV-1 protease inhibitors may have synergistic effects on the processing of Pr55gag.