Role of acyl-CoAs and acyl-CoA-binding protein in regulation of carbon supply for fatty acid biosynthesis

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.     

Evidence that oleoyl-CoA and ATP-dependent elongations coexist in rapeseed (Brassica napus L.).

The elongation of different substrates was studied using several subcellular fractions from Brassica napus rapeseed. In the presence of malonyl-CoA, NADH and NADPH, very-long-chain fatty acid (VLCFA) synthesis was observed from either oleoyl-CoA (acyl-CoA elongation) or endogenous primers (ATP-dependent elongation). No activity was detected using oleic acid as precursor. Acyl-CoA and ATP-dependent elongation activities were mainly associated with the 15 000 g/25 min membrane fraction. Reverse-phase TLC analysis showed that the proportions of fatty acids synthesized by these activities were different. Acyl-CoA elongation increased up to 60 microM oleoyl-CoA, and ATP-dependent elongation was maximum at 1 mM ATP. Both activities increased with malonyl-CoA concentration (up to 200 microM). Under all conditions tested, acyl-CoA elongation was higher than ATP-dependent elongation, and, in the presence of both ATP and oleoyl-CoA, the elongation activity was always lower. ATP strongly inhibited acyl-CoA elongation, whereas ATP-dependent elongation was slightly stimulated by low oleoyl-CoA concentrations (up to 15 microM) and decreased in the presence of higher concentrations. CoA (up to 150 microM) had no effect on the ATP-dependent elongation, whereas it inhibited the acyl-CoA elongation. These marked differences strongly support the presence in maturing rapeseed of two different elongating activities differently modulated by ATP and oleoyl-CoA.     

Fatty acid biosynthesis in Erlich cells. The mechanism of short term control by exogenous free fatty acids.

We have examined the mechanism by which extracellular free fatty acids regulate fatty acid biosynthesis in Ehrlich ascites tumor cells. De novo biosynthesis in intact cells was inhibited by stearate greater than oleate greater than palmitate greater than linoleate. The amount of citrate and long chain acyl-CoA in the cells was not changed appreciably by the addition of free fatty acids to the incubation medium, indicating than free fatty acids do not regulate fatty acid biosynthesis by changing the total intracellular content of these metabolites. By measuring the incorporation of labeled free fatty acids into acyl-CoA, however, it was determined that the fatty acid composition of the acyl-CoA poolwas changed dramatically to reflect the composition of the exogenous free fatty acids. The relative inhibitory effects of different free fatty acids appear to depend on the ability of their acyl-CoA derivatives to regulate acyl-CoA carboxylase activity. The acyl-CoA concentration needed to produce 50% inhibition of purified Ehrlich cell carboxylase was found to be 0.68 mum for stearoyl-CoA, 1.6 mum for oleoyl-CoA, 2.2 mum for palmitoyl-CoA, 23 mum for myristoyl-CoA, 30 mum for lauroyl-CoA, and 37 mum for linoleoyl-CoA. In contrast to their effects on de novo synthesis, all of the free fatty acids added except stearate stimulated chain elongation in intact cells. Microsomal chain elongation, the major system for elongation in Ehrlich cells, also was regulated by the composition of the cellular acyl-CoA pool. Lauroyl-CoA, myristoyl-CoA, and palmitoyl-CoA were good substrates for elongation by isolated microsomes; oleoyl-CoA, and linoleoyl-CoA were intermediate; and stearoyl-CoA was a very poor substrate. We conclude that free fatty acids regulate fatty acid biosynthesis by changing the composition of the cellular acyl-CoA pool. These changes control the rate of malonyl-CoA production and, because of the acyl-CoA substrate specificity of the microsomal elongation system, modulate the amount of malonyl-CoA used for chain elongation.     

Fatty acyl coenzyme A-sensitive adenine nucleotide transport in a reconstituted liposome system

The adenine nucleotide translocase was purified from bovine heart mitochondria and incorporated into membranes of phospholipid liposomes. The rate of transport of the adenine nucleotides was competitively inhibited by oleoyl coenzyme A with an approximate Ki of 1.0 microM. Significant inhibition was limited to those fatty acyl coenzyme A esters which are carnitine dependent for their oxidation in isolated mitochondria. Octanoyl coenzyme A was almost completely inactive as was palmitic acid and palmitoyl carnitine. By comparing the inhibitory characteristics of carboxyatractylate and bongkrekic acid with those of oleoyl-CoA, it was determined that the fatty acyl-CoA esters could produce inhibition whether the carrier was inserted into the liposome in either the conventional (65%) or reverse (30%) orientation. The results demonstrate that the interaction of long chain fatty acyl-CoA esters with the ADP/ATP carrier in a purified reconstituted system mimics their effects with isolated mitochondria and inverted submitochondrial particles. In general, these findings are consistent with the role of acyl-CoA esters acting as natural ligands and biological effectors of the translocator.     

Fatty Acyl-CoA Inhibits 1-Alkyl-sn-Glycero-3-Phosphate Acetyltransferase in Microsomes of Immature Rabbit Cerebral Cortex: Control of the First Committed Step in the De Novo Pathway of Platelet-Activating Factor Synthesis

Abstract: Microsomal fractions of cerebral cortices of 15-day-old rabbits were used to study the 1-alkyl- sn -glycero-3-phosphate (AGP) acetyltransferase that generates 1-alkyl-2-acetyl- sn -glycero-3-phosphate in the de novo path of platelet-activating factor synthesis. The AGP acetyltransferase activity was inhibited by small concentrations of medium-long chain fatty acyl-CoA thioesters. In contrast, the AGP acyltransferase used oleoyl-CoA as substrate and was not inhibited by the presence of acetyl-CoA in high molar excess. The inhibition of AGP acetyltransferase was seen at concentrations of oleoyl-CoA as low as 0.5 µ M using 12.5 µ M AGP and 200 µ M acetyl-CoA. The inhibition by oleoyl-CoA was noncompetitive for the acetyl-CoA substrate. However, there was evidence that the oleoyl-CoA was competing with AGP in the acetyltransferase reaction, as the inhibition was lessened by increasing the AGP substrate concentration. Several acyl-CoA thioesters were effective as inhibitors of the AGP acetyltransferase, including oleoyl-, palmitoyl-, lauroyl-, and octanoyl-CoA. Propionyl- and butyryl-CoA were less effective as inhibitors, and propionyl-CoA was found to be a competitive inhibitor for acetyl-CoA. We have noted earlier that MgATP is an effective inhibitor of the AGP acetyltransferase and here we show that the inhibition by oleoyl-CoA can be increased by the presence of 0.1 m M MgATP. In brain ischemia, a decline in ATP levels would likely lead to a corresponding fall in acyl-CoA concentrations, thereby relieving the inhibition of AGP acetyltransferase and permitting the flow of AGP into the de novo pathway of platelet-activating factor synthesis.     

Complementarity in the regulation of phosphoglucomutase, phosphofructokinase and hexokinase; the role of glucose 1,6-bisphosphate.

ATP and citrate, the well known inhibitors of phosphofructokinase (ATP: D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11), were found to inhibit the activities of the multiple forms of phosphoglucomutase (alpha-D-glucose 1,6-bisphosphate: alpha-D-glucose 1-phosphate phosphotransferase, EC 2.7.5.1) from rat muscle and adipose tissue. This inhibition could be reversed by an increase in the glucose 1,6-bisphosphate (Glc-1,6-P2) concentration. Other known activators (deinhibitors) of phosphofructokinase, viz. cyclic AMP, AMP, ADP or Pi, had no direct deinhibitory action on the ATP or citrate inhibited multiple phosphoglucomutases. Cyclic AMP and AMP, could however lead indirectly to deinhibition of the phosphoglucomutases, by activating phosphofructokinase which catalyzes the ATP-dependent phosphorylation of glucose 1-phosphate to form Glc-1,6-P2, the la-ter then released the multiple phosphoglucomutases from ATP or citrate inhibition. The Glc-1,6-P2 was also found to exert a selective inhibitory effect on hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) type II, the predominant form in skeletal muscle. This selective inhibition by Glc-1,6-P2 was demonstrated on the multiple hexokinases which were resolved by cellogel electrophoresis or isolated by chromatography on DEAE-cellulose. Based on the in vitro studies it is suggested that during periods of highly active epinephrine-induced glycogenolysis in muscle, the Glc-1,6-P2, produced by the cyclic AMP-stimulated reaction of phosphofructokinase with glucose 1-phosphate, will release the phosphoglucomutases from ATP or citrate inhibition, and will depress the activity of muscle type II hexokinase.     

Coordinate changes in carbon partitioning and plastidial metabolism during the development of oilseed rape embryos

Measurements of metabolic fluxes in whole embryos and isolated plastids have revealed major changes in the pathways of carbon utilization during cotyledon filling by oilseed rape (Brassica napus L.) embryos. In the early cotyledon stage (stage A), embryos used sucrose (Suc) predominantly for starch synthesis. Plastids isolated from these embryos imported glucose-6-phosphate (Glc-6-P) and partitioned it to starch and fatty acids synthesis and to the oxidative pentose phosphate pathway in the ratio of 2:1:1 on a hexose basis. Of the substrates tested, Glc-6-P gave the highest rates of fatty acid synthesis by the plastids and pyruvate was used weakly. By the mid- to late-cotyledon stage (stage C), oil accumulation by the embryos was rapid, as was their utilization of Suc for oil synthesis in vitro. Plastids from C-stage embryos differed markedly from those of stage-A embryos: (a) pyruvate uptake and utilization for fatty acid synthesis increased by respectively 18- and 25-fold; (b) Glc-6-P partitioning was predominantly to the oxidative pentose phosphate pathway (respective ratios of 1:1:3); and (c) the rate of plastidial fatty acid synthesis more than doubled. This increased rate of fatty synthesis was dependent upon the increase in pyruvate uptake and was mediated through the induction of a saturable transporter activity.     

Biosynthesis in Escherichia coli of sn-glycerol 3-phosphate, a precursor of phospholipid. Palmitoyl-CoA inhibition of the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

Homogeneous biosynthetic sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) of Escherichia coli was potently inhibited by palmitoyl-CoA and other long chain acyl-CoA thioesters. The concentration dependence of this inhibition was not cooperative. Enzyme activity was inhibited 50% at 1 microM palmitoyl-CoA; thus, this inhibition occurred at concentrations below the critical micellar concentration of palmitoyl-CoA. Palmitoyl-CoA was a reversible, noncompetitive inhibitor with respect to both NADPH and dihydroxyacetone phosphate. Palmitoyl-CoA did not affect the quaternary structure of the enzyme. This inhibition could be prevented or reversed by the addition of phospholipid vesicles prepared from E. coli phospholipids. Palmitoyl-CoA did not alter the kinetics of inhibition by sn-glycerol 3-phosphate, which is a proven physiological regulator of this enzyme. Decanoyl-CoA, dodecanoyl-CoA, myristoyl-CoA, palmitoyl-(1,N6-etheno)CoA, stearoyl-CoA, and oleoyl-CoA inhibited sn-glycerol-3-phosphate dehydrogenase at concentrations below their critical micellar concentrations. Palmitate inhibited sn-glycerol-3-phosphate dehydrogenase activity 50% at 200 microM. Palmitoyl-carnitine, deoxycholate, taurocholate, and dodecyl sulfate were more potent inhibitors than Triton X-100, Tween-20, or Tween-80. Palmitoyl-acyl carrier protein at concentrations up to 50 microM had no effect on sn-glycerol-3-phosphate dehydrogenase activity. The possible physiological role of long chain fatty acyl-CoA thioesters in the regulation of sn-glycerol 3-phosphate and phospholipid biosynthesis in E. coli is discussed.     

Loss of the major isoform of phosphoglucomutase results in altered calcium homeostasis in Saccharomyces cerevisiae

Phosphoglucomutase (PGM) is a key enzyme in glucose metabolism, where it catalyzes the interconversion of glucose 1-phosphate (Glc-1-P) and glucose 6-phosphate (Glc-6-P). In this study, we make the novel observation that PGM is also involved in the regulation of cellular Ca(2+) homeostasis in Saccharomyces cerevisiae. When a strain lacking the major isoform of PGM (pgm2Delta) was grown on media containing galactose as sole carbon source, its rate of Ca(2+) uptake was 5-fold higher than an isogenic wild-type strain. This increased rate of Ca(2+) uptake resulted in a 9-fold increase in the steady-state total cellular Ca(2+) level. The fraction of cellular Ca(2+) located in the exchangeable pool in the pgm2Delta strain was found to be as large as the exchangeable fraction observed in wild-type cells, suggesting that the depletion of Golgi Ca(2+) stores is not responsible for the increased rate of Ca(2+) uptake. We also found that growth of the pgm2Delta strain on galactose media is inhibited by 10 microM cyclosporin A, suggesting that activation of the calmodulin/calcineurin signaling pathway is required to activate the Ca(2+) transporters that sequester the increased cytosolic Ca(2+) load caused by this high rate of Ca(2+) uptake. We propose that these Ca(2+)-related alterations are attributable to a reduced metabolic flux between Glc-1-P and Glc-6-P due to a limitation of PGM enzymatic activity in the pgm2Delta strain. Consistent with this hypothesis, we found that this "metabolic bottleneck" resulted in an 8-fold increase in the Glc-1-P level compared with the wild-type strain, while the Glc-6-P and ATP levels were normal. These results suggest that Glc-1-P (or a related metabolite) may participate in the control of Ca(2+) uptake from the environment.     

Inhibition of diacylglycerol acyltransferase by 2-bromooctanoate in cultured rat hepatocytes

Triacylglycerol synthesis in cultured rat hepatocytes was inhibited by 2-bromooctanoate with a concomitant accumulation of diacylglycerols. 2-Bromooctanoate inhibition could be ascribed to its thioesterification by medium chain fatty acyl-CoA synthase (Raaka, B.M., and Lowenstein, J.M. (1979) J. Biol. Chem. 254, 6755-6762) with 2-bromooctanoyl-CoA acting as a competitive inhibitor of diacylglycerol acyltransferase. The Ki of 2-bromooctanoyl-CoA was 1.5 microM compared with a Km of 25 microM for the palmitoyl-CoA substrate. Diacylglycerol esterification was also inhibited by C12-C16 2-bromo fatty acids. However, inhibition of triacylglycerol synthesis by long chain 2-bromo fatty acids was accompanied by decreased overall neutral lipid synthesis as a result of inhibition of the long chain fatty acyl-CoA synthase. Since 2-bromooctanoate was a poor inhibitor of the long chain fatty acyl-CoA synthase, it appears to function selectively as an inhibitor of diacylglycerol acyltransferase in cultured rat hepatocytes.     

Metabolism of oleoyl-CoA in rat brain synaptosomes

The hydrolysis of acyl-CoA by acyl-CoA hydrolase (EC 3.1.2.2.) in brain synaptosomes was inhibited by calcium. This inhibition was partly due to interaction of Ca²⁺ with the acyl-CoA, which was present in the soluble form, and partly due to complex formation among acyl-CoA, Ca²⁺ and membrane phospholipids. The inhibition of acyl-CoA hydrolase activity, as well as the complex formation. could be reversed if incubation was carried out in the presence of Ca²⁺ chelating agents. Synaptosomes isolated from brain samples after 1 min of postdecapitative treatment showed a decrease in oleoyl-CoA hydrolase activity. The physiological implication of acyl-CoA metabolism in relation to synaptic function is discussed.Abbreviations FFA Free fatty acids - GPC glycerophosphocholines - GPE glycerophosphoethanolamines - GPI glycerophosphoinositols - GPS glycerophosphoserines     

Eicosapentaenoic acid reduces hepatic synthesis and secretion of triacylglycerol by decreasing the activity of acyl-coenzyme A:1,2-diacylglycerol acyltransferase

The mechanism for the reduced hepatic production of triacylglycerol in the presence of eicosapentaenoic acid was explored in short-term experiments using cultured parenchymal cells and microsomes from rat liver. Oleic, palmitic, stearic, and linoleic acids were the most potent stimulators of triacyl[3H]glycerol synthesis and secretion by hepatocytes, whereas erucic, alpha-linolenic, gamma-linolenic, arachidonic, docosahexaenoic, and eicosapentaenoic acids (in decreasing order) were less stimulatory. There was a linear correlation (r = 0.85, P less than 0.01) between synthesis and secretion of triacyl[3H]glycerol for the fatty acids examined. The extreme and opposite effects of eicosapentaenoic and oleic acids on triacylglycerol metabolism were studied in more detail. With increasing number of free fatty acid molecules bound per molecule of albumin, the rate of synthesis and secretion of triacyl[3H]glycerol increased, most markedly for oleic acid. Cellular uptake of the two fatty acids was similar, but more free eicosapentaenoic acid accumulated intracellularly. Eicosapentaenoic acid caused higher incorporation of [3H]water into phospholipid and lower incorporation into triacylglycerol and cholesteryl ester as compared to oleic acid. No difference was observed between the fatty acids on incorporation into cellular free fatty acids, monoacylglycerol and diacylglycerol. The amount of some 16- and 18-carbon fatty acids in triacylglycerol was significantly higher in the presence of oleic acid compared with eicosapentaenoic acid. Rat liver microsomes in the presence of added 1,2-dioleoyl-glycerol incorporated eicosapentaenoic acid and eicosapentaenoyl-CoA into triacylglycerol to a lesser extent than oleic acid and its CoA derivative. Decreased formation of triacylglycerol was also observed when eicosapentaenoyl-CoA was given together with oleoyl-CoA, whereas palmitoyl-CoA, stearoyl-CoA, linoleoyl-CoA, linolenoyl-CoA, and arachi-donoyl-CoA had no inhibitory effect. In conclusion, inhibition of acyl-CoA:1,2-diacylglycerol O-acyltransferase (EC 2.3.1.20) by eicosapentaenoic acid may be important for reduced synthesis and secretion of triacylglycerol from the liver.     

Effect of ATP Depletion on the Palmitoylation of Myelin Proteolipid Protein in Young and Adult Rats

The present study was designed to determine whether the palmitoylation of the hydrophobic myelin proteolipid protein (PLP) is dependent on cellular energy. To this end, brain slices from 20- and 60-day-old rats were incubated with [3H]palmitate for 1 h in the presence or absence of various metabolic poisons. In adult rats, the inhibition of mitochondrial ATP production with KCN (5 mM), oligomycin (10 microM), or rotenone (10 microM) reduced the incorporation of [3H]palmitate into fatty acyl-CoA and glycerolipids by 50-60%, whereas the labeling of PLP was unaltered. Incubation in the presence of rotenone (10 microM) plus NaF (5 mM) abolished the synthesis of acyl-CoA and lipid palmitoylation, but the incorporation of [3H]palmitate into PLP was still not different from that in controls. In rapidly myelinating animals, the inhibition of both mitochondrial electron transport and glycolysis obliterated the palmitoylation of lipids but reduced that of PLP by only 40%. PLP acylation was reduced to a similar extent when slices were incubated for up to 3 h, indicating that exogenously added palmitate is incorporated into PLP by ATP-dependent and ATP-independent mechanisms. Determination of the number of PLP molecules modified by each of these reactions during development suggests that the ATP-dependent process is important during the formation and/or compaction of the myelin sheath, whereas the ATP-independent mechanism is likely to play a role in myelin maintenance, perhaps by participating in the periodic repair of thioester linkages between the fatty acids and the protein.     

Arachidonoyl-Coenzyme A Synthetase and Nonspecific AcylCoenzyme A Synthetase Activities in Purified Rat Brain Microvessels

Purified rat brain microvessels were prepared to demonstrate the occurrence of acyl-CoA (EC 6.2.1.3) synthesis activity in the microvasculature of rat brain. Both arachidonoyl-CoA and palmitoyl-CoA synthesis activities showed an absolute requirement for ATP and CoA. This activity was strongly enhanced by magnesium chloride and inhibited by EDTA. The apparent Km values for acyl-CoA synthesis by purified rat brain microvessels were 4.0 microM and 5.8 microM for palmitic acid and arachidonic acid, respectively. The apparent Vmax values were 1.0 and 1.5 nmol X min-1 X mg protein-1 for palmitic acid and arachidonic acid, respectively. Cross-competition experiments showed inhibition of radiolabelled arachidonoyl-CoA formation by 15 microM unlabelled arachidonic acid, with a Ki of 7.1 microM, as well as by unlabelled docosahexaenoic acid, with a Ki of 8.0 microM. Unlabelled palmitic acid and arachidic acid had no inhibitory effect on arachidonoyl-CoA synthesis. In comparison, radiolabelled palmitoyl-CoA formation was inhibited competitively by 15 microM unlabelled palmitic acid, with a Ki of 5.0 microM and to a much lesser extent by arachidonic acid (Ki, 23 microM). The Vmax of palmitoyl-CoA formation obtained on incubation in the presence of the latter fatty acids was not changed. Unlabelled arachidic acid and docosahexaenoic acid had no inhibitory effect on palmitoyl-CoA synthesis. Both arachidonoyl-CoA and palmitoyl-CoA synthesis activities were thermolabile. Arachidonoyl-CoA formation was inhibited by 75% after 7 min at 40 degrees C whereas a 3-min heating treatment was sufficient to produce the same relative inhibition of palmitoyl-CoA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of a plant SNF1-related protein kinase by glucose-6-phosphate

One of the major protein kinases (PK(III)) that phosphorylates serine-158 of spinach sucrose-phosphate synthase (SPS), which is responsible for light/dark modulation of activity, is known to be a member of the SNF1-related family of protein kinases. In the present study, we have developed a fluorescence-based continuous assay for measurement of PK(III) activity. Using the continuous assay, along with the fixed-time-point (32)P-incorporation assay, we demonstrate that PK(III) activity is inhibited by glucose-6-phosphate (Glc-6-P). Relative inhibition by Glc-6-P was increased by decreasing pH from 8. 5 to 5.5 and by reducing the concentration of Mg(2+) in the assay from 10 to 2 mM. Under likely physiological conditions (pH 7.0 and 2 mM Mg(2+)), 10 mM Glc-6-P inhibited kinase activity approximately 70%. Inhibition by Glc-6-P could not be ascribed to contaminants in the commercial preparations. Other metabolites inhibited PK(III) in the following order: Glc-6-P > mannose-6-P, fructose-1,6P(2) > ribose-5-P, 3-PGA, fructose-6-P. Inorganic phosphate, Glc, and AMP were not inhibitory, and free Glc did not reverse the inhibition by Glc-6-P. Because SNF1-related protein kinases are thought to function broadly in the regulation of enzyme activity and gene expression, Glc-6-P inhibition of PK(III) activity potentially provides a mechanism for metabolic regulation of the reactions catalyzed by these important protein kinases.     

Fatty acid metabolism by a particulate fraction from germinating peas

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.     

Mitochondrial hexokinase from differentiated and undifferentiated HT29 colon cancer cells: effect of some metabolites on the bound/soluble equilibrium

1. Solubilization of mitochondrial bound hexokinase (HK), which represents 75-80% of the total enzyme activity in the cells, was investigated in freshly isolated mitochondria from undifferentiated (Glc+) or differentiated (Glc-) HT29 adenocarcinoma cells. In both models, the bound HK is almost completely released in vitro by 100 microM glucose 6-P (G 6-P). 2. Free ATP (5 mM) or palmitate (800 microM) produce a partial solubilization of bound HK, more markedly in the case of Glc- mitochondria. 3. Glucose or glucose 1-P are found unable to solubilize bound HK. Glucose 1,6-P2, 2-deoxyglucose 6-P or glucosamine 6-P can solubilize the enzyme but are less efficient than G 6-P. 4. Mg2+ and Pi are found to counteract the glucose 6-P induced solubilization of HK in both types of mitochondria. Taking into account the intracellular concentrations of these ions, this could in part explain why, in HT29 cells, HK is predominantly bound to the mitochondria.     

Synthesis and metabolism of arachidonyl- and eicosapentaenoyl-CoA in rat aorta

In studies on the metabolism of polyunsaturated fatty acids, acyl-CoA synthetase for 5,8,11,14-20:4 (arachidonic acid) and 5,8,11,14,17-20:5 (eicosapentaenoic acid) and the incorporation of these fatty acids into complex lipids and their conversion to CO2 were investigated in rat aorta. The activity of acyl-CoA synthetase was 35.9 for arachidonic acid and 63.0 for eicosapentaenoic acid (nmol/mg protein per 10 min) and the apparent Km values were 45 microM for arachidonic acid and 56 microM for eicosapentaenoic acid. Inhibition of eicosapentaenoyl-CoA synthesis by arachidonic acid was stronger than that of arachidonyl-CoA synthesis by eicosapentaenoic acid. Arachidonic acid and eicosapentaenoic acid were mostly incorporated into phospholipids. The incorporation of these fatty acids into cholesterol ester and their conversion to CO2 were less than those of palmitic acid, but their incorporation into triacyglycerol was greater. The incorporation of these fatty acids into phosphatidylserine + phosphatidylinositol and phosphatidylethanolamine was also greater than that of palmitic acid. The patterns of incorporation of arachidonic acid and eicosapentaenoic acid were similar. The physiological roles of these polyunsaturated fatty acids and the interference of eicosapentaenoic acid in arachidonic acid metabolism are discussed on the basis of these results.     

Chain elongation in the formation of polyunsaturated fatty acids by brain: Some properties of the microsomal system

Chain elongation of polyunsaturated acids has been investigated using microsomes from developing rat brain. With 18:3(n ? 6) in 0.05% detergent as an acceptor and [2-¹⁴C]malonyl-coenzyme A (CoA) as a two-carbon donor, incorporation of radioactivity into 20:3 was optimal (and incorporation into other acyl chains was minimal) in the presence of 100 μm substrate, 200 μmp-bromophenacylbromide and 10 mm KCN. Up to 30% of the labeled products were incorporated into phospholipids and triacylglycerol. Maximal microsomal elongation activity was observed at 3–4 weeks of age. Several other fatty acid or acyl-CoA acceptors tested in this system were elongated at slower rates compared to 18:3(n ? 6) [e.g., 16:0-CoA, 75%; 20:4(n ? 6), 57%; 18:3(n ? 3), 13%; 18:2(n ?6), 10%; 20:3(n ? 6), 6%]. The rate of elongation of chemically synthesized 18:3-CoA was only 50% of the detergent-suspended acid and was optimal at 6 μm substrate; inhibition above 6 μm 18:3-CoA was reduced by bovine serum albumin, but incorporation of label into palmitate was greatly stimulated. CoA markedly inhibited elongation of 18:3(n ? 6) or 18:3-CoA; N-ethylmaleimide at equimolar amounts reversed this CoA inhibition but did not alter the inhibition caused by concentrations of 18:3-CoA above 6 μm. ATP was absolutely required for elongation of either the free acid or the acyl-CoA derivative, whereas exogenous MgCl₂ had little effect.