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.     

Evidence for two distinct lysophospholipase activities that degrade lysophosphatidylcholine and lysophosphatidic acid in neuronal nuclei of cerebral cortex.

Neuronal nuclei were isolated from immature rabbit cerebral cortex and nuclear lysophospholipase activities studied using two different 1-acyl lysophospholipids: lysophosphatidylcholine (lysoPC) and lysophosphatidic acid (lysoPA). Our interest in these two lysolipids arose from the observation that lysoPA could promote the acetylation of lysoPC by substantially inhibiting a very active nuclear lysoPC lysophospholipase activity, in a competitive manner (R.R. Baker, H. -y. Chang, Mol. Cell. Biochem. (1999) in press). As there was also evidence for nuclear lysoPA deacylation, it was of interest to see whether one activity could possibly utilize both lysolipid substrates. We now have evidence for two separate lysophospholipase activities in neuronal nuclei. The lysoPC lysophospholipase activity was the more active, more highly enriched in the neuronal nuclei, and showed optimal activity at pH 8.4-9, while the lysoPA lysophospholipase activity was maintained over a much broader pH range. The lysoPC activity was substantially inhibited by free fatty acid, and showed considerable stimulation by serum albumin, while the activity utilizing lysoPA was much less affected by these agents. When lysoPC was added to incubations containing radioactive lysoPA, there was no significant inhibition found in rates of release of radioactive fatty acid, indicating that the lysoPA lysophospholipase activity did not utilize the lysoPC substrate. In incubations with lysoPC, MgATP and CoA brought about a sizable formation of phosphatidylcholine whose radioactivity was equally distributed between the sn-1 and sn-2 positions suggesting labelling both directly from the lysoPC substrate and from fatty acid produced by the lysophospholipase activity. By comparison, with the radioactive lysoPA substrate, MgATP and CoA promoted relatively lower levels of phosphatidic acid formation whose principal labelling came directly from the radioactive lysoPA. Largely because of the high activity of the nuclear lysoPC lysophospholipase, there is considerable potential in the neuronal nucleus to limit the use of lysoPC in other reactions, such as the formation of acylPAF (1-acyl analogue of platelet activating factor). It is of interest that conditions associated with brain ischaemia such as increased free fatty acid levels, falling pH and declines in MgATP may allow a preservation of neuronal nuclear lysoPC levels for acetylation. The existence of a separate lysophospholipase activity for lysoPA allows an independent control of lysoPA which can serve as an important regulator of the nuclear lysoPC lysophospholipase.     

Evidence for two distinct lysophospholipase activities that degrade lysophosphatidylcholine and lysophosphatidic acid in neuronal nuclei of cerebral cortex

Neuronal nuclei were isolated from immature rabbit cerebral cortex and nuclear lysophospholipase activities studied using two different 1-acyl lysophospholipids: lysophosphatidylcholine (lysoPC) and lysophosphatidic acid (lysoPA). Our interest in these two lysolipids arose from the observation that lysoPA could promote the acetylation of lysoPC by substantially inhibiting a very active nuclear lysoPC lysophospholipase activity, in a competitive manner (R.R. Baker, H.-y. Chang, Mol. Cell. Biochem. (1999) in press). As there was also evidence for nuclear lysoPA deacylation, it was of interest to see whether one activity could possibly utilize both lysolipid substrates. We now have evidence for two separate lysophospholipase activities in neuronal nuclei. The lysoPC lysophospholipase activity was the more active, more highly enriched in the neuronal nuclei, and showed optimal activity at pH 8.4–9, while the lysoPA lysophospholipase activity was maintained over a much broader pH range. The lysoPC activity was substantially inhibited by free fatty acid, and showed considerable stimulation by serum albumin, while the activity utilizing lysoPA was much less affected by these agents. When lysoPC was added to incubations containing radioactive lysoPA, there was no significant inhibition found in rates of release of radioactive fatty acid, indicating that the lysoPA lysophospholipase activity did not utilize the lysoPC substrate. In incubations with lysoPC, MgATP and CoA brought about a sizable formation of phosphatidylcholine whose radioactivity was equally distributed between the sn-1 and sn-2 positions suggesting labelling both directly from the lysoPC substrate and from fatty acid produced by the lysophospholipase activity. By comparison, with the radioactive lysoPA substrate, MgATP and CoA promoted relatively lower levels of phosphatidic acid formation whose principal labelling came directly from the radioactive lysoPA. Largely because of the high activity of the nuclear lysoPC lysophospholipase, there is considerable potential in the neuronal nucleus to limit the use of lysoPC in other reactions, such as the formation of acylPAF (1-acyl analogue of platelet activating factor). It is of interest that conditions associated with brain ischaemia such as increased free fatty acid levels, falling pH and declines in MgATP may allow a preservation of neuronal nuclear lysoPC levels for acetylation. The existence of a separate lysophospholipase activity for lysoPA allows an independent control of lysoPA which can serve as an important regulator of the nuclear lysoPC lysophospholipase.     

A metabolic path for the degradation of lysophosphatidic acid, an inhibitor of lysophosphatidylcholine lysophospholipase, in neuronal nuclei of cerebral cortex

Neuronal nuclei isolated from rabbit cerebral cortex were found to be enriched in an NEM-insensitive lysophosphatidic acid (lysoPA) phosphohydrolase activity. LysoPA is an inhibitor of the nuclear lysophosphatidylcholine (lysoPC) lysophospholipase, and by preserving lysoPC levels, lysoPA boosted the nuclear production of the acyl analogue of platelet-activating factor by promoting the acetylation of lysoPC (Baker and Chang, Mol. Cell Biochem., 1999, in press). The nuclear phosphohydrolase converts lysoPA to 1-monoacylglycerol, and thus eliminates this lysoPA inhibition of lysoPC lysophospholipase. The nuclear lysoPA phosphohydrolase specific activity was more than three times that observed for the nuclear lysoPA lysophospholipase (Baker and Chang, Biochim. Biophys. Acta 1438 (1999) 253-263) and represents a more active route for nuclear lysoPA removal. The neuronal nuclear lysoPA phosphohydrolase was inhibited at acidic pH, and also inhibited by calcium ions. The 1-monoacylglycerol product of the phosphohydrolase is rapidly degraded by neuronal monoacylglycerol lipase, an enzyme some sevenfold more active than the phosphohydrolase and sensitive to inhibition by arachidonoyl trifluoromethyl ketone (AACOCF(3)). Both acidic pH and free fatty acid inhibited the lipase. In the absence of AACOCF(3), production of fatty acid from lysoPA substrate could be largely attributed to the sequential actions of the nuclear phosphohydrolase and lipase. This facilitates fatty acid recycling back into phospholipid by lysophospholipid acylation when ATP levels are restored following periods of brain ischemia. At relatively low concentrations, sphingosine-1-phosphate, and alkylglycerophosphate were the most effective phosphohydrolase inhibitors while phosphatidic acid, alkylacetylglycerophosphate and ceramide were without effect. LysoPA is an interesting regulatory molecule that can potentially preserve lysophosphatidylcholine within the nuclear membrane for use in acetylation reactions. Thus conditions relevant to brain ischemia such as falling pH, falling ATP concentrations, rising fatty acid and intracellular calcium levels may, by slowing this metabolic path for lysoPA loss, promote the production of acyl PAF and contribute to the increased levels of the acetylated lipids noted in ischemia.     

The properties and regulation of pantothenate kinase from rat heart

Pantothenate kinase (ATP:D-pantothenate 4'-phosphotransferase, EC 2.7.1.33), the first enzyme in the pathway of CoA synthesis, was partially purified from rat heart. A study of the properties of the kinase showed that it possesses a broad pH optimum between 6 and 9, is activated or inhibited nonspecifically by various anions, and has MgATP as the nucleotide substrate. The Km for MgATP is 0.6 mM and that for pantothenate is 18 microM. CoA and acyl esters of CoA are inhibitors of the kinase with the inhibition by acetyl-CoA being only slightly greater than that by free CoA. The inhibition by free CoA is uncompetitive with respect to pantothenate concentration, with a Ki for inhibition of 0.2 microM. L-Carnitine was found to be a nonessential activator of the kinase. This compound had no effect by itself but specifically reversed the inhibition of the kinase by CoA. The Ka for deinhibition by L-carnitine is 0.27 mM. Free carnitine content was measured in perfused hearts and is found to vary in correlation with perfusion conditions that are known to alter rates of intracellular phosphorylation of pantothenate. These properties of pantothenate kinase provide a potential mechanism for the control of CoA synthesis. The enzyme is regulated by feedback inhibition by CoA and its acyl esters and this inhibition is modified by changes in the concentration of free carnitine.     

Evidence for the reversibility of the acyl-CoA:lysophosphatidylcholine acyltransferase in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons and rat liver.

Acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine occurs in the microsomal preparations of developing safflower cotyledons. Evidence is presented to show that the acyl exchange is catalysed by the combined back and forward reactions of an acyl-CoA:lysophosphatidylcholine acyltransferase (EC 2.3.1.23). The back reaction of the enzyme was demonstrated by the stimulation of the acyl exchange with free CoA and by the observation that the added CoA was acylated with acyl groups from position 2 of sn-phosphatidylcholine. Re-acylation of the, endogenously produced, lysophosphatidylcholine with added acyl-CoA occurred with the same specificity as that observed with added palmitoyl lysophosphatidylcholine. A similar acyl exchange, catalysed by an acyl-CoA:lysophosphatidylcholine acyltransferase, occurred in microsomal preparations of rat liver. The enzyme from safflower had a high specificity for oleate and linoleate, whereas arachidonate was the preferred acyl group in the rat liver microsomal preparations. The rate of the back reaction was 3-5% and 0.2-0.4% of the forward reaction in the microsomal preparations of safflower and rat liver respectively. Previous observations, that the acyl exchange in safflower microsomal preparations was stimulated by bovine serum albumin and sn-glycerol 3-phosphate, can now be explained by the lowered acyl-CoA concentrations in the incubation mixture with albumin and in the increase in free CoA in the presence of sn-glycerol 3-phosphate (by rapid acylation of sn-glycerol 3-phosphate with acyl groups from acyl-CoA to yield phosphatidic acid). Bovine serum albumin and sn-glycerol 3-phosphate, therefore, shift the equilibrium in acyl-CoA:lysophosphatidylcholine acyltransferase-catalysed reactions towards the rate-limiting step in the acyl exchange process, namely the removal of acyl groups from phosphatidylcholine. The possible role of the acyl exchange in the transfer of acyl groups between complex lipids is discussed.     

Fatty acids inhibit N-methylation of phosphatidylethanolamine in rat hepatocytes and liver microsomes

Supplementation of rat hepatocytes with various fatty acids in the culture medium reduced the conversion of [3H]phosphatidylethanolamine into phosphatidylcholine. Unsaturated fatty acids were the most effective inhibitors of phospholipid methylation. The inhibition of phosphatidylethanolamine methylation by oleate (2 mM) was reversed within 1 h after replacement with fatty acid-deficient medium. Fatty acids and their CoA derivatives (0.15-0.5 mM) produced 50% inhibition of phosphatidylethanolamine methyltransferase in rat liver microsomes. The first methylation reaction was the site of fatty acid inhibition, as methylation of phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine was not reduced in the presence of oleate. The inhibition by oleate was reversed by inclusion of bovine serum albumin or by addition of phospholipid liposomes. Thus, while fatty acids stimulate phosphatidylcholine biosynthesis in hepatocytes via the CDP-choline pathway, the methylation pathway is inhibited.     

Lysophosphatidic acid,alkylglycerophosphate and alkylacetylglycerophosphate increase the neuronal nuclear acetylation of 1-acyl lysophosphatidylcholine by inhibition of lysophospholipase

Neuronal nuclei were isolated from rabbit cerebral cortex, and lipid acetylation reactions were studied because of the high nuclear concentration of acetyltransferases that generate platelet activating factor (PAF) and its acyl analogue AcylPAF. The neuronal nuclear acetylation of 1-palmitoyl lysophosphatidylcholine (lyso PC) was found to be increased more than two fold when low concentrations of lyso PC were incubated in acetylation assays in the presence of 1-palmitoyl lysophosphatidic acid (lyso PA) or 1-hexadecyl glycerophosphate (AGP). This effect was not found for a variety of other acidic and neutral 1-acyl lysoglycerophospholipids. At 4 M concentrations, AGP was the more effective in increasing rates of lyso PC acetylation, while lyso PA was more effective at 25-35 M. 1-Stearoyl, 1-alkenyl and 1-decanoyl analogues of lyso PA were all less effective than 1-palmitoyl lyso PA. Phosphatidic acid was considerably less effective than lyso PA, while the acetylated analogue of AGP, AAcGP (alkylacetylglycerophosphate), increased rates of lyso PC to maxima similar to those seen with lyso PA or AGP. In addition, AAcGP promoted these maxima at considerably lower concentrations (2-4 M). A mechanism for these effects was suggested when nuclear envelopes (NE), isolated in the presence of PMSF, showed these maximal acetylation rates at low lyso PC concentrations, and these rates were not elevated by the presence of lyso PA. PMSF is a protease inhibitor but can also inhibit lysophospholipase activity. We found a nuclear lysophospholipase that degraded lyso PC at rates more than 13 times those of nuclear lyso PC acetylation. PMSF did inhibit this nuclear lysophospholipase, as did lyso PA, AGP and AAcGP. Kinetic analyses of the effects of lyso PA, AGP and AAcGP on lyso PC lysophospholipase indicated that these three lipids acted as competitive inhibitors for the lyso PC substrate. It is possible that low rates of lyso PC acetylation seen in neuronal nuclei at low lyso PC concentrations, are caused by lyso PC loss mediated by a very strong nuclear lysophospholipase. The effects of lyso PA, AGP and AAcGP in boosting rates of lyso PC acetylation likely come from the inhibition of nuclear lysophospholipase and a preservation of lyso PC concentrations. Competing neuronal nuclear reactions for low endogenous levels of lyso PC may regulate the formation of AcylPAF, and rising lyso PA, AGP or AAcGP concentrations can increase rates of nuclear AcylPAF synthesis.     

Inhibition of palmitoyl CoA of EDTA- and Mg2+-ATPase of heavy meromyosin from rabbit skeletal muscle

Palmitoyl CoA inhibited EDTA-ATPase of heavy meromyosin (HMM) prepared from rabbit skeletal muscle. The concentration for half maximum inhibition of EDTA-ATPase was about 18 microM. Myristoyl CoA, the other long chain fatty acyl CoA, also inhibited EDTA-HMM ATPase, but CoA and short chain CoA thioesters, such as butyryl CoA, acetoacetyl CoA and acetyl CoA, at 40 microM hardly inhibited EDTA-ATPase. Less than 20% inhibition of EDTA-HMM ATPase was obtained with Na-palmitate and Na-myristate at 40 microM, whereas about 90% inhibition of the enzyme occurred in the presence of 40 microM palmitoyl CoA and myristoyl CoA. Palmitoyl carnitine, as well as carnitine, failed to inhibit EDTA-HMM-ATPase. The inhibition of palmitoyl CoA of EDTA-ATPase was reversed by bovine serum albumin and spermine. Mg2+-HMM ATPase activity was enhanced by palmitoyl CoA at 2, 5, and 10 microM. About a 25% increase in Mg2+-HMM ATPase activity was obtained at 5 and 10 microM. At higher concentrations than 20 microM, the enzyme was inhibited by palmitoyl CoA and the degree of inhibition was related to the concentration of the CoA thioester. At 80 microM, the activity was about 15% of the maximum value. The efficacy of myristoyl CoA on Mg2+-ATPase was almost the same as that of palmitoyl CoA. Mg2+-ATPase activity was not enhanced by CoA, butyryl CoA, acetoacetyl CoA, Na-myristate, Na-palmitate, palmitoyl carnitine, or carnitine at 10 microM, and was hardly reduced by these substances at 40 microM. Serum albumin and spermine also canceled, to some extent, these effects of palmitoyl CoA on Mg2+-ATPase.     

Involvement of acyl exchange between acyl-CoA and phosphatidylcholine in the remodelling of phosphatidylcholine in microsomal preparations of rat lung

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.     

Sepharose-stearate as substrate for rat liver long-chain fatty acyl-CoA synthetase

Stearic acid coupled covalently to Sepharose 6B serves as substrate for thioesterification catalyzed by rat liver long-chain fatty acyl-CoA synthetase (ATP-forming) (EC 6.2.1.3). Availability as substrate is dependent upon the conservation of the free omega-terminal in addition to that of the free carboxyl function. The enzymatic overall formation of matrix-acyl-CoA in the presence of ATP and CoA as cosubstrates conforms to the stoichiometry reported for thioesterification of the free long-chain fatty acyl substrate. The preformed matrix-acyl-CoA serves as substrate for the backward synthetase reaction in the presence of AMP and PPi. The apparent Km values for ATP and CoA in the presence of the acyl matrix are similar to the respective Km values observed in the presence of the free acid substrate. The apparent Km for the acyl matrix is 10-fold higher (0.5 mM) than the apparent Km value for the free acid. The feasibility of enzymatic thioesterification of bound long-chain fatty acids implies that the exact nature of the bulky chain situated between the carboxy and omega-terminal plays a secondary role in defining the fatty acyl substrate specificity for long-chain fatty acyl-CoA synthetase. Also, dissociation of bound long-chain fatty acids does not constitute an obligatory preliminary step to fatty acid thioesterification.     

In vivo and in vitro intervention with L-carnitine prevents abnormal energy metabolism in isolated diabetic rat heart: chemical and phosphorus-31 NMR evidence

The beneficial effects of in vivo injections (200 mg/kg, twice daily) or in vitro perfusion (5.0 mM) of L-carnitine on an intrinsic abnormality in energy metabolism was investigated in isolated, perfused diabetic rat heart. Hearts were aerobically perfused for 60 min with elevated fatty acid substrate to simulate diabetic conditions. Phosphorus-31 nuclear magnetic resonance spectroscopy revealed a temporal decline in myocardial ATP levels (to approx 82%) during perfusion of diabetic hearts, but not in control hearts. This reduction was prevented by prior treatment in vivo with L-carnitine or by providing L-carnitine acutely in the perfusion medium. Chemical analysis of tissue extracts indicated that L-carnitine injections were effective in replenishing the decrease in total myocardial carnitine content which was present in diabetic hearts and in preventing the accumulation of long chain fatty acyl CoA. Perfusion with L-carnitine also attenuated the elevation of long chain fatty acyl CoA in diabetic hearts. This study gives additional support to the hypothesis that decreases in ATP which occur in the isolated, perfused diabetic heart are correlated with a concomitant elevation in long chain fatty acyl CoA, a known inhibitor of adenine nucleotide translocase. In the presence of elevated exogenous fatty acids, a primary deficiency in the total myocardial carnitine pool would result in elevations in tissue concentrations of long chain fatty acyl CoA since carnitine is a required carrier for transport of fatty acids into mitochondria. Replenishment of the carnitine in vivo was shown to be sufficient to prevent subsequent alteration in long chain fatty acyl CoA and ATP in isolated perfused diabetic hearts despite the burden of elevated fatty acid substrates.     

Inhibition by palmitoyl CoA of dynein ATPase from sea urchin spermatozoa

ATPase of 14S dynein, extracted from spermatozoa of the sea urchin, Hemicentrotus pulcherrimus, and partially purified by sucrose density gradient centrifugation, was inhibited non-competitively by palmitoyl CoA at concentrations higher than 20 microns, and was stimulated at concentrations between 2 microns and 10 microns. The effects of palmitoyl CoA on dynein ATPase were reversed by bovine serum albumin (1 mg/ml) and spermine (0.1 and 1 mM). Myristoyl CoA exerted effects similar to those of palmitoyl CoA. Short chain fatty acyl CoAs, such as butyryl CoA, propionyl CoA and acetyl CoA, CoA, Na-palmitate, Na-myristate, and palmitoyl carnitine had hardly any effect on dynein ATPase. Palmitoyl CoA failed to inhibit purified CF1 ATPase from chloroplasts of spinach, ATPase of rat liver mitochondria and alkaline phosphatase from calf intestine.     

Biosynthesis of Long-Chain Alcohols by Developing and Regenerating Rat Sciatic Nerve

Abstract: Cell-free preparations of rat sciatic nerve were found to catalyze the reduction of fatty acid to alcohol in the presence of NADPH as reducing cofactor. The reductase was membrane-bound and associated primarily with the microsomal fraction. When fatty acid was the substrate, ATP, coenzyme A (CoA), and Mg²⁺ were required, indicating the formation of acyl CoA prior to reduction. When acyl CoA was used as substrate, the presence of albumin was required to inhibit acyl CoA hydro-lase activity. Fatty acid reductase activity was highest with palmitic and stearic acids, and somewhat lower with lauric and myristic acids. It was inhibited by sulfhydryl reagents, indicating the participation of thiol groups in the reduction. Only traces of long-chain aldehyde could be detected or trapped as semicarbazone. Fatty acid reductase activity in rat sciatic nerve was highest between the second and tenth days after birth and decreased substantially thereafter. Microsomal preparations of sciatic nerve from 10-day-old rats exhibited about four times higher fatty acid reductase activity than brain or spinal cord microsomes from the same animals. Wallerian degeneration and regeneration of adult rat sciatic nerve resulted in enhanced fatty acid reductase activity, which reached a maximum at about 12 days after crush injury.     

The roles of magnesium ions in the reaction catalysed by phosphofructokinase from Trypanosoma brucei.

The involvement of Mg2+ ions in the reaction catalysed by phosphofructokinase from Trypanosoma brucei was studied. The true substrate for the enzyme was shown to be the MgATP2-complex, and free Mg2+ ions are also required for enzyme activity. At concentrations of MgATP2- of 2.92 mM and greater, and a fructose 6-phosphate concentration of 1 mM and in the presence of EDTA as a Mg2+ buffer, the Km value for Mg2+ was determined to be 294 +/- 18 microM. Neither MgATP nor free ATP is an inhibitor of the enzyme, although apparent inhibition by the latter can be observed as a consequence of the decrease in free Mg2+ by chelation.     

Identification of alpha-1 acid glycoprotein as a lysophospholipid binding protein: a complementary role to albumin in the scavenging of lysophosphatidylcholine

Alpha-1 acid glycoprotein (AGP, orosomucoid), a major acute phase protein in plasma, displays potent cytoprotective and anti-inflammatory activities whose molecular mechanisms are largely unknown. Because AGP binds various exogenous drugs, we have searched for endogenous ligands for AGP. We found that AGP binds lysophospholipids in a manner discernible from albumin in several ways. First, mass spectrometric analyses showed that AGP isolated from plasma and serum contained lysophosphatidylcholine (LPC) enriched in mono and polysaturated acyl chains, whereas albumin contained mostly saturated LPC. Second, AGP bound LPC in a 1:1 molar ratio and with a higher affinity than free fatty acids, whereas albumin bound LPC in a 3:1 ratio but with a lower affinity than that of free fatty acids. Consequently, free fatty acids displaced LPC more avidly from albumin than from AGP. Competitive ligand displacement indicated the highest affinity for AGP to LPC20:4, 18:3, 18:1, and 16:0 (150-180 nM), lysophosphatidylserine (Kd 190 nM), and platelet activating factor (PAF) (Kd 235 nM). The high affinity of AGP to LPC in equilibrium was verified by stopped-flow kinetics, which implicated slow dissociation after fast initial binding, being consistent with an induced-fit mechanism. AGP also bound pyrene-labeled phospholipids directly from vesicles and more efficiently than albumin. AGP prevented LPC-induced priming and PAF-induced activation of human granulocytes, thus indicating scavenging of the cellular effects of the lipid ligands. The results suggest that AGP complements albumin as a lysophospholipid scavenging protein, particularly in inflammatory conditions when the capacity of albumin to sequester LPC becomes impaired.     

Regulation of eicosanoid synthesis in microvessel endothelium: glucocorticoids do not affect arachidonyl CoA synthetase activity

The properties of acyl coenzyme A (CoA) synthetase activity were characterized in cultured rabbit coronary microvessel endothelial cells. We report here that microvessel endothelial cells contain two long-chain acyl CoA synthetases. One shows activity with a variety of fatty acids, similar to long-chain non-selective fatty acyl CoA synthetases described previously. The other activity was selective for arachidonic acid and other structurally related substrates. Both activities required ATP, Mg2+ and CoA for optimal activity. The arachidonyl CoA and the non-selective acyl CoA synthetases showed different thermolabilities. Arachidonyl CoA formation was inhibited by greater than 50% after 1 min at 45 degrees C, whereas a 15 min heating treatment was necessary to produce the same relative inhibition of oleoyl CoA synthesis. Glucocorticoid pretreatment (10(-7) M dexamethasone) of the RCME cells did not affect the apparent Km or Vmax, nor the fatty acid selectivity for either acyl CoA synthetase. Therefore, although fatty acyl CoA synthetases may be involved in limiting eicosanoid formation, these activities do not appear to be glucocorticoid-responsive.     

Fatty acid synthesis in pea root plastids is inhibited by the action of long-chain acyl- coenzyme as on metabolite transporters.

The uptake in vitro of glucose (Glc)-6-phosphate (Glc-6-P) into plastids from the roots of 10- to 14-d-old pea (Pisum sativum L. cv Puget) plants was inhibited by oleoyl-coenzyme A (CoA) concentrations in the low micromolar range (1--2 microM). The IC(50) (the concentration of inhibitor that reduces enzyme activity by 50%) for the inhibition of Glc-6-P uptake was approximately 750 nM; inhibition was reversed by recombinant rapeseed (Brassica napus) acyl-CoA binding protein. In the presence of ATP (3 mM) and CoASH (coenzyme A; 0.3 mM), Glc-6-P uptake was inhibited by 60%, due to long-chain acyl-CoA synthesis, presumably from endogenous sources of fatty acids present in the preparations. Addition of oleoyl-CoA (1 microM) decreased carbon flux from Glc-6-P into the synthesis of starch and through the oxidative pentose phosphate (OPP) pathway by up to 73% and 40%, respectively. The incorporation of carbon from Glc-6-P into fatty acids was not detected under any conditions. Oleoyl-CoA inhibited the incorporation of acetate into fatty acids by 67%, a decrease similar to that when ATP was excluded from incubations. The oleoyl-CoA-dependent inhibition of fatty acid synthesis was attributable to a direct inhibition of the adenine nucleotide translocator by oleoyl-CoA, which indirectly reduced fatty acid synthesis by ATP deprivation. The Glc-6-P-dependent stimulation of acetate incorporation into fatty acids was reversed by the addition of oleoyl-CoA.     

The effects of bovine serum albumin and oleic acid on rat pancreatic lipase and bovine milk lipoprotein lipase

The effects of bovine serum albumin on rat pancreatic lipase and bovine milk lipoprotein lipase were studied in a system of triacylglycerol emulsions stabilized by 1 1 mg/ml albumin. At concentrations greater than 1 mg/ml, albumin inhibited the activity of pancreatic lipase and interfered with enzyme binding to emulsified triacylglycerol particles. These effects could be countered by occupying five fatty acid binding sites on albumin with oleic acid. Following an initial lag period which increased with albumin concentrations, enzyme activity escaped from inhibition presumably due to saturation of fatty acid sites on albumin with oleic acid. Pancreatic lipase was active at 1 mg/ml albumin and 1 mM emulsion-bound oleic acid in the system. The effects of albumin on lipoprotein lipase were diametrically opposed to the above; enzyme activity was completely inhibited by 0.1 mM oleic acid, it increased with increasing fatty acid-free albumin concentrations and decreased as the fatty acid sites on albumin were filled. At 1 mM oleic acid and no added albumin the enzyme failed to bind at the oil water interface, whereas fatty acid-free or saturated albumin had no effect on binding. It is concluded that if the inhibition of pancreatic lipase by albumin is due to the inaccessibility of the enzyme to an oil-water interface blocked by denatured albumin, then albumin saturated with oleic acid would seem to be protected from unfolding at the interface and more readily displaced by the lipase. Pancreatic lipase and lipoprotein lipase, although sharing a number of common features, are distinct enzymes both functionally and mechanistically.     

Differential interaction of fatty acids and fatty acyl CoA esters with the purified/reconstituted brown adipose tissue mitochondrial uncoupling protein

Proteoliposomes containing highly purified uncoupling protein generated by a modified purification/reconstitution procedure carried out active GDP dependent proton conductance. It was further established that long chain acyl CoA esters as well as fatty acids stimulated proton influx by the uncoupling protein, and, moreover, that the acyl CoA esters were partially effective in overcoming the inhibition by GDP. GDP binding to the purified uncoupling protein was inhibited by acyl CoA esters but not fatty acids. Phenylglyoxal which prevents GDP binding to the uncoupling protein eliminated the acyl CoA but not the fatty acid effect on proton conductance. These results substantiate the fact that nucleotides and acyl CoA esters act at the same regulatory site on the uncoupling protein, whereas, fatty acids act at a separate site. The properties of the purified/reconstituted uncoupling protein confirm they are identical to those inherent in brown adipose tissue mitochondria.