Effect of cis-unsaturated fatty acids on aortic protein kinase C activity.

Long-chain cis-unsaturated fatty acids could substitute for phosphatidylserine and activate bovine aortic protein kinase C in assays with histone as substrate. The optimal concentration was 24-40 microM for oleic, linoleic and arachidonic acids. With arachidonic acid, the Ka for Ca2+ was 130 microM and kinase activity was maximal at 0.5 mM-Ca2+. Diolein only slightly activated the oleic acid-stimulated enzyme at low physiological Ca2+ concentrations (0.1 and 10 microM). Oleic acid also stimulated kinase C activity, determined with a Triton X-100 mixed-micellar assay. Under these conditions, the fatty acid activation was absolutely dependent on the presence of diolein, but a Ca2+ concentration of 0.5 mM was still required for maximum kinase C activity. The effect of fatty acids on protein kinase C activity was also investigated with the platelet protein P47 as a substrate, since the properties of kinase C can be influenced by the choice of substrate. In contrast with the results with histone, fatty acids did not stimulate the phosphorylation of P47 by the aortic protein kinase C. Activation of protein kinase C by fatty acids may allow the selective phosphorylation of substrates, but the physiological significance of fatty acid activation is questionable because of the requirement for high concentrations of Ca2+.     

Protein kinase C activation by cis-fatty acid in the absence of Ca2+ and phospholipids

Protein kinase C has been shown to be a phospholipid/Ca2+-dependent enzyme activated by diacylglycerol (Nishizuka, Y. (1984) Nature 308, 693-697; Nishizuka, Y. (1984) Science 225, 1365-1370). We have reported that unsaturated fatty acids (oleic acid and arachidonic acid) can activate protein kinase C independently of Ca2+ and phospholipid (Murakami, K., and Routtenberg, A. (1985) FEBS Lett. 192, 189-193). This study shows that other cis-fatty acids such as linoleic acid also fully activate protein kinase C in the same manner. None of the saturated fatty acids (C:4 to C:18) nor the detergents (sodium dodecyl sulfate and Triton X-100) tested here were as effective as oleic acid. Unlike oleic acid, these detergents strongly inhibited protein kinase C activity induced by Ca2+/phosphatidylserine (PS) and diacylglycerol. Lowering the critical micelle concentration of oleic acid by increasing ionic strength also strongly inhibited oleic acid activation of protein kinase C activity. Dioleoylphosphatidylserine activated protein kinase C effectively (Ka = 7.2 microM). On the other hand, dimyristoylphosphatidylserine, which contains saturated fatty acids at both acyl positions, failed to activate protein kinase C even in the presence of Ca2+. These observations suggest that: protein kinase C activation by free fatty acid is specific to the cis-form and is not due to their detergent-like action, cis-fatty acid activation is due to the direct interaction of protein kinase C with the monomeric form of cis-fatty acids and not with the micelles of fatty acids, and cis-fatty acids at acyl positions in PS are also important for Ca2+/PS activation of protein kinase C.     

22-Carbon polyenoic acids. Incorporation into platelet phospholipids and the synthesis of these acids from 20-carbon polyenoic acid precursors by intact platelets

The types of unsaturated fatty acids found in platelet phospholipids must be regulated by a series of controls which include specificity for activation and acylation as well as modification of circulating fatty acids by platelets prior to incubation into phospholipids. In this study we show that washed human platelets not only incorporate [1-14C]6,9,12-18:3, [1-14C]6,9,12,15-18:4, [1-14C]5,8,11-20:3, [1-14C]5,8,11,14-20:4, and [1-14C]5,8,11,14,17-20:5 into their phospholipids but also chain elongate each of these acids with subsequent acylation of the chain elongated products into phospholipids. Platelets incubated alone with 1-14C-labeled 5,8,11-20:3, 5,8,11,14-20:4, 5,8,11,14,17-20:5, 7,10,13,16,19-22:5, or 4,7,10,13,16,19-22:6 incorporated each of these acids into individual phosphoglycerides with phosphatidylinositol having the highest specific activity followed by phosphatidylcholine with phosphatidylserine approximately equal to phosphatidylethanolamine. The incorporation specificity of 4,7,10,13,16,19-22:6 was atypical since it was a relatively poor substrate for acylation into all phospholipids except phosphatidylethanolamine. The 20-carbon acids were better substrates for incorporation into phospholipids than were the 22-carbon compounds. Simultaneous incubation of 10 microM [1-14C]5,8,11,14-20:4 with increasing levels (5 to 15 microM) of each of the above five other 1-14C-labeled acids showed a concentration-dependent increase in the amount of the second fatty acid incorporated into platelet phospholipids. Dietary fat modification thus has the potential of increasing the plasma pool of 22-carbon acids for incorporation into platelets. In addition the activation of 20-carbon eicosanoid precursors by the high affinity platelet activating enzyme (Wilson, D. B., Prescott, S. M. and Majerus, P. W. (1982) J. Biol. Chem. 257, 3510-3515) will yield an acyl-CoA for both acylation and chain elongation followed by subsequent incorporation of 22-carbon acids into phosphoglycerides.     

Do rat hepatic microsomes contain multiple NADPH-supported fatty acid chain elongation pathways or a single pathway?

[2-14C]-trans-2-hexadecenoyl CoA (16:1) and [2-14C]-trans-2-cis-8,11,14-eicosatetraenoyl CoA (20:4) were chemically synthesized and employed as competitive substrates for the liver microsomal trans-2-enoyl CoA reductase component of the fatty acid chain elongation system. Both 7.5 microM and 15 microM 20:4 competitively inhibited the reduction of 16:1 CoA to palmitoyl CoA. In addition, the reduction of both substrates was identically inhibited to the same extent by the acetylenic derivative, dec-2-ynoyl CoA. Furthermore, trypsin, chymotrypsin and subtilisin inhibited trans-2-enoyl CoA reductase activity when three different substrates were employed--16:1, 20:4 and trans-2-cis-11-octadecadienoyl CoA (18:2). These results are consistent with the hypothesis of multiple condensing enzymes connected to a single elongation pathway.     

Characterization of bovine aortic protein kinase C with histone and platelet protein P47 as substrates.

A Ca2+- and phospholipid-dependent protein kinase (protein kinase C) was partially purified from the media of bovine aortas by chromatography on DEAE-Sephacel and phenyl-Sepharose. Enzyme activity was characterized with both histone and a 47 kDa platelet protein (P47) as substrates, because the properties of protein kinase C can be modified by the choice of substrate. Both phosphatidylserine and Ca2+ were required for kinase activity. With P47 as substrate, protein kinase C had a Ka for Ca2+ of 5 microM. Addition of diolein to the enzyme assay caused a marked stimulation of activity, especially at low Ca2+ concentrations, but the Ka for Ca2+ was shifted only slightly, to 2.5 microM. With histone as substrate, the enzyme had a very high Ka (greater than 50 microM) for Ca2+, which was substantially decreased to 3 microM-Ca2+ by diolein. A Triton X-100 mixed-micelle preparation of lipids was also utilized to assay protein kinase C with histone as the substrate. Under these conditions kinase activity was almost totally dependent on the presence of diolein; again, diolein caused a large decrease in the Ka for Ca2+, from greater than 100 microM to 2.5 microM. The increased sensitivity of protein kinase C to Ca2+ with P47 rather than histone, and the ability of diacylglycerol to activate protein kinase C without shifting the Ka for Ca2+, when P47 is the substrate, illustrate that the mechanism of protein kinase C activation is influenced by the exogenous substrate used to assay the enzyme.     

The glyoxysomal beta-oxidation system in cucumber seedlings: identification of enzymes required for the degradation of unsaturated fatty acids

Fat-degrading cotyledons from cucumber seedlings were investigated with respect to the enzymes metabolizing cis-unsaturated fatty acids. Isolated glyoxysomes degrade linoleic acid, the dominating fatty acid in the storage tissue of the seed. Glyoxysomes were shown to be the sole intracellular site of enzymes responsible for the degradation of unsaturated fatty acids. All three auxiliary enzyme activities discussed for the degradation of polyunsaturated fatty acids, 2,4-dienoyl-CoA reductase, enoyl-CoA isomerase, and 3-hydroxyacyl-CoA epimerase were localized within the matrix of glyoxysomes. They were not found in mitochondria. Separation of glyoxysomal matrix proteins on CM-cellulose revealed that epimerase activity was attributable to the multifunctional protein and also to another protein which apparently exhibited no other beta-oxidation activity. Furthermore, on the basis of the high epimerase activity present in glyoxysomes compared to a much lower 2,4-dienoyl-CoA reductase activity, the metabolism of unsaturated fatty acids via delta 2-cis-enoyl-CoA is considered as alternative to the reductase-dependent pathway.     

Arachidonic acid and related methyl ester mediate protein kinase C activation in intact platelets through the arachidonate metabolism pathways

Unlike unsaturated fatty acids, which almost fully activated purified brain protein kinase C in a phosphatidylserine- and Ca2(+)-free reaction, related methyl esters were poorly active in vitro. In contrast, methyl arachidonate was revealed to be as potent as arachidonic acid in activating protein kinase C in intact platelets. Arachidonic acid-mediated activation peaked at 20 s while methyl arachidonate-mediated activation plateaued at 2 min when both lipids were added at 50 microM. At concentrations higher than 0.3 mM, all tested unsaturated fatty acids and related methyl esters were weak activators of the enzyme, with the exception of linolenic acid and methyl linolenate which evoked strong enzyme activation. However, inhibitors of arachidonate metabolism blocked both arachidonic-acid and methyl-arachidonate-induced responses. At 5 microM arachidonic acid and methyl arachidonate, protein kinase C activation was due to a cyclooxygenase product(s) whereas at 50 microM the lipoxygenase pathway was mostly involved in the reaction. Therefore, arachidonic acid and its methyl ester activate protein kinase C in platelets mainly through action of their metabolites and eicosanoid synthesis. It is suggested that such indirect protein kinase C activation may account for the tumor-promoting activity of unsaturated fatty acids and related methyl esters.     

Cis-unsaturated fatty acids induce both lipogenesis and calcium binding in adipocytes

The addition of 0.4-3 mM of cis-unsaturated fatty acids such as oleic acid (18:1) or linoleic acid (18:2) to intact rat adipocytes stimulated lipogenesis at 37 degrees C. Saturated or trans-unsaturated fatty acids were ineffective. Fluorescence photobleaching recovery studies performed under similar conditions indicated that the cis-unsaturated fatty acids do not alter lateral mobility of either a lipid probe or a general protein marker in the plasma membrane. A high concentration (7 mM) of Ca2+, which by itself has some stimulatory effect on lipogenesis, significantly potentiated the effect of oleic acid on this insulin-like activity. Measurement of 45Ca2+ binding by fat cells has indicated that cis-unsaturated (but not saturated) fatty acids increased 12- to 20-fold the amount of Ca2+ associated with the cells. The dependence of this effect on the fatty acid concentration correlates well with the effect of the fatty acid on the induction of lipogenesis. Our results suggest that cis-unsaturated fatty acids affect membrane organization in a manner which induces a significant increase in membrane associated or intracellular Ca2+. This increase may be responsible for inducing exocytotic-like processes which facilitate translocation of glucose transport activity from storage sites to the plasma membrane and thus produce an insulin-like effect.     

Inhibition of topoisomerases by fatty acids

The inhibitory effects of various fatty acids on topoisomerases were examined, and their structure activity relationships and mechanism of action were studied. Saturated fatty acids (C6:0 to C22:0) did not inhibit topoisomerase I, but cis-unsaturated fatty acids (C16:1 to C22:1) with one double bond showed strong inhibition of the enzyme. The inhibitory potency depended on the carbon chain length and the position of the double bond in the fatty acid molecule. The trans-isomer, methyl ester and hydroxyl derivative of oleic acid had no or little inhibitory effect on topoisomerases I and II. Among the compounds studied petroselinic acid and vaccenic acid (C18:1) with a cis-double bond were the potent inhibitors. Petroselinic acid was a topoisomerase inhibitor of the cleavable complex-nonforming type and acted directly on the enzyme molecule in a noncompetitive manner without DNA intercalation.     

Isolation and characterization of the calcium- and phospholipid-dependent protein kinase (protein kinase C) subtypes from bovine heart

Protein kinase C was isolated from bovine heart by chromatography on DEAE-Sephacel, phenyl-Sepharose, poly(L-lysine) agarose, and hydroxylapatite. Estimates based upon enzyme recovery indicate 10-20 nmol/min of protein kinase C activity per gram of bovine ventricular myocardium. Hydroxylapatite column chromatography resolved the preparation into two peaks of calcium- and phospholipid-dependent protein kinase activity. By Western blot analysis, peaks 1 and 2 contained subtypes II (beta 2) and III (alpha), respectively. No cross-reactivity was observed, indicating that separation was complete. Type III, the major subtype detected, was subsequently purified to apparent homogeneity by chromatography on phosphatidylserine (PS) acrylamide. Type II activity could not be recovered following phosphatidylserine affinity chromatography. Phospho amino acid analysis showed that type III autophosphorylated at serine residues, whereas type II autophosphorylated at both serine and threonine residues. Among the various phospholipids tested for activity, PS was the most effective. Both subtypes were activated by 1-stearoyl-2-arachidonylglycerol (SAG) in the presence of phosphatidylserine and calcium. Activation of both subtypes occurred at calcium concentrations of less than 1 microM. In addition to several similarities, these two subtypes showed differences in activation and kinetic properties: type II was activated by cardiolipin, 1,2-and 1,3-dioleoylglycerol, and both cis- and trans-unsaturated fatty acids. Type III was activated to a lesser degree by cardiolipin and showed no response to 1,3-dioleoylglycerol. Type III was activated to a greater extent by 1,2-diacylglycerols and by cis-unsaturated fatty acids. In the presence of PS and SAG, type II exhibited substantial activity in the presence of 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) without added calcium. Activation of types II and III by unsaturated fatty acids was independent of phospholipid and showed a lower apparent calcium affinity than that observed for activation by phosphatidylserine. These results show that cardiac protein kinase C subtypes II and III were functionally distinguishable and may play unique roles in the regulation of cardiac function.     

Long-chain acyl-coenzyme A synthetase from rat brain microsomes. Kinetic studies using [1-14C]docosahexaenoic acid substrate

The activation of docosahexaenoic acid by rat brain microsomes was studied using an assay method based on the extraction of unreacted [1-14C]docosahexaenoic acid and the insolubility of [1-14C]docosahexaenoyl-CoA in heptane. This reaction showed a requirement for ATP, CoA, and MgCl2 and exhibited optimal activity at pH 8.0 in the presence of dithiothreitol and when incubated at 45 degrees C. The apparent Km values for ATP (185 microM), CoA (4.88 microM), MgCl2 (555 microM) and [1-14C]docosahexaenoic acid (26 microM) were determined. The presence of bovine serum albumin or Triton X-100 in the incubation medium caused a significant decrease in the Km and Vm values for [1-14C]docosahexaenoic acid. The enzyme was labile at 45 degrees C (t1/2:3.3 min) and 37 degrees C (t1/2:26.5 min) and lost 36% of its activity after freezing and thawing. The transition temperature (Tc) obtained from Arrhenius plot was 27 degrees C with the activation energies of 74 kJ/mol between 0 degrees C and 27 degrees C and 30 kJ/mol between 27 degrees C and 45 degrees C. [1-14C]Palmitic acid activation in rat brain and liver microsomes showed apparent Km values of 25 microM and 29 microM respectively, with V values of 13 and 46 nmol X min-1 X mg protein-1. The presence of Triton X-100 (0.05%) in the incubation medium enhanced the V value of the liver enzyme fourfold without affecting the Km value. Brain palmitoyl-CoA synthetase, on the other hand, showed a decreased Km value in the presence of Triton X-100 with unchanged V. The Tc obtained were 25 degrees C and 28 degrees C for brain and liver enzyme with an apparent activation energy of 109 and 24 kJ/mol below and above Tc for brain enzyme and 86 and 3.3 kJ/mol for liver enzyme. The similar results obtained for the activation of docosahexaenoate and palmitate in brain microsomes suggest the possible existence of a single long-chain acyl-CoA synthetase. The differences observed in the activation of palmitate between brain and liver microsomes may be due to organ differences. Fatty acid competition studies showed a greater inhibition of labeled docosahexaenoic and palmitic acid activation in the presence of unlabeled unsaturated fatty acids. The Ki values for unlabeled docosahexaenoate and arachidonate were 38 microM and 19 microM respectively for the activation of [1-14C]docosahexaenoate. In contrast, the competition of unlabeled saturated fatty acids for activation of labeled docosahexaenoate is much less than that for activation of labeled palmitate.(ABSTRACT TRUNCATED AT 400 WORDS)     

A soluble fatty acyl-acyl carrier protein synthetase from the bioluminescent bacterium Vibrio harveyi

An enzyme catalyzing the ligation of long chain fatty acids to bacterial acyl carrier protein (ACP) has been detected and partially characterized in cell extracts of the bioluminescent bacterium Vibrio harveyi. Acyl-ACP synthetase activity (optimal pH 7.5-8.0) required millimolar concentrations of ATP and Mg2+ and was slightly activated by Ca2+, but was inhibited at high ionic strength and by Triton X-100. ACP from either Escherichia coli (apparent Km = 20 microM) or V. harveyi was used as a substrate. Of the [14C]fatty acids tested as substrates (8-18 carbons), a preference for fatty acids less than or equal to 14 carbons in length was observed. Vibrio harveyi acyl-ACP synthetase appears to be a soluble hydrophilic enzyme on the basis of subcellular fractionation and Triton X-114 phase partition assay. The enzyme was not coinduced with luciferase activity or light emission in vivo during the late exponential growth phase in liquid culture. Acyl-ACP synthetase activity was also detected in extracts from the luminescent bacterium Vibrio fischeri, but not Photobacterium phosphoreum. The cytosolic nature and enzymatic properties of V. harveyi acyl-ACP synthetase indicate that it may have a different physiological role than the membrane-bound activity of E. coli, which has been implicated in phosphatidylethanolamine turnover. Acyl-ACP synthetase activity in V. harveyi could be involved in the intracellular activation and elongation of exogenous fatty acids that occurs in this species or in the reactivation of free myristic acid generated by luciferase.     

Activation of protein kinase C by myristate and its requirements of Ca2+ and phospholipid

Myristate (C14:0) was found to significantly activate partially purified rat brain Ca(2+)- and phospholipid-dependent protein kinase (PKC). The Ka value, the concentration needed for half maximum activation, for C14:0 in the presence of 1 microM Ca2+ and 20 microM phosphatidylserine (PS) was 20 microM. This activation required Ca2+ and acidic phospholipid and was associated with a decreased Ka for Ca2+ of the enzyme to 10 microM in an analogous fashion as dioleoylglycerol (DO) or phorbol myristate acetate (PMA). The phospholipid requirement for the activation was concentration dependent and was inhibited by 1-(5-isoquinolinesulfonyl)-methylpiperazine dihydrochloride (H-7), a inhibitor of this enzyme. The concentration of H-7 required for half inhibition of the enzyme was about 15 microM and maximum inhibition was about 75%. The concentration profile of cytoplasmic proteins phosphorylated by C14:0-activated PKC was similar to that by PMA-activated PKC. The 47 kDa protein of guinea pig neutrophil was also phosphorylated by the C14:0-activated PKC. It is further discussed whether PKC can function as signal transduction for stimulus-mediated generation of superoxide in neutrophils.     

Characterization of the CoA ligases of human liver mitochondria catalyzing the activation of short- and medium-chain fatty acids and xenobiotic carboxylic acids.

Two distinct forms of xenobiotic/medium-chain fatty acid:CoA ligase (XM-ligase) were isolated from human liver mitochondria. They were referred to as HXM-A and HXM-B based on their order of elution from a DEAE-cellulose column. Activity of the two ligases was determined toward 15 different carboxylic acids. HXM-A represented 60-80% of the benzoate activity in the lysate, and kinetic analysis revealed that benzoate was the best substrate (highest V(max)/K(m)). The enzyme also had medium-chain fatty acid:CoA ligase activity. HXM-B had the majority of the hexanoate activity and hexanoate was its best substrate. It was, however, also active toward many xenobiotic carboxylic acids. Comparison of these two human XM-ligases with the previously characterized bovine XM-ligases indicated that they were kinetically distinct. When assayed with benzoic acid as substrate, both HXM-A and HXM-B had an absolute dependence on either Mg(2+) or Mn(2+) for activity. Further, addition of monovalent cation (K(+), Rb(+), or NH(4)(+)) stimulated HXM-A activity by >30-fold and HXM-B activity by 4-fold. For both forms, activity toward straight-chain fatty acids was stimulated less by K(+) than was activity toward benzoate or phenylacetate. A 60 kDa short-chain fatty acid:CoA ligase was also isolated. It had activity toward propionate and butyrate, but not acetate, hexanoate or benzoate. The K(m)(app) values were high but similar for propionate and butyrate (285 microM and 250 microM, respectively) but the V(max)(app) was nearly 6-fold greater with propionate as substrate. While the K(m) values are somewhat high, the enzyme is still more efficient with these substrates than either of the XM-ligases.     

Comparative biochemical studies of the murine fatty acid transport proteins (FATP) expressed in yeast

The fatty acid transport protein (FATP) family is a group of proteins that are predicted to be components of specific fatty acid trafficking pathways. In mammalian systems, six different isoforms have been identified, which function in the import of exogenous fatty acids or in the activation of very long-chain fatty acids. This has led to controversy as to whether these proteins function as membrane-bound fatty acid transporters or as acyl-CoA synthetases, which activate long-chain fatty acids concomitant with transport. The yeast FATP orthologue, Fat1p, is a dual functional protein and is required for both the import of long-chain fatty acids and the activation of very long-chain fatty acids; these activities intrinsic to Fat1p are separable functions. To more precisely define the roles of the different mammalian isoforms in fatty acid trafficking, the six murine proteins (mmFATP1-6) were expressed and characterized in a genetically defined yeast strain, which cannot transport long-chain fatty acids and has reduced long-chain acyl-CoA synthetase activity (fat1Delta faa1Delta). Each isoform was evaluated for fatty acid transport, fatty acid activation (using C18:1, C20:4, and C24:0 as substrates), and accumulation of very long-chain fatty acids. Murine FATP1, -2, and -4 complemented the defects in fatty acid transport and very long-chain fatty acid activation associated with a deletion of the yeast FAT1 gene; mmFATP3, -5, and -6 did not complement the transport function even though each was localized to the yeast plasma membrane. Both mmFATP3 and -6 activated C20:4 and C20:4, while the expression of mmFATP5 did not substantially increase acyl-CoA synthetases activities using the substrates tested. These data support the conclusion that the different mmFATP isoforms play unique roles in fatty acid trafficking, including the transport of exogenous long-chain fatty acids.     

Inhibition of phospholipase A2 by cis-unsaturated fatty acids: evidence for the binding of fatty acid to enzyme.

Calcium-dependent phospholipases A2 are markedly inhibited in vitro by cis-unsaturated fatty acids (CUFAs) and to a much lesser extent by trans-unsaturated or saturated fatty acids. Thus, CUFAs may function as endogenous suppressors of lipolysis. To better understand the mechanism of inhibition, kinetic analysis, fluorescence spectroscopy and gel permeation chromatography were employed to demonstrate that CUFAs interact with a highly purified Ca(2+)-dependent phospholipase A2 from Naja mossambica mossambica venom. Arachidonate inhibited hydrolysis of both [1-14C]oleate-labelled, autoclaved Escherichia coli and [1-14C]linoleate-labelled phosphatidylethanolamine in an apparent competitive manner. When subjected to gel permeation chromatography, [3H]arachidonate, but not [3H]palmitate, comigrated with the enzyme. Arachidonic and other CUFAs increased the fluorescence intensity of the enzyme almost 2-fold in a dose-dependent fashion (50 microM = 180% of control); methyl arachidonate was without effect. Saturated fatty acids had only a modest effect on enzyme fluorescence (50 microM = 122% of control). Concentrations of arachidonate that inhibited in vitro enzymatic activity by almost 80% did not alter binding of phospholipase A2 to the E. coli substrate. Collectively, these data demonstrate that, while CUFAs selectively bind to the enzyme, they do not influence phospholipase A2-substrate interaction. Inhibition of in vitro phospholipase A2 activity by CUFAs may be mediated by the formation of an enzymatically inactive enzyme-substrate-inhibitor complex.     

Mechanistic studies of the long chain acyl-CoA synthetase Faa1p from Saccharomyces cerevisiae

Long chain acyl-CoA synthetase (ACSL; fatty acid CoA ligase: AMP forming; EC 6.2.1.3) catalyzes the formation of acyl-CoA through a process, which requires fatty acid, ATP and coenzymeA as substrates. In the yeast Saccharomyces cerevisiae the principal ACSL is Faa1p (encoded by the FAA1 gene). The preferred substrates for this enzyme are cis-monounsaturated long chain fatty acids. Our previous work has shown Faa1p is a principal component of a fatty acid transport/activation complex that also includes the fatty acid transport protein Fat1p. In the present work hexameric histidine tagged Faa1p was purified to homogeneity through a two-step process in the presence of 0.1% eta-dodecyl-beta-maltoside following expression at 15 degrees C in Escherichia coli. In order to further define the role of this enzyme in fatty acid transport-coupled activation (vectorial acylation), initial velocity kinetic studies were completed to define the kinetic parameters of Faa1p in response to the different substrates and to define mechanism. These studies showed Faa1p had a Vmax of 158.2 nmol/min/mg protein and a Km of 71.1 microM oleate. When the concentration of oleate was held constant at 50 microM, the Km for CoA and ATP were 18.3 microM and 51.6 microM respectively. These initial velocity studies demonstrated the enzyme mechanism for Faa1p was Bi Uni Uni Bi Ping Pong.     

Arachidonic and cis-unsaturated fatty acids induce selective platelet substrate phosphorylation through activation of cytosolic protein kinase C

The ability of arachidonic acid and other fatty acids to induce phosphorylation of endogenous substrates and the role of protein kinase C in mediating these effects were examined. In a cell-free cytosolic system derived from human platelets, arachidonic, oleic, and other cis-unsaturated fatty acids induced a dose-dependent phosphorylation of several endogenous substrates. These substrates form a subset of phorbol ester-induced phosphorylations. Multiple lines of evidence suggested the direct involvement of protein kinase C in mediating fatty acid-induced phosphorylations. These observations suggest that arachidonic acid and other unsaturated fatty acids are capable of activating protein kinase C in a physiologic environment resulting in the phosphorylation of multiple endogenous substrates.