Fatty acid structural requirements for leukotriene biosynthesis

Utilizing a variety of fatty acids, differing in chain length, degree and position of unsaturation, we investigated the substrate specificity for the enzymatic production of biologically active slow reacting substances (SRS) and of the other leukotrienes. A cellfree enzyme system obtained from RBL-1 cells was used in this study. The primary structural requirement observed for the conversion by this lipoxygenase enzyme system was a Δ^5,8,11 unsaturation in a polyenoic fatty acid. Such fatty acids as 20:4 (5,8,11,14), 20:5 (5,8,11,14,17), 20:3 (5,8,11), 19:4 (5,8,11,14) and 18:4 (5,8,11,14) were readily converted to compounds that comigrated with 5-HETE and 5,12-DiHETE and to biologically active SRS. Chain length did not have an influence on the formation of these hydroxyacids. Fatty acids with the initial unsaturation at Δ⁴, Δ⁶, Δ⁷ or Δ⁸ were a poor substrate for the leukotriene enzyme system. Therefore, this lipoxygenase pathway in leukocytes is quite different from the lipoxygenase in platelets which does not exhibit this specificity.     

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.     

Reticulocyte lipoxygenase exhibits both n-6 and n-9 activities

Purified reticulocyte lipoxygenase converts arachidonic acid to both 15- and 12-hydroxyperoxyeico-satetraenoic acids. The proportion of the two reaction products does not change during the purification procedure as shown by HPLC analysis. By means of isoelectric focusing it was not possible to separate the n-6 and n-9 activities. Reticulocyte lipoxygenase was completely inactivated by both 5,8,11-eicosatriynoic and 5,8,11,14-eicosatetraynoic acids in contrast to soybean lipoxygenase-1 which was inactivated only by 5,8,11,14-eicosatetraynoic acid. These results indicate that reticulocyte lipoxygenase exhibits both n-6 and n-9 activities. A contamination of the enzyme preparation with other lipoxygenases, e.g., the n-9 lipoxygenase from thrombocytes appears to be excluded.     

Slow reacting substance (SRS) from ionophore A23187-stimulated peritoneal mast cells of the normal rat. II. Evidence for a precursor role of arachidonic acid and further purification.

The generation of slow reacting substance (SRS) from ionophore A23187-stimulated rat peritoneal mast cells was enhanced by arachidonic acid (AA). This SRS generation was inhibited by 5,8,11,14-eicosatetraynoic acid (ETYA), an acetylenic analogue of AA and an inhibitor of both fatty acid cyclooxygenase and lipoxygenase. Indomethacin, a fatty acid cyclooxgenase inhibitor, had an enhancing effect upon SRS generation. This suggests SRS generation occurred through an ETYA sensitive step--perhaps a lipoxygenase. Radiolabel from [14C]-AA was incorporated into SRS with comigration of radioactivity and bioreactivity in silicic acid and thin layer chromatographies. Upon silicic acid chromatography, the active principle was eluted in the methanol fraction. Two-dimensional thin layer chromatography revealed chromatographic separation from other known spasmogenic substances and phospholipids. Mast cell SRS was found to display physiochemical properties similar to those of rat basophilic leukemia cell SRS, namely: that mast cell SRS generation was 1) enhanced by arachidonic acid; 2) inhibited by ETYA but not by indomethacin; 3) incorporation of [14C]-AA into the active principle; and 4) similar behavior during purification in silicic acid and thin layer chromatographies.     

Substrate specificity of the agonist-stimulated release of polyunsaturated fatty acids from vascular endothelial cells

Stimulation of vascular endothelial cells with agonists such as histamine and thrombin results in release of arachidonic acid from membrane lipids and subsequent eicosanoid synthesis. As shown previously, the agonist-stimulated deacylation is specific for arachidonate, eicosapentaenoate, and 5,8,11-eicosatrienoate. This study has utilized radiolabeled fatty acids differing in chain length and position of double bonds to further elucidate the fatty acyl specificity of agonist-stimulated deacylation. Replicate wells of confluent human umbilical vein endothelial cells were incubated with 14C-labeled fatty acids and then challenged with histamine, thrombin, or the calcium ionophore A23187. Comparison of the results obtained with isomeric eicosatetraenoic fatty acids with initial double bonds at carbons 4, 5, or 6 indicated that the deacylation induced by all three agonists exhibited marked specificity for the cis-5 double bond. Lack of stringent chain length specificity was indicated by agonist-stimulated release of 5,8,11,14- tetraenoic fatty acids with 18, 19, 20, and 21 carbons. Release of 5,8,14-[14C]eicosatrienoate was two-to threefold that of 5,11,14-[14C]eicosatrienoate, thus indicating that the cis-8 double bond may also contribute to the stringent recognition by the agonist-sensitive phospholipase. The present study has also demonstrated that histamine, thrombin, and A23187 do not stimulate release of docosahexaenoate from endothelial cells.     

Fatty acid structural requirements for activity of arachidonoyl-CoA synthetase

We have examined the fatty acid substrate specificity of arachidonoyl-CoA synthetase from human platelet membranes. A variety of positional isomers and chain-length analogs of arachidonic acid [20:4(5, 8, 11, 14)] were synthesized, and assayed for their ability to inhibit arachidonoyl-CoA formation or to serve as substrates for the synthetase. The chain-length specificity of the synthetase for delta 8,11,14 trienoic fatty acids was C19 greater than C18 = C20 much greater than C21 greater C22. Inhibition activity by positional isomers of arachidonate was 20:4(5, 8, 11, 14) approximately equal to 20:4(6, 9, 12, 15) = 20:4(7, 10, 13, 16) much greater than 20:4(4, 7, 10, 13), however, Vmax for arachidonate was greater than that for 20:4(6, 9, 12, 15). The enzyme apparently "counts" double bonds from the carboxyl terminus. As counted from the methyl terminus we found that several n-6,-9,-12 fatty acids were ineffective as inhibitors [18:3(6, 9, 12); 19:4)4, 7, 10, 13); 21:3(9, 12, 15)], whereas all methylene-interrupted tri- and tetraenoic fatty acids which contained delta 8 and delta 11 double bonds were potent inhibitors. The delta 11 double bond was best associated with optimal inhibition: 20:3(5, 11, 14) had a lower Ki than 20:3(5, 8, 14). 13-Methyl-20:3(8, 11, 14) did not inhibit the enzyme. Partially purified enzyme from calf brain, depleted of nonspecific long-chain acyl-CoA synthetase, exhibited the same fatty acid specificity as crude platelet enzyme.     

Substrate specificity and positional preference of a lipoprotein lipase

The substrate specificity and positional preference of a membrane-bound lipoprotein lipase with and without reconstitution in liposomes were studied. The enzyme showed the same preference for the acyl groups in the 1- and 3-positions of triglycerides, while its activity toward the 2-position was one-third of that toward the 1- or 3-positions. The substrate specificity of the enzyme toward fatty acids of different chain length and degree of unsaturation was in the order of C:14>C:16>C:18:1≥C:18. The enzyme with and without reconstitution showed the same positional preference and substrate specificity.     

Monohydroxylated fatty acid substrate specificity of human leukocyte 5-lipoxygenase and omega-hydroxylase.

Various monohydroxylated fatty acids were synthesized from eicosapolyenoic acids, namely arachidonic (20:4 omega-6), timnodonic (20:5 omega-3), dihomogammalinolenic (20:3 omega-6) and mead (20:3 omega-9) acids. 12-Hydroxy derivatives, as well as 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), were produced with platelets as the enzyme source, and 15-hydroxy derivatives were produced by soya bean lipoxygenase treatment. Each monohydroxylated fatty acid was incubated with human leukocytes in the presence or absence of the calcium ionophore A23187, and dihydroxylated products were analysed by h.p.l.c. 12-Hydroxy derivatives of 20:4 omega-6, 20:5 omega-3 and 20:3 omega-9 were similarly oxygenated by both the 5-lipoxygenase and the omega-hydroxylase. As expected, the 12-hydroxy derivative of 20:3 omega-6 was not a substrate for 5-lipoxygenase, but surprisingly, omega-6 oxygenated products, like 15-OH-20:4 or HHT, were not converted by the enzyme, although being potential substrates because of the presence of two double bonds at C-5 and C-8. omega-6 oxygenated derivatives were also poorly converted by leukotriene B4 omega-hydroxylase, a cytochrome P-450-dependent enzyme. It is concluded that both leukocyte 5-lipoxygenase and omega-hydroxylase exhibit a substrate specificity towards monohydroxylated fatty acids with respect to their double bonds and/or the carbon position of the alcohol function.     

Tumour-necrosis-factor-mediated cytotoxicity is correlated with phospholipase-A2 activity, but not with arachidonic acid release per se.

L929, a murine fibrosarcoma cell line highly sensitive to the anti-proliferative and cytotoxic action of tumour necrosis factor (TNF), was used as a target cell in our studies. We [Suffys et al. (1987) Biochem. Biophys. Res. Commun. 149, 735-743], as well as others, have previously provided evidence that a phospholipase (PL), most probably a PL-A2-type enzyme, is likely to be involved in TNF-mediated cell killing. We now further document this conclusion and provide suggestive evidence that the enzyme activity specifically involved in TNF cytotoxicity differs from activities associated with the eventual cell death process itself or with non-toxic serum treatment. We also show that the 5,8,11,14-icosatetraenoic acid (arachidonic acid, delta 4 Ach) released by PL, and possibly metabolized, is unlikely to be a key mediator of the TNF-mediated cytotoxicity. These conclusions are based on the following experimental findings. 1. TNF treatment of cells, prelabelled for 24 h with [3H] delta 4Ach or [14C] delta 3Ach (delta 3Ach identical to 5,8,11-icosatrienoic acid) resulted in an early, time-dependent and concentration-dependent release of radioactivity in the supernatant preceding actual cell death. The extent of this response was moderate, albeit reproducible and significant. Analysis of the total lipid fraction from cells plus supernatant revealed that only release of arachidonic acid from phospholipids, but not its metabolization was induced by TNF. However, the release of less unsaturated fatty acids, such as linoleic acid (Lin) or palmitic acid (Pam), was not affected during the first hours after TNF addition. 2. An L929 subclone, selected for resistance to TNF toxicity, was found to be defective in TNF-induced delta 4Ach libration. 3. Interleukin-1 (IL1) was not cytotoxic for L929 and did not induce release of delta 4Ach. 4. Release of delta 4Ach was not restricted to TNF; the addition of serum to the cells also induced release of fatty acids into the medium. In this case, however, there was no specificity, as all fatty acids tested, including Lin and Pam, were released. 5. Inhibition of PL-A2 activity by appropriate drugs markedly diminished TNF-induced delta 4Ach release and resulted also in a strong decrease in TNF-induced cytotoxicity. 6. Other drugs, including serine protease inhibitors, which strongly inhibit TNF-induced cytotoxicity, also decreased the TNF-induced delta 4Ach release, whereas LiCl potentiated both TNF-mediated effects. 7. Protection of cells against TNF toxicity by means of various inhibitors was not counteracted by addition of exogenous fatty acids, including delta 4Ach.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stereospecific induction of starfish oocyte maturation by (8R)-hydroxyeicosatetraenoic acid

Oocyte maturation (meiosis reinitiation) in starfish is induced by the natural hormone 1-methyladenine. This induction of meiotic divisions can be triggered also by four fatty acids: 5,8,11-20:3; 5,8,11,14-20:4 (arachidonic acid); 6,9,12,15-20:4; 5,8,11,14,17-20:5, all other fatty acids being completely inactive. This maturation triggered by eicosanoids occurs in the micromolar range and is facilitated by the presence of calcium. A variety of arachidonic acid derivatives (esters, epoxides, etc.) and metabolites (cyclooxygenase and lipoxygenase products) has been tested; the biological activity is restricted to 8-hydroxyeicosatetraenoic acid (8-HETE), other mono- and poly-HETEs being completely inactive. Maturation triggered by 8-HETE occurs around 10 nM and is insensitive to the presence of calcium. 8-HETE methyl ester and 8-hydroperoxyeicosatetraenoic acid are able to induce maturation at higher concentrations. Both (8S) and (8R) stereoisomers have been tested; the biological activity is strictly restricted to the (8R) isomer. 8-HETE triggers a complete maturation, i.e. maturation-promoting factor appearance, germinal vesicle breakdown, emission of the polar bodies, and formation of a female pronucleus. (8R)-HETE, but not (8S)-HETE, triggers the typical decrease in cyclic AMP concentration induced by 1-methyladenine and the burst of protein phosphorylation associated with maturation. Starfish oocytes oxidize exogenous arachidonic acid into 8-HETE and other HETEs. 8-HETE was identified, after high pressure liquid chromatography purification, by gas chromatography mass spectrometry. Furthermore, it was found that the starfish oocytes only produce the (8R)-HETE isomer. This highly stereospecific induction of oocyte maturation by (8R)-HETE suggests that this fatty acid, or a very closely related fatty acid, may play a role in the transduction of the 1-methyladenine message at the plasma membrane level.     

Metabolism of polyunsaturated fatty acids by rabbit reticulocytes

Rabbit reticulocytes obtained by repeated bleeding metabolize exogenous [1-14C]linoleic acid and [1-14C]arachidonic acid by three different pathways. 1. Incorporation into cellular lipids: 50% of the fatty acids metabolized are incorporated into phospholipids, mainly phosphatidylcholine (32.8%) but also into phosphatidylethanolamine (12%), whereas about 10% of the radioactivity was found in the neutral lipids (mono- di- and triacylglycerols, but not cholesterol esters). 2. Formation of lipoxygenase products: 30% of the fatty acids metabolized are converted via the lipoxygenase pathway mainly to hydroxy fatty acids. Their formation is strongly inhibited by lipoxygenase inhibitors such as 5,8,11,14-eicosatetraynoic acid or nordihydroguaiaretic acid. Inhibition of the lipoxygenase pathway results in an increase of the incorporation of the fatty acids into cellular lipids. 15-Hydroxy-5,8,11,13(Z,Z,Z,E)eicosatetraenoic acid and 13-hydroxy-9,11(Z,E)-octadecadienoic acid are incorporated by reticulocytes into cellular lipids and also are metabolized via beta-oxidation. The metabolism of arachidonic acid and linoleic acid is very similar except for a higher incorporation of linoleic acid into neutral lipids. 3. beta-Oxidation of the exogenous fatty acids: about 10% of the polyenoic fatty acids are metabolized via beta-oxidation to 14CO2. Addition of 5,8,11,14-eicosatetraynoic acid strongly increased the 14CO2 formation from the polyenoic fatty acids whereas antimycin A completely abolished beta-oxidation. Erythrocytes show very little incorporation of unsaturated fatty acids into phospholipids and neutral lipids. Without addition of calcium and ionophore A23187 lipoxygenase metabolites could not be detected.     

少根根霉Δ^6-脂肪酸脱氢酶基因在毕赤酵母中的表达

Δ^6-脂肪酸脱氢酶是一种膜整合蛋白,也是多不饱和脂肪酸合成途径中的限速酶。在前期工作中,通过RT-PCR和RACE技术,从少根根霉NK300037中克隆到一个潜在编码Δ^6-脂肪酸脱氢酶的序列,序列和功能分析结果表明该序列具有一个长度为1377bp、编码由458个氨基酸组成、大小为52kD的新的Δ^6-肪酸脱氢酶基因。把少根根霉Δ^6-脂肪酸脱氢酶基因（RAD6）亚克隆到表达载体pPIC3．5K,构建重组表达载体pPICRAD6,并转化到毕赤酵母菌株GS115进行表达。提取酵母细胞总脂肪酸和进行甲酯化,经气相色谱和气相色谱-质谱连用分析表明,目的基因的编码产物能将C16：1、C17：1、C18：1、亚油酸和α-亚麻酸在△6和7位间特异性脱氢而引入一个新的双键,生成更高不饱和的脂肪酸,该催化反应没有链长特异性,只有键位特异性。此外,按Kozak序列特点,改变目的基因转译起始密码子周边序列结构,并把改变后序列导入毕赤酵母GS115中进行功能表达分析,结果表明在毕赤酵母中这种改变同样能提高目的基因的表达水平。综合所有分析结果表明,巴斯德毕赤酵母更适合用来综合分析Δ^6-脂肪酸脱氢酶基因的功能。     

Substrate specificity for the synthesis of cyclic Fatty acids by a flaxseed extract

12-Oxo-cis-10,15-phytodienoic acid is an enzymic product obtained from incubations of (9, 12, 15)-linolenic acid with extracts of flaxseed (Linum usitatissimum L.). 13-l-Hydroperoxy-cis-9, 15-trans-11-octadecatrienoic acid, a product of lipoxygenase catalysis, was an intermediate in the enzymic synthesis of 12-oxo-cis-10, 15-phytodienoic acid from (9, 12, 15)-linolenic acid. Substrate specificity studies showed that n-3,6,9 unsaturation was an absolute requirement for conversion of polyunsaturated fatty acids into analogous products containing a cyclopentenone ring. Fatty acids with 18, 20, or 22 carbons that satisfied this requirement were effective substrates. The optimum activity of the enzyme from flaxseed was at pH 7.2.     

A unique cardiac cytosolic acyltransferase with preferential selectivity for fatty acids that form cyclooxygenase/lipoxygenase metabolites and reverse essential fatty acid deficiency

The rabbit heart contains a cytosolic enzyme which selectively incorporates polyunsaturated fatty acids into phosphatidylcholine. This unique acyltransferase is selective for fatty acids, thus far tested, that are substrates for cyclooxygenase or lipoxygenase (i.e., arachidonic, eicosapentaenoic, linoleic and dihomo-gamma-linoleic acids) or which reverse the symptoms of essential fatty acid deficiency (columbinic acid). On the other hand, palmitic, oleic, 5,8,11-eicosatrienoic (n-9, Mead acid), and docosatetraenoic acid (n-6, adrenic acid) were not incorporated in phospholipids by the cytosolic acyltransferase. No such fatty acid selectivity was exhibited by the cytosolic acyl-CoA synthetase or by the acyltransferase activities present in cardiac microsomes and mitochondria.     

Fatty acyl CoA synthetase from Antarctic notothenioid fishes may influence substrate specificity of fat oxidation

Antarctic notothenioid fishes possess large lipid stores that are important fuels for aerobic metabolism. Oxidative muscle tissues of these animals oxidize long-chain mono-unsaturated fatty acids more readily than saturated fatty acids. The mechanistic basis(es) for the substrate specificity of their fatty acid-oxidizing pathway is unknown. We examined the substrate specificity of fatty acyl coenzyme A synthetase (FACS) to determine whether the enzyme contributes to targeting unsaturated fatty acids for preferential transport into mitochondria as fuels for beta-oxidation. Maximal activities of FACS were measured in isolated mitochondria from Notothenia coriiceps and Chaenocephalus aceratus oxidative skeletal muscles in the presence of fatty acids differing in chain lengths and degrees of unsaturation. With the exception of C(22:6), maximal activities were greater with unsaturated substrates than with C(16:0), a saturated fatty acid. Monoenoic fatty acids did not produce the highest activities. Predicted amino acid sequences of FACS from Antarctic C. aceratus, Gobionotothen gibberifrons, and N. coriiceps and sub-Antarctic Notothenia angustata and Eleginops maclovinus were determined to identify amino acid candidates that may be important for determining the substrate specificity of FACS. Substitutions cysteine548 and polar threonine552 within the putative fatty acid binding pocket may contribute to preference for unsaturated fatty acyl substrates compared to saturated fatty acids.     

Role of lipoxygenase in the O2-dependent activation of soluble guanylate cyclase from rat lung.

Guanylate cyclase activity in rat lung supernatant fractions is stimulated 3-4 fold by aerobic incubation at 30 degrees C for approx. 30 min ('O2-dependent activation'). This stimulation was blocked by 20 microM-eicosa-5,8,11,14-tetraynoic acid (ETYA), an inhibitor of lipoxygenase and cyclo-oxygenase, but not by aspirin or indomethacin, which are cyclo-oxygenase inhibitors. The enzyme activator(s) is presumed to be the fatty acid hydroperoxide(s) formed by lipoxygenase. Removal of lipoxygenase from the supernatant fraction by chromatography on Amberlite XAD-4 also prevented activation, which was restored by the addition of soya-bean lipoxygenase. Bovine serum albumin prevented O2-dependent activation or activation by soya-bean lipoxygenase, through its ability to bind the unsaturated fatty acid substrate of lipoxygenase. The lipoxygenase in the supernatant fraction is inhibited by endogenous glutathione peroxidase plus reduced glutathione (GSH); removal of GSH de-inhibits lipoxygenase and activates guanylate cyclase. This was effected by autoxidation, by cumene hydroperoxide (with GSH peroxidase) and by titration with N-ethylmaleimide (NEM). Activation by NEM was inhibited by serum albumin or ETYA, as was activation by low concentrations (less than 50 microM) of cumene hydroperoxide. Activation by higher concentrations was not so inhibited; therefore, cumene hydroperoxide can also activate by a direct effect on guanylate cyclase. A hypothesis for physiological activation is proposed.     

Studies on polyenoic acid incorporation into human platelet lipid stores: interactions with linoleic and arachidonic acids

Several polyunsaturated fatty acids (C18-C22 acids) have been compared in their uptake by human platelets and their acylation into glycerophospholipid subclasses. This was also studied in the presence of linoleic and/or arachidonic acids, the main fatty acids of plasma free fatty acid pool. Amongst C20 fatty acids, dihomogamma linolenic acid (20:3(n-6)), 5,8,11-icosatrienoic acid (20:3(n-9)) and arachidonic acid (20:4(n-6)) were better incorporated. The uptake of 5,8,11,14,17-icosapentaenoic acid (20:5(n-3)) was significantly lower and comparable to that of C22 fatty acids (7,10,13,16-docosatetraenoic acid (22:4(n-6)) and 4,7,10,13,16,19-docosahexaenoic acid (22:6(n-3)) and linoleic acid (18:2(n-6)). In this respect, linolenic acid (18:3(n-3)) appeared the poorest substrate. The bulk of each acid was acylated into glycerophospholipids although the presence of linoleic and/or arachidonic acids diverted a part towards neutral lipids. This was prominent for 18:3(n-3) and C22 fatty acids. The glycerophospholipid distribution of each acid differed substantially and was not affected by the presence of linoleic and or arachidonic acids, except for 18:3(n-3) and 22:6(n-3) that were strongly diverted towards phosphatidylethanolamine (PE) at the expense of phosphatidylcholine (PC). The main features were an efficient acylation of 20:3(n-9) into phosphatidylinositol (PI) followed by 20:3(n-6) and 20:4(n-6), then by 20:5(n-3) and 22:4(n-6), and finally 22:6(n-3) and C18 fatty acids. This was reciprocal to the acylation into PE and to a lesser extent into PC which remained the main storage species in all cases. We conclude that human platelets may exhibit a certain specificity for taking up polyunsaturated fatty acids both in terms of total uptake and glycerophospholipid subclass distribution. Also the presence of polyunsaturated fatty acids of normal plasma, like linoleic and arachidonic acids, may interact specifically with such an uptake and distribution.     

Is ATP a substrate for 15-lipoxygenase?

Lipoxygenases catalyze peroxidation of polyunsaturated fatty acids containing the 1-cis, 4-cis pentadiene structure. Linoleic (18:2), linolenic (18:3), and arachidonic (20:4) acids are the predominant substrates for this class of enzymes. Effects of 15-lipoxygenase on the hydrolysis of adenosine 5'-triphosphate were investigated in vitro using soybean lipoxygenase and adenosine 5'-[gamma-32P]triphosphate. The amount of inorganic phosphate released from adenosine 5'-triphosphate was dependent upon enzyme as well as substrate concentrations, pH, and the duration of incubation. The ATPase activity with a Vmax value of 3.3 mumol.mg protein-1.h-1 and a Km value of 5.9 mM was noted in the presence of different concentrations of ATP at pH = 7.4. Phenidone, a lipoxygenase inhibitor, had no effect on this reaction. These findings suggest that soybean lipoxygenase catalyzes the release of inorganic phosphate from ATP primarily via hydrolysis.     

Fatty acid composition and lipoxygenase metabolism in blood cells of the lesser spotted dogfish, Scyliorhinus canicula.

1. The fatty acid composition of erythrocytes and leucocytes of the elasmobranch, Scyliorhinus canicula, was determined so as to indicate substrate availability for eicosanoid formation. 2. Leucocytes showed a greater degree of fatty acid unsaturation than the erythrocytes, with particularly high levels of docosahexaenoic acid (22:6,n-3). 3. The major eicosanoid precursors, arachidonic acid (20:4,n-6) and eicosapentaenoic acid (20:5,n-3), represented 13.9% and 5.2% of the total fatty acid, respectively, in erythrocytes compared with 10.7% and 6% in leucocytes. 4. Whole blood and isolated leucocytes were stimulated with calcium ionophore, A23187 and the resulting lipoxygenase products separated by reverse phase high performance liquid chromatography. 5. The main lipoxygenase products formed were 6-trans-leukotriene B4, 6-trans-12-epi-leukotriene B4, 5(S),6(R) dihydroxyeicosatetraenoic acid and 5- and 15-hydroxyeicosatetraenoic acid. 6. No leukotriene B4, leukotriene B5, or lipoxins were detected.     

Arachidonic acid-related elicitors of the hypersensitive response in potato and enhancement of their activities by glucans from Phytophthora infestans (Mont.) deBary

The dose response for elicitation of the hypersensitive reaction in potato tuber discs by arachidonic acid (AA) suggested saturation at higher concentrations. Glucans from Phytophthora infestans, inactive themselves as elicitors of the hypersensitive reaction, enhanced sesquiterpene accumulation and hypersensitive browning elicited by AA. Significant activity (seven times control values) was observed with 33 pmol AA/3.0-cm potato disc in the presence of glucans. Glucans did not affect accumulation of steroid glycoalkaloids, influence the timing or relative amounts of sesquiterpenes which accumulate, or affect recovery of AA added to potato discs. Glucans enhanced activity whether added to potato discs 18 h prior to AA, at the same time as AA, or 18 h after AA. Elicitor activity in the presence of glucans was evident with 20-carbon unsaturated fatty acids that had little or no elicitor activity in the absence of glucans. The position of double bonds had considerable influence on the specific activity of unsaturated fatty acids. The most active had a minimum of three double bonds in a methylene-interrupted series beginning with delta 5, e.g., delta 5,8,11. A delta 5 double bond conferred significant activity even if it was not part of a methylene-interrupted series. The 20-carbon chain length appeared optimal for elicitor activity. The 22-carbon chain acids had low activity, and 16- and 18-carbon acids were inactive. A free carboxyl group or easily transesterified group appeared necessary for activity. Arachidonyl alcohol had very low activity and arachidonyl cyanide was inactive. AA-containing phosphatidylcholine, lysophosphatidylcholine and monoacylglycerol were at least as active as free AA, AA-containing diacylglycerols were slightly less active than free AA, and triarachidonyl glycerol was inactive.