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J R Falck

Publications and source records attributed to J R Falck.

At least 127 records · Page 7Linked to original sources
PubMed1998

Structure-activity analysis of the interaction of curacin A, the potent colchicine site antimitotic agent, with tubulin and effects of analogs on the growth of MCF-7 breast cancer cells.

Originally purified as a major lipid component of a strain of the cyanobacterium Lyngbya majuscula isolated in Curaçao, curacin A is a potent inhibitor of cell growth and mitosis, binding rapidly and tightly at the colchicine site of tubulin. Because its molecular structure differs so greatly from that of colchicine and other colchicine site inhibitors, we prepared a series of curacin A analogs to determine the important structural features of the molecule. These modifications include reduction and E-to-Z transitions of the olefinic bonds in the 14-carbon side chain of the molecule; disruption of and configurational changes in the cyclopropyl moiety; disruption, oxidation, and configurational reversal in the thiazoline moiety; configurational reversal and substituent modifications at C13; and demethylation at C10. Inhibitory effects on tubulin assembly, the binding of colchicine to tubulin, and the growth of MCF-7 human breast carcinoma cells were examined. The most important portions of curacin A required for its interaction with tubulin seem to be the thiazoline ring and the side chain at least through C4, the portion of the side chain including the C9-C10 olefinic bond, and the C10 methyl group. Only two modifications totally eliminated the tubulin-drug interaction. The inactive compounds were a segment containing most of the side chain, including its two substituents, and analogs in which the methyl group at the C13 oxygen atom was replaced by a benzoate residue. Antiproliferative activity comparable with that observed with curacin A was only reproduced in compounds that were potent inhibitors of the binding of colchicine to tubulin. Molecular modeling and quantitative structure-activity relationship studies demonstrated that most active analogs overlapped extensively with curacin A but failed to provide an explanation for the apparent structural analogy between curacin A and colchicine.

Animals↗
PubMed1998

Human platelets and polymorphonuclear leukocytes synthesize oxygenated derivatives of arachidonylethanolamide (anandamide): their affinities for cannabinoid receptors and pathways of inactivation.

Arachidonylethanolamide (AEA), the putative endogenous ligand of the cannabinoid receptor, has been shown to be a substrate for lipoxygenase enzymes in vitro. One goal of this study was to determine whether lipoxygenase-rich cells metabolize AEA. [14C]AEA was converted by human polymorphonuclear leukocytes (PMNs) to two major metabolites that comigrated with synthetic 12(S)- and 15(S)-hydroxy-arachidonylethanolamide (HAEA). Human platelets convert [14C]AEA to 12(S)-HAEA. 12(S)-HAEA binds to both CB1 and CB2 receptors with approximately the same affinity as AEA. 12(R)-HAEA, which is not produced by PMNs, has 2-fold lower affinity for the CB1 receptor and 10-fold lower affinity for the CB2 receptor than 12(S)-HAEA. 15-HAEA has a lower affinity than AEA for both receptors, with Ki values of 738 and >1000 nM for CB1 and CB2 receptors, respectively. The addition of a hydroxyl group at C20 of AEA resulted in a ligand with the same affinity for the CB1 receptor but a 4-fold lower affinity for the CB2 receptor than AEA. 12(S)-HAEA and 15-HAEA are poor substrates for AEA amidohydrolase and do not bind to the AEA uptake carrier. In conclusion, the addition of a hydroxyl group at C12 of the arachidonate backbone of AEA does not affect binding to CB receptors but is likely to increase its half-life. The addition of hydroxyl groups at other positions affects ligand affinity for CB receptors; both the position of the hydroxyl group and the configuration of the remaining double bonds are determinants of affinity.

Amidohydrolases↗
PubMed1998

Contribution of 20-HETE to the vasodilator actions of nitric oxide in renal arteries.

The present study examined the contribution of elevations in cGMP versus inhibition of cytochrome P-4504A enzymes and the production of the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE) to the vasodilator actions of NO in renal arterioles. The NO donor sodium nitroprusside (SNP) at 10(-5), 10(-4), and 10(-3) M reduced the production of 20-HETE in microsomes prepared from renal arterioles to 80 +/- 2, 43 +/- 5, and 7 +/- 1% of control, respectively (n = 4). In other experiments, the vasodilator response to SNP (10(-7) to 10(-3) M) was examined in rat renal interlobular arteries (<90 micron ID), preconstricted with phenylephrine (1 microM) under control conditions and after blockade of the cGMP and P-4504A pathways. Inhibition of guanylyl cyclase with 1H-[1,2, 4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) (10 microM, n = 6) or of cGMP-dependent protein kinase with 8R,9S, 11S-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cycloocta-(c, d, e)-trinden-1-one (KT-5823, 1 microM; n = 5) attenuated the vasodilator response to SNP by 26 and 30%, respectively. In contrast, inhibition of the endogenous production of 20-HETE with a suicide substrate, irreversible inhibitor [17-octadecynoic acid (17-ODYA), 1 microM, n = 5], or a selective, competitive inhibitor of 20-HETE formation (dibromo-dodecenyl-methylsulfimide, 25 microM, n = 5) markedly impaired the vasodilator response to SNP by 76 and 78%, respectively. Similarly, when 20-HETE levels were fixed at 100 nM (n = 6), the response to SNP was attenuated by 73%. Blockade of both pathways with ODQ and 17-ODYA completely abolished the response to SNP (n = 6). These results indicate that the vasodilator response to NO is largely cGMP independent and that inhibition of 20-HETE formation contributes to the cGMP-independent effects of NO in the renal microcirculation.

Alkaloids↗
PubMed1998

20-HETE relaxes bovine coronary arteries through the release of prostacyclin.

Neutrophils respond to ischemic injury by infiltrating the myocardium via the vascular wall. During this process, neutrophils are activated and release inflammatory mediators. Some of these mediators are metabolites of arachidonic acid. We have reported that neutrophils metabolize arachidonic acid to 20-HETE, a cytochrome P450 metabolite. We investigated the effects of 20-HETE on coronary vascular tone by examining 20-HETE-induced changes in isometric tension in bovine coronary artery rings precontracted with the thromboxane-mimetic, U46619. 20-HETE relaxed precontracted coronary rings in a concentration-dependent manner (EC50 of 3 x 10(-7) mol/L). Pretreatment with indomethacin, a cyclooxygenase inhibitor, shifted the concentration-response curve to the right (EC50 of 1 x 10(-6) mol/L); maximal relaxations were not affected. This suggested that 20-HETE-induced relaxations were, in part, dependent on the cyclooxygenase pathway. Relaxations to 20-HETE were not significantly changed in endothelium-denuded rings. To determine whether metabolism of 20-HETE to a vasoactive compound might explain the relaxations caused by 20-HETE, rings of coronary artery were incubated with [3H] 20-HETE. The incubation buffer was extracted and the [3H] products resolved on reverse-phase HPLC. Both denuded and intact arteries failed to metabolize [3H] 20-HETE. To investigate whether 20-HETE-induced relaxations were related to release of prostacyclin, we measured the release of 6-keto PGF1alpha, the stable metabolite of prostacyclin, from bovine coronary arteries. 20-HETE (1 x 10(-6) mol/L) stimulated an increase in 6-keto PGF1alpha in intact vessels (908 +/- 138 pg/mL versus 1402 +/- 157 pg/mL, basal versus stimulated). Thus, 20-HETE-induced relaxations are due, in part, to the stimulation of the release of the dilatory prostanoid, prostacyclin.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1998

Cytochrome P450-derived arachidonic acid metabolism in the rat kidney: characterization of selective inhibitors.

We characterized the inhibitory activity of several acetylenic and olefinic compounds on cytochrome P450 (CYP)-derived arachidonic acid omega-hydroxylation and epoxidation using rat renal cortical microsomes and recombinant CYP proteins. Among the acetylenic compounds, 6-(2-propargyloxyphenyl)hexanoic acid (PPOH) and N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide were found to be potent and selective inhibitors of microsomal epoxidation with IC50 values of 9 and 13 microM, respectively. On the other hand, 17-octadecynoic acid inhibited both omega-hydroxylation and epoxidation of arachidonic acid with IC50 values of 7 and 5 microM, respectively. The olefinic compounds N-methylsulfonyl-12, 12-dibromododec-11-enamide (DDMS) and 12, 12-dibromododec-11-enoic acid (DBDD) exhibited a high degree of selectivity inhibiting microsomal omega-hydroxylation with an IC50 value of 2 microM, whereas the IC50 values for epoxidation were 60 and 51 microM for DDMS and DBDD, respectively. Studies using recombinant rat CYP4A isoforms showed that PPOH caused a concentration-dependent inhibition of omega-hydroxylation and 11, 12-epoxidation by CYP4A3 or CYP4A2 but had no effect on CYP4A1-catalyzed omega-hydroxylase activity. On the other hand, DDMS inhibited both CYP4A1- and CYP4A3- or CYP4A2-catalyzed arachidonic acid oxidations. Inhibition of microsomal activity by PPOH, but not DDMS, was time- and NADPH-dependent, a result characteristic of a mechanism-based irreversible inhibitor. These studies provide information useful for evaluating the role of the CYP-derived arachidonic acid metabolites in the regulation of renal function and blood pressure.

Animals↗
PubMed1998

Epoxygenase metabolites of arachidonic acid affect electrophysiologic properties of rat tracheal epithelial cells1.

Epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids, products of the cytochrome P450 arachidonic acid epoxygenase pathway, have been shown to affect electrolyte transport in the kidney; however, the effects of these compounds on airway epithelial ion transport have not been investigated. Intact rat tracheas and primary cultures of rat tracheal epithelial cells were mounted in Ussing chambers to monitor changes in transepithelial voltage (Vt), short circuit current (Isc) and electrical resistance (Rt), with or without the addition of increasing concentrations (10(-9)-10(-6) M) of arachidonic acid, each of the four regioisomeric EETs and each of the corresponding dihydroxyeicosatrienoic acids. In intact tracheas, 11,12-EET caused dose-dependent decreases in Vt and Isc (DeltaVt = 0. 4 +/- 0.1 mV, DeltaIsc = -16.9 +/- 5.4 microA/cm2 at 10(-6) M, P < . 05 vs. vehicle), whereas changes in Rt were not significantly different than vehicle alone. 11,12-dihydroxyeicosatrienoic acid caused less impressive decreases in Vt and Isc, although arachidonic acid and the other compounds tested were without significant effects. 11,12-EET induced similar changes in cultured tracheal epithelial cell electrical parameters at concentrations as low as 10(-9) M. The effects of 11,12-EET were highly stereoselective, with activity limited to 11(R),12(S)-EET, the least abundant rat lung enantiomer. Pretreatment with amiloride or mucosal exposure to sodium free media did not significantly alter the 11,12-EET-induced changes in Vt. In contrast, pretreatment with bumetanide abolished the 11,12-EET electrophysiologic effects, suggesting that these effects may be mediated through inhibition of a chloride conductive pathway. We conclude that arachidonic acid epoxygenase metabolites cause significant changes in rat airway electrical parameters and may be involved in the control of lung fluid and electrolyte transport.

8,11,14-Eicosatrienoic Acid↗
PubMed1998

A method for the determination of 5,6-EET using the lactone as an intermediate in the formation of the diol.

The 5,6 epoxyeicosatrienoic acid (5,6-EET) exhibits a range of biological activities but the functional significance of this labile eicosanoid is unknown due, in part, to difficulties of quantitation in biological samples. We have developed a sensitive and specific method to measure 5,6-EET utilizing its selective capacity to form a lactone. The initial conversion of 5,6-EET and 5,6-dihydroxyeicosatrienoic acid (5,6-DHT) to 5,6-delta-lactone is followed by selective purification using reverse phase high performance liquid chromatography (HPLC), reconversion to 5,6-DHT and quantitation by gas chromatography-mass spectrometry (GCMS). In oxygenated Krebs' buffer, 5,6-EET degrades to 5,6-delta-lactone and 5,6-DHT with a t1/2 approximately 8 min. In the presence of camphorsulfonic acid, 5,6-EET and 5,6-DHT convert to a single HPLC peak (lambda = 205) comigrating with 5,6-delta-lactone. Incubation of 5,6-delta-lactone with triethylamine resulted in a single HPLC peak with the retention time of 5,6-DHT. In the perfusate from the isolated kidney, release of 5,6-EET (20 +/- 5 pg/ml), measured indirectly via conversion to 5,6-DHT, was approx. 6-fold less than that reported for prostaglandin E2 (PGE2) and 20-HETE. The coronary perfusate concentration of 5,6 EET was 9 +/- 2 pg/ml. 5,6-EET recovered from renal and coronary perfusates was increased 2-fold to 45.5 +/- 5.5 pg/ml and 21.6 +/- 6.3 pg/ml, respectively, by arachidonic acid.

8,11,14-Eicosatrienoic Acid↗
PubMed1998

Hypoxia stimulates the synthesis of cytochrome P450-derived inflammatory eicosanoids in rabbit corneal epithelium.

The corneal epithelium metabolizes arachidonic acid by a cytochrome P450-(CYP) mediated pathway to 12(R)hydroxy-5,8,10,14-eicosatrienoic acid [12(R)-HETE] and 12(R)hydroxy-5,8,14-eicosatrienoic acid [12(R)-HETrE]. Both metabolites possess potent inflammatory properties with 12(R)-HETrE being a powerful angiogenic factor and assume the role of inflammatory mediators in hypoxia- and chemical-induced injury in the cornea, in vivo. We developed an in vitro model of corneal organ culture to characterize the biochemical and molecular events involved in the increased synthesis of these metabolites. These cultured corneas exhibit epithelial cytochrome P450 CYP-dependent 12(R)-HETE and 12(R)-HETrE synthesis as indicated by chiral analysis and by the ability of CYP enzyme inhibitors to repress their synthesis. Hypoxia greatly and selectively stimulated the synthesis of 12(R)-HETE (7-fold over control normoxic conditions) and 12(R)-HETrE. The bacterial endotoxin, lipopolysaccharide, also increased the synthesis of these eicosanoids, substantiating the notion that this activity may function as an inflammatory pathway. These metabolites were detected in the culture medium by gas chromatography/mass spectroscopy (GC/MS) analysis and their levels significantly increased in hypoxia-treated corneas, further indicating their endogenous formation in response to injury. This in vitro model provides an excellent preparation for studying factors regulating the synthesis of these inflammatory eicosanoids and for isolating, identifying and characterizing the CYP protein responsible for their synthesis.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1997

Molecular cloning, expression, and functional significance of a cytochrome P450 highly expressed in rat heart myocytes.

A cDNA encoding a P450 monooxygenase was amplified from reverse transcribed rat heart and liver total RNA by polymerase chain reaction using primers based on the 5'- and 3'-end sequences of two rat pseudogenes, CYP2J3P1 and CYP2J3P2. Sequence analysis revealed that this 1,778-base pair cDNA contained an open reading frame and encoded a new 502 amino acid protein designated CYP2J3. Based on the deduced amino acid sequence, CYP2J3 was approximately 70% homologous to both human CYP2J2 and rabbit CYP2J1. Recombinant CYP2J3 protein was co-expressed with NADPH-cytochrome P450 oxidoreductase in Sf9 insect cells using a baculovirus expression system. Microsomal fractions of CYP2J3/NADPH-cytochrome P450 oxidoreductase-transfected cells metabolized arachidonic acid to 14,15-, 11,12-, and 8, 9-epoxyeicosatrienoic acids and 19-hydroxyeicosatetraenoic acid as the principal reaction products (catalytic turnover, 0.2 nmol of product/nmol of cytochrome P450/min at 37 degrees C). Immunoblotting of microsomal fractions prepared from rat tissues using a polyclonal antibody raised against recombinant CYP2J2 that cross-reacted with CYP2J3 but not with other known rat P450s demonstrated abundant expression of CYP2J3 protein in heart and liver. Immunohistochemical staining of formalin-fixed paraffin-embedded rat heart tissue sections using the anti-CYP2J2 IgG and avidin-biotin-peroxidase detection localized expression of CYP2J3 primarily to atrial and ventricular myocytes. In an isolated-perfused rat heart model, 20 min of global ischemia followed by 40 min of reflow resulted in recovery of only 44 +/- 6% of base-line contractile function. The addition of 5 microM 11, 12-epoxyeicosatrienoic acid to the perfusate prior to global ischemia resulted in a significant 1.6-fold improvement in recovery of cardiac contractility (69 +/- 5% of base line, p = 0.01 versus vehicle alone). Importantly, neither 14,15-epoxyeicosatrienoic acid nor 19-hydroxyeicosatetraenoic acid significantly improved functional recovery following global ischemia, demonstrating the specificity of the biological effect for the 11, 12-epoxyeicosatrienoic acid regioisomer. Based on these data, we conclude that (a) CYP2J3 is one of the predominant enzymes responsible for the oxidation of endogenous arachidonic acid pools in rat heart myocytes and (b) 11,12-epoxyeicosatrienoic acid may play an important functional role in the response of the heart to ischemia.

8,11,14-Eicosatrienoic Acid↗
PubMed1997

High affinity binding of inositol phosphates and phosphoinositides to the pleckstrin homology domain of RAC/protein kinase B and their influence on kinase activity.

The influence of inositol phosphates and phosphoinositides on the alpha isoform of the RAC-protein kinase B (RAC/PKB) was studied using purified wild type and mutant kinase preparations and a recombinant pleckstrin homology (PH) domain. Binding of inositol phosphates and phosphoinositides to the PH domain was measured as the quenching of intrinsic tryptophan fluorescence. Inositol phosphates and D3-phosphorylated phosphoinositides bound with affinities of 1-10 microM and 0.5 microM, respectively. Similar values were obtained using RAC/PKB expressed and purified from baculovirus-infected Sf9 cells in the fluorescence assay. The influence of synthetic dioctanoyl derivatives of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate on the activity of RAC/PKB purified from transfected COS-1 cells was studied. Phosphatidylinositol 3,4,5-trisphosphate was found to inhibit the RAC/PKB kinase activity with half-maximal inhibition at 2.5 microM. In contrast, phosphatidylinositol 3, 4-bisphosphate stimulated kinase activity (half-maximal stimulation at 2.5 microM). A mutant RAC/PKB protein lacking the PH domain was not affected by D3-phosphorylated phosphoinositides. These results demonstrate that the PH domain of RAC/PKB binds inositol phosphates and phosphoinositides with high affinity, and suggest that the products of the phosphatidylinositide 3-kinase can act as both a membrane anchor and modulator of RAC/PKB activity. The data also provide further evidence for a link between phosphatidylinositide 3-kinase and RAC/PKB regulation.

Amino Acid Sequence↗
PubMed1997

Determination of 14,15-epoxyeicosatrienoic acid and 14,15-dihydroxyeicosatrienoic acid by fluoroimmunoassay.

A fluoroimmunoassay (FIA) for 14,15-epoxyeicosatrienoic acid (14,15-EET) and 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), cytochrome P450 epoxygenase products of arachidonic acid, was developed using fluorescence polarization. 14-15-EET was hydrolyzed and analyzed as 14,15-DHET. 14,15-DHET was conjugated to thyroglobulin and a specific antibody was raised in rabbits. Both [3H8]14,15-DHET in radioimmunoassay or fluorescein-labeled 14,15-DHET (14, 15-DHET*) in FIA bound to this antibody and were competitively displaced by 14,15-DHET. The binding activity and cross-reactivity of 14,15-DHET antibody were also studied by RIA compared to FIA. The antibody cross-reacted < or = 1% with 11,12-DHET and 14,15-EET and < 0.1% with other regioisomeric DHETs and arachidonic acid metabolites. The detection limit of 14,15-DHET was 2 pg/0.6 ml by FIA. Using this method, we found that A23187 stimulated the production of 14,15-EET by endothelial cells by angiotensin II stimulated 14,15-EET release from zona glomerulosa cells. The production of 14,15-EET in these samples was confirmed by gas chromatography/mass spectrometry. These studies demonstrate a sensitive and specific FIA for 14,15-EET and 14,15-DHET and that agonists stimulate the release of these eicosanoids in two cell types, bovine coronary artery endothelial cells and bovine zona glomerulosa cells.

8,11,14-Eicosatrienoic Acid↗
PubMed1997

Regulation of AP-3 function by inositides. Identification of phosphatidylinositol 3,4,5-trisphosphate as a potent ligand.

As part of the growing effort to understand the role inositol phosphates and inositol lipids play in the regulation of vesicle traffic within nerve terminals, we determined whether or not the synapse-specific clathrin assembly protein AP-3 can interact with inositol lipids. We found that soluble dioctanoyl-phosphatidylinositol 3,4,5-trisphosphate (DiC8PtdIns(3,4, 5)P3) was only 7.5-fold weaker a ligand than D-myo-inositol hexakisphosphate in assays that measured the displacement of D-myo-[3H]inositol hexakisphosphate. In functional assays we found that both of these ligands inhibited clathrin assembly, but DiC8-PtdIns(3,4,5)P3 was more potent and exhibited a larger maximal effect. We also examined the structural features of DiC8-PtdIns(3,4, 5)P3 that establish specificity. Dioctanoyl-phosphatidylinositol 3, 4-bisphosphate, which does not have a 5-phosphate, and 4, 5-O-bisphosphoryl-D-myo-inosityl 1-O-(1, 2-O-diundecyl)-sn-3-glycerylphosphate, which does not have a 3-phosphate, were, respectively, 2-fold and 4-fold less potent than DiC8-PtdIns(3,4,5)P3 as inhibitors of clathrin assembly. Deacylation of DiC8-PtdIns(3,4,5)P3 reduced its affinity for AP-3 almost 20-fold, and also dramatically lowered its ability to inhibit clathrin assembly. The deacylated products of the soluble derivatives of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 4, 5-bisphosphate were both not significant inhibitors of clathrin assembly. It therefore appears that the interactions of inositides with AP-3 should not be considered simply in terms of electrostatic effects of the highly charged phosphate groups. Ligand specificity appears also to be mediated by hydrophobic interactions with the fatty-acyl chains of the inositol lipids.

Acylation↗
PubMed1997

The lipid products of phosphoinositide 3-kinase increase cell motility through protein kinase C.

Phosphoinositide 3-kinase has been implicated as an activator of cell motility in a variety of recent studies, yet the role of its lipid product, phosphatidylinositol 1,4,5-trisphosphate (PtdIns-3,4,5-P3), has yet to be elucidated. In this study, three independent preparations of PtdIns-3,4,5-P3 were found to increase the motility of NIH 3T3 cells when examined utilizing a microchemotaxis chamber. Dipalmitoyl L-alpha-phosphatidyl-D-myo-inositol 3,4,5-triphosphate (Di-C16-PtdIns-3,4,5-P3) also produced actin reorganization and membrane ruffling. Cells pretreated with 12-O-tetradecanoylphorbol-13-acetate to cause down-regulation of protein kinase C (PKC) exhibited complete inhibition of cell motility induced by Di-C16-PtdIns-3,4,5-P3. These results are consistent with previous observations that PtdIns-3,4,5-P3 activates Ca2+-independent PKC isoforms in vitro and in vivo and provide the first demonstration of an in vivo role for the lipid products of the phosphoinositide 3-kinase. PtdIns-3,4,5-P3 appears to directly initiate cellular motility via activation of a PKC family member.

3T3 Cells↗
PubMed1997

Diphospho-myo-inositol phosphates from Dictyostelium identified as D-6-diphospho-myo-inositol pentakisphosphate and D-5,6-bisdiphospho-myo-inositol tetrakisphosphate.

Two diphospho-myo-inositol phosphates from Dictyostelium were recently investigated by two-dimensional 1H/31P NMR analysis and assigned to be either D-4-diphospho-myo-inositol pentakisphosphate (D-4-PP-InsP5) and D-4,5-bisdiphospho-myo-inositol tetrakisphosphate (D-4,5-bis-PP-InsP4) or their corresponding enantiomers D-6-PP-InsP5 and D-5,6-bis-PP-InsP4. In the present study the naturally occurring enantiomers were identified by using defined synthetic PP-InsP5 isomers as substrates for a partially purified PP-InsP5 5-kinase from Dictyostelium. This enzyme specifically phosphorylates the naturally occurring PP-InsP5 and the synthetic D-6-PP-InsP5, leading to D-5,6-bis-PP-InsP4. In contrast, neither D-4-PP-InsP5 nor D-1-PP-InsP5 or D-3-PP-InsP5 are converted by the enzyme.

Animals↗
PubMed1997

An active site substitution, F87V, converts cytochrome P450 BM-3 into a regio- and stereoselective (14S,15R)-arachidonic acid epoxygenase.

Cytochrome P450 BM-3 catalyzes the high turnover regio- and stereoselective metabolism of arachidonic and eicosapentaenoic acids. To map structural determinants of productive active site fatty acid binding, we mutated two amino acid residues, arginine 47 and phenylalanine 87, which flank the surface and heme ends of the enzyme's substrate access channel, respectively. Replacement of arginine 47 with glutamic acid resulted in a catalytically inactive mutant. Replacement of arginine 47 with alanine yielded a protein with reduced substrate binding affinity and arachidonate sp3 carbon hydroxylation activity (72% of control wild type). On the other hand, arachidonic and eicosapentaenoic acid epoxidation was significantly enhanced (154 and 137%, of control wild type, respectively). As with wild type, the alanine 47 mutant generated (18R)-hydroxyeicosatetraenoic, (14S,15R)-epoxyeicosatrienoic, and (17S,18R)-epoxyeicosatetraenoic acids nearly enantiomerically pure. Replacement of phenylalanine 87 with valine converted cytochrome P450 BM-3 into a regio- and stereoselective arachidonic acid epoxygenase ((14S,15R)-epoxyeicosatrienoic acid, 99% of total products). Conversely, metabolism of eicosapentaenoic acid by the valine 87 mutant yielded a mixture of (14S,15R)- and (17S,18R)-epoxyeicosatetraenoic acids (26 and 69% of total, 94 and 96% optical purity, respectively). Finally, replacement of phenylalanine 87 with tyrosine yielded an inactive protein. We propose that: (a) fatty acid oxidation by P450 BM-3 is incompatible with the presence of residues with negatively charged side chains at the surface opening of the substrate access channel or a polar aromatic side chain in the vicinity of the heme iron; (b) the high turnover regio- and stereoselective metabolism of arachidonic and eicosapentaenoic acids involves charge-dependent anchoring of the fatty acids at the mouth of the access channel by arginine 47, as well as steric gating of the heme-bound oxidant by phenylalanine 87; and (c) substrate binding coordinates, as opposed to oxygen chemistries, are the determining factors responsible for reaction rates, product chemistry, and, thus, catalytic outcome.

Animals↗
PubMed1997

Production of 3R-hydroxy-polyenoic fatty acids by the yeast Dipodascopsis uninucleata.

Various fatty acids were fed to the yeast Dipodascopsis uninucleata UOFS Y 128, and the extracted samples were analyzed for the accumulation of 3-hydroxy metabolites with the help of electron impact gas chromatography-mass spectrometry. Fatty acids containing of 5Z,8Z-diene system (5Z,8Z,11Z-eicosatrienoic, 5Z,8Z,11Z,14Z-eicosatetraenoic, and 5Z,8Z,11Z,14Z,17Z-eicosapentaenoic acids) yielded the corresponding 3-hydroxy-all-Z-eicosapolyenoic acids. Moreover, linoleic acid (9Z,12Z-octadecadienoic acid) and 11Z,14Z,17Z-eicosatrienoic acid were converted to the 3-hydorxylated metabolites of shorter chain length, e,g., 3-hydroxy-5Z,8Z-tetradecadienoic acid and 3-hydroxy-5Z,8Z,11Z-tetradecatrienoic acid, respectively. In contrast, no accumulation of a 3-hydroxy metabolite was observed with oleic acid (9Z-octadecenoic acid), linolelaidic acid (9E,12E-octadecadienoic acid), gamma-linolenic acid (6Z,9Z,12Z-octadecatrienoic acid), and eicosanoic acid as substrate. These findings pinpoint that the 3-hydroxylation of a fatty acid in Dipodascopsis uninucleata requires a 5Z,8Z-diene system either directly or following initial incomplete beta-oxidation. Following analysis of the enantiomer composition, the arachidonic acid metabolite was identified as 3R-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid, which rules out a normal beta-oxidation as biosynthetic route to this new class of oxylipins.

Arachidonic Acids↗
PubMed1997

Post-receptor signal transduction and regulation of 14(R),15(S)-epoxyeicosatrienoic acid (14,15-EET) binding in U-937 cells.

14(R),15(S)-epoxyeicosatrienoic acid (14,15-EET), a cytochrome P-450 monooxygenase (epoxygenase) metabolite of arachidonic acid has been reported to induce adhesion of a monocyte cell line (U-937) to cultured endothelial cells. In this study, we identified a population of specific, high affinity binding sites for 14(R),15(S)-EET in U-937 cell surface with Kd of 13.84 +/- 2.58 nM and Bmax of 3.54 +/- 0.28 pmol/10(6) cells. The specific binding of [3H]-14,15-EET on U-937 cells is more effectively displaced by 14(R),15(S)-EET than the 14(S),15(R)-isomer thus indicating stereospecificity. The binding was sensitive to various protease treatments suggesting the binding site is protein in nature. 14,15-EET binding in U937 cells is attenuated by cholera toxin (CT) and dibutyryl cAMP. Mean binding site density (Bmax) decreased 31.61% and 34.8% by the pretreatment with cholera toxin (200 micrograms/ml) and dibutyryl cAMP (300 nM), respectively, without affecting the dissociation constant. Under similar conditions, pertussis toxin (20-200 ng/ml) was less effective as compared to CT and dibutyryl cAMP. The down regulation of 14,15-EET binding caused by dibutyryl cAMP in U-937 cell was reversed by a specific protein kinase A (PKA) inhibitor, H-89, but not by the PKC inhibitor K252a. Thus, the results suggest that the specific binding site of 14,15-EET in U-937 cells is associated with a receptor that could be down regulated through an increase in intracellular cAMP and activation of a PKA signal transduction mechanism. We propose that the signal transduction mechanism of 14,15-EET begins with the binding of the receptor, which leads to the increase of intracellular cAMP levels and the activation of PKA, and finally with the down regulation of 14,15-EET receptor binding.

8,11,14-Eicosatrienoic Acid↗