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F A Fitzpatrick

Publications and source records attributed to F A Fitzpatrick.

At least 19 recordsLinked to original sources
PubMed1992

Mechanism-based inactivation of leukotriene A4 hydrolase/aminopeptidase by leukotriene A4. Mass spectrometric and kinetic characterization.

"Suicide" inactivation of leukotriene (LT) A4 hydrolase/aminopeptidase occurs via an irreversible mechanism-based process which is saturable, of pseudo firstorder, and dependent upon catalysis. Data obtained with either recombinant enzyme or enzyme purified from human leukocytes were similar. Apparent binding constants and inactivation rate constants are equivalent, compatible with a single type of substrate-enzyme complex which partitions between two fates, turnover and inactivation. Both catalytic functions are inactivated, consistent with an overlapping active site for this bifunctional enzyme. The partition ratio (turnover/inactivation) for the LTA4-enzyme complex is 129 +/- 16 for LTA4 hydrolase activity and 124 +/- 10 for aminopeptidase activity. The pH dependence for turnover and inactivation are indistinguishable with a maximum at pH 8. L-Proline p-nitroanilide, a weak substrate with a high Km for the aminopeptidase affords only partial protection against inactivation by LTA4. However, two potent competitive inhibitors, bestatin and captopril, protect both catalytic processes from inactivation, consistent with an active-site specificity for the suicide event. Electrospray ionization mass spectrometry indicates that the molecular weight of pure recombinant enzyme is 69,399 +/- 4 and that covalent modification accompanies catalysis, producing an LTA4:enzyme adduct with a molecular weight 69,717 +/- 4 and a 1:1 stoichiometry. In agreement with kinetic data, electrospray ionization mass spectrometry shows that bestatin inhibits the covalent modification of enzyme by LTA4 and that the extent of modification is proportional to the loss of enzymatic activity.

Binding, Competitive↗
PubMed1992

Epoxyeicosatrienoic acids inhibit Ca2+ entry into platelets stimulated by thapsigargin and thrombin.

The epoxyeicosatrienoic acids derived from the cytochrome P-450 pathway of arachidonic acid metabolism have a unique platelet antiaggregatory profile. This prompted us to examine their influence on cellular Ca2+ mobilization. 14,15-cis-Epoxyeicosatrienoic acid and related compounds inhibited the rise in cytosolic Ca2+ following agonist stimulation of platelets by thapsigargin, a receptor-independent agonist, and thrombin, a receptor-dependent agonist. The epoxyeicosatrienoic acids selectively inhibited the entry of Ca2+ from the exterior of the platelets but did not alter Ca2+ discharge from intracellular pools. The magnitude of inhibition by 14,15-cis-epoxyeicosatrienoic acid was proportional to the rate of Ca2+ entry. 14,15-cis-Epoxyeicosatrienoic acid also inhibited the rate of influx of Mn2+, a cation which enters platelets via pathways similar to Ca2+. The magnitude of inhibition was proportional to the rate of Mn2+ entry, suggesting that epoxyeicosatrienoic acids act on divalent cation channels in a fashion which depends on the state of opening of the channel. Selective inhibition of Ca2+ entry into platelets may account for the antiaggregatory effects of the epoxyeicosatrienoic acids. We are unaware of other endogenous compounds exhibiting this property, suggesting that epoxyeicosatrienoic acids may be useful to probe agonist-stimulated Ca2+ mobilization in nonexcitable cells.

8,11,14-Eicosatrienoic Acid↗
PubMed1992

Albumins activate peptide hydrolysis by the bifunctional enzyme LTA4 hydrolase/aminopeptidase.

Albumins from several species activated the bifunctional, Zn2+ metalloenzyme amino-peptidase/leukotriene A4 hydrolase (EC 3.3.2.6). Bovine serum albumin, 1 mg/mL, increased hydrolysis of L-proline-p-nitroanilide and leucine-enkephalin by 12-fold and 7-fold, respectively. The apparent Km for L-proline-p-nitroanilide was inversely proportional to the albumin concentration from 0 to 1 mg/mL, declining from 9.4 to 0.7 mM without an appreciable change in apparent Vmax. These data imply a random activation process in which the enzyme-activator complex is catalytically dominant. Hill plots indicated a 1:1 stoichiometric relationship between albumin and enzyme. Secondary plots of slope versus the reciprocal of albumin concentration indicated that it binds to the enzyme with an affinity constant of 0.9 microM. The pH optimum of the nonactivated enzyme occurred at pH 8; the albumin-activated enzyme had an optimum near pH 7. Neither ultrafiltration nor dialysis of albumin altered its activating effect, but boiling abolished it. Albumin did not affect other cytosolic or microsomal leucine aminopeptidases, or gamma-glutamyltransferase. Albumin functions as a nonessential activator, since enzymatic activity was always detectable in its absence. Chloride ions, which activate other Zn2+ metalloenzymes, also activated leukotriene A4 hydrolase/aminopeptidase with an EC50 = 50 mM, increasing its initial velocity 2.2-fold in the absence of albumin. Zn2+ activated the enzyme, increasing its apparent Vmax but not its apparent Km, suggesting it replaced Zn2+ lost from the active site, especially at acidic pH. At concentrations greater than 30-50 microM, Zn2+ was inhibitory. Albumin mitigated the effect of chloride, but not the effect of Zn2+ or that of the competitive inhibitor, captopril.(ABSTRACT TRUNCATED AT 250 WORDS)

Albumins↗
PubMed1992

Incorporation and distribution of epoxyeicosatrienoic acids into cellular phospholipids.

The different regioisomers of epoxyeicosatrienoic acids derived from cytochrome P-450 monooxygenase are readily esterified into phospholipids of mastocytoma cells. Incorporation of 14,15-epoxyeicosatrienoic acid was concentration-dependent, with Km = 1.1 microM and Vmax = 36 pmol/min/10(7) cells. Half-maximal incorporation occurred in 30 min, reaching a steady-state concentration of 470 pmol/10(6) cells. This was slightly lower than the values for arachidonic acid (665 pmol/10(6) cells) or 5-hydroxyeicosatetraenoic acid (554 pmol/10(6) cells). The distribution of 14,15-epoxyeicosatrienoic acid was preferential in the order phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylinositol greater than phosphatidyl serine much greater than neutral lipids plus fatty acids. This contrasted with 5(S)-hydroxyeicosatetraenoic acid, which was distributed primarily into phosphatidylcholine. Fast atom bombardment/tandem mass spectrometry facilitated identification of molecular species containing epoxyeicosatrienoic acids without relying on radioisotopes. Phosphatidylethanolamine plasmalogens with 16:1 or 18:2 at the sn-1 position, or an 18:0 acyl group, and phosphatidylcholine with 16:0 alkyl ether or an acyl group at the sn-1 position incorporated all possible epoxyeicosatrienoic acid regioisomers. Under basal conditions, cells eliminated 14,15-cis-epoxyeicosatrienoic acid slowly with a half-life of 34.9 +/- 7 h. Cells stimulated with calcium ionophore A23187 eliminated 14,15-epoxyeicosatrienoic acid rapidly. It was notable that its rate of release from phosphatidylcholine and phosphatidylinositol exceeded that for arachidonic acid. A coenzyme A-independent transacylase also catalyzed the transfer of epoxyeicosatrienoic acids from mastocytoma cell membranes into 1-palmitoyl-2-lysophosphatidylcholine. The cellular incorporation, release, and distribution of epoxyeicosatrienoic acids is distinctive and contrasts with most other eicosanoids, suggesting that these compounds may have both autocoid and nonautocoid functions.

8,11,14-Eicosatrienoic Acid↗
PubMed1992

Opioid peptides are substrates for the bifunctional enzyme LTA4 hydrolase/aminopeptidase.

We determined if any naturally occurring peptides could act as substrates or inhibitors of the bifunctional, Zn2+ metalloenzyme LTA4 hydrolase/aminopeptidase (E.C.3.3.2.6). Several opioid peptides including met5-enkephalin, leu5-enkephalin, dynorphin1-6, dynorphin1-7, and dynorphin1-8 competitively inhibited the hydrolysis of L-proline-p-nitroanilide by leukotriene A4 hydrolase/aminopeptidase, consistent with an interaction at its active site. The enzyme catalyzed the N-terminal hydrolysis of tyrosine from met5-enkephalin with Km = 450 +/- 58 microM and Vmax = 4.9 +/- 0.6 nmol-hr-1-ug-1 and from leu5-enkephalin with Km = 387 +/- 90 microM and Vmax = 6.2 +/- 2.5 nmol-hr-1-ug-1. Bestatin, captopril and carnosine inhibited the hydrolysis of the enkephalins. It is noteworthy that the bifunctional catalytic traits of this enzyme include generation of an hyperalgesic substance, LTB4, and inactivation of analgesic opioid peptides.

Amino Acid Sequence↗
PubMed1992

Indirect actions of thapsigargin on human platelets: activation of eicosanoid biosynthesis and cellular signaling pathways.

Thapsigargin, which acts by inhibition of a Ca(++)-ATPase on the dense tubule system in platelets, is a pharmacological tool to study the effects of increases in intracellular Ca++. Secondary consequences of thapsigargin treatment in platelets include extensive thromboxane B2 formation (493 +/- 106 ng/10(8) platelets) and [3H]5-hydroxytryptamine secretion (80.7 +/- 8.0%). Inhibition of cyclooxygenase by ibuprofen prevents thromboxane B2 formation (0.1 +/- 0.04 ng/10(8) platelets) and dense tubule secretion (6.5 +/- 3.8%). Aggregation in response to thapsigargin is rapid and maximal, but the rate and extent of aggregation are lowered by ibuprofen or aspirin. Mobilization of intracellular Ca++ is also significantly attenuated when eicosanoid formation is prevented, indicating the dependence of thapsigargin actions on endogenous lipid mediator formation. These studies also support the idea that formation of endogenous thromboxane A2/prostaglandin H2 is self-amplifying; thromboxane receptor antagonists inhibit endogenous thromboxane B2 formation, indicating that Ca(++)-dependent activation of phospholipase A2 is only partially responsible for eicosanoid production. Our data indicate the importance of distinguishing secondary effects of thapsigargin, especially because it may influence eicosanoid formation.

Blood Platelets↗
PubMed1991

Thromboxane A2 synthase. Modification during "suicide" inactivation.

Thromboxane synthase is a ferrihemoprotein which undergoes mechanism-based inactivation during catalysis. This "suicide" process may be an important factor for limiting thromboxane A2 biosynthesis in cells. Although the kinetics have been characterized for purified enzyme and platelets, the chemical basis for inactivation has remained unclear. Protein modification or alteration of the heme prosthetic group is each compatible with the irreversible nature of suicide inactivation of thromboxane synthase. We have investigated these two possibilities using enzyme purified to homogeneity. Our data show that the Soret absorbance spectrum of thromboxane synthase is unaltered by additions of prostaglandin endoperoxide H2 which cause enzymatic inactivation. Using a coupled cyclooxygenase/thromboxane synthase system and polyacrylamide gel electrophoresis we have demonstrated that the enzyme retains radiolabel under nondenaturing gel conditions. Label incorporation is reduced by the competitive thromboxane synthase inhibitor U63557, an agent that also protects the enzyme from inactivation. Under denaturing conditions the radiolabel localizes with the released heme prosthetic group. In addition, interaction of the heme prosthetic group with cyanide was prevented by inactivating the enzyme with prostaglandin H2. In similar experiments, the lipid hydroperoxide 15(S)-hydroperoxyeicosatetraenoic acid inactivated thromboxane synthase with concurrent bleaching of the Soret spectrum. Labeling studies with a coupled soybean lipoxygenase/thromboxane synthase system indicate that, in this case, the apoenzyme is modified. These results suggest that the mechanism of thromboxane synthase inactivation during thromboxane A2 biosynthesis involves a tight, nondestructive association of substrate or product with the prosthetic heme group. Inactivation by hydroperoxides, however, appears to result from apoenzyme modification. These reactions may have important implications for cellular physiology and pathophysiology of thrombosis.

Arachidonate 15-Lipoxygenase↗
PubMed1991

Thromboxane A2 synthesis in human erythroleukemia cells.

Human erythroleukemia cells transformed arachidonic acid and prostaglandin endoperoxide H2 into thromboxane A2. Stimulation of these cells with A23187 or thrombin, however, produced no thromboxane. Similarly, cells labeled with [3H]-arachidonic acid released no detectable label upon stimulation. Data suggest that human erythroleukemia cells contain the enzymatic capacity for thromboxane formation from exogenous precursors, but lack the endogenous mechanisms for arachidonate release. The presence of thromboxane synthase messenger RNA was verified using the polymerase chain reaction. Amplification and sequence analysis of a 528 bp cDNA demonstrated virtually 100% identity to a published thromboxane synthase cDNA fragment.

Amino Acid Sequence↗
PubMed1991

Inhibition of leukotriene A4 hydrolase/aminopeptidase by captopril.

Captopril ((2S)-1-(3-mercapto-2-methyl-propionyl)-L-proline) inhibited the bifunctional, Zn(2+)-containing enzyme leukotriene A4 hydrolase/aminopeptidase reversibly and competitively with Ki = 6.0 microM for leukotriene B4 formation and Ki = 60 nM for L-lysine-p-nitroanilide hydrolysis at pH 8. Inhibition was independent of pH between pH 7 and 8, the optimum range for each catalytic activity. Half-maximal inhibition of leukotriene B4 formation by intact erythrocytes and neutrophils required 50 and 88 microM captopril, respectively. In neutrophils and platelets neither 5(S)-hydroxyeicosatetraenoic acid, 12(S)-hydroxyeicosatetraenoic acid, nor leukotriene C4 formation were reduced, indicating selective inhibition of leukotriene A4 hydrolase/aminopeptidase, not 5-lipoxygenase, 12-lipoxygenase, or leukotriene C4 synthase. In whole blood, captopril inhibited leukotriene B4 formation with an accompanying redistribution of substrate toward formation of cysteinyl leukotrienes. The decrease in leukotriene B4 was more substantial than the corresponding increase in cysteinyl leukotrienes suggesting that nonenzymatic hydration predominates over transcellular metabolism of leukotriene A4 by platelets during selective inhibition of leukotriene A4 hydrolase. Enalapril dicarboxylic acid and Glu-Trp-Pro-Arg-ProGln-Ile-Pro-Pro which inhibit angiotensin-converting enzyme: angiotensin I, bradykinin, and N-[3-(2-furyl)acryloyl]Phe-Gly-Gly which are substrates; and chloride ions which activate angiotensin-converting enzyme did not modulate leukotriene A4 hydrolase/aminopeptidase activity. The results indicate that: (i) the sulfhydryl group of captopril is an important determinant for inhibition of leukotriene A4 hydrolase/aminopeptidase, probably by binding to an active site Zn2+; (ii) aminopeptidase and leukotriene A4 hydrolase display differential susceptibility to inhibition; (iii) there is minimal functional similarity between angiotensin-converting enzyme (peptidyl dipeptidase) and leukotriene A4 hydrolase/aminopeptidase; (iv) captopril may be a useful prototype to identify more potent and selective leukotriene A4 hydrolase inhibitors.

Amino Acid Sequence↗
PubMed1991

Leukotriene A4 hydrolase. Inhibition by bestatin and intrinsic aminopeptidase activity establish its functional resemblance to metallohydrolase enzymes.

Bestatin, an inhibitor of aminopeptidases, was also a potent inhibitor of leukotriene (LT) A4 hydrolase. On isolated enzyme its effects were immediate and reversible with a Ki = 201 +/- 95 mM. With erythrocytes it inhibited LTB4 formation greater than 90% within 10 min; with neutrophils it inhibited LTB4 formation by only 10% during the same period, increasing to 40% in 2 h. Bestatin inhibited LTA4 hydrolase selectively; neither 5-lipoxygenase nor 15-lipoxygenase activity in neutrophil lysates was affected. Purified LTA4 hydrolase exhibited an intrinsic aminopeptidase activity, hydrolyzing L-lysine-p-nitroanilide and L-leucine-beta-naphthylamide with apparent Km = 156 microM and 70 microM and Vmax = 50 and 215 nmol/min/mg, respectively. Both LTA4 and bestatin suppressed the intrinsic aminopeptidase activity of LTA4 hydrolase with apparent Ki values of 5.3 microM and 172 nM, respectively. Other metallohydrolase inhibitors tested did not reduce LTA4 hydrolase/aminopeptidase activity, with one exception; captopril, an inhibitor of angiotensin-converting enzyme, was as effective as bestatin. The results demonstrate a functional resemblance between LTA4 hydrolase and certain metallohydrolases, consistent with a molecular resemblance at their putative Zn2(+)-binding sites. The availability of a reversible, chemically stable inhibitor of LTA4 hydrolase may facilitate investigations on the role of LTB4 in inflammation, particularly the process termed transcellular biosynthesis.

Aminopeptidases↗
PubMed1991

Inhibitors of arachidonic acid metabolism: effects on rat striatal dopamine release and uptake.

The purpose of this study was to investigate the possibility that arachidonic acid metabolites mediate D-2 dopamine (DA) receptor inhibition of striatal DA release. The phospholipase A2 inhibitor p-bromophenacyl bromide (BPB; 10 microM) increased electrically evoked overflow of endogenous DA from rat striatal slices and appeared to partially block the modulatory effects of the D-2 DA receptor agonist N-0437 on this release. However, BPB also increased spontaneous DA outflow in a dose-dependent manner. U-73122 (10 microM), another phospholipase A2 inhibitor, decreased evoked overflow of DA, did not affect the action of N-0437 but also increased spontaneous outflow of DA. In contrast, arachidonic acid (30 microM) produced no effects. In slices prelabeled with [3H]DA, exposure to BPB, U-73122 and nordihydroguaiaretic acid (a lipoxygenase inhibitor) significantly increased spontaneous outflow of tritium whereas the cyclooxygenase inhibitors aspirin and indomethacin did not. In low micromolar concentrations, BPB, U-73122 and nordihydroguaiaretic acid, but not aspirin and indomethacin, inhibited uptake of [3H]DA into striatal synaptosomes and binding of [3H]mazindol to the DA transporter. Only U-73122 affected D-2 DA receptor binding. Taken together, these results suggest that it is unlikely that arachidonic acid metabolites mediate the actions of release-modulating D-2 DA autoreceptors in the striatum. However, the results also suggest that certain inhibitors of arachidonic acid metabolism are relatively potent DA uptake blockers/releasers and that this action is unrelated to their inhibition of enzymes in the arachidonic acid cascade. Caution should be used when using BPB and nordihydroguaiaretic acid to study mechanisms involved in DA release, because these compounds may increase DA release and thereby appear to antagonize the effects of activation of presynaptic receptors.

Acetophenones↗
PubMed1991

14, 15-cis-episulfide-eicosatrienoic acid, an 'epoxygenase' eicosanoid analog, inhibits ionophore- but not thrombin-induced platelet aggregation.

An 'epoxygenase' eicosanoid analog, 14, 15-cis-episulfide-eicosatrienoic acid, has several unique pharmacological effects on platelets. These include (i) inhibition of ionophore A23187- but not thrombin-induced activation, (ii) inhibition of thromboxane B2 biosynthesis derived from endogenous but not exogenous arachidonic acid, and (iii) attenuation of ionophore-mediated increases in cytosolic Ca2+ when extracellular or membrane Ca2+ is available but not when these pools are excluded. Neither elevation of cyclic AMP levels, a potent inhibitory process, nor direct antagonism of the prostaglandin H2/thromboxane A2 receptor is responsible for the actions of 14, 15-cis-episulfide-eicosatrienoic acid. These properties distinguish 14, 15-cis-episulfide-eicosatrienoic acid from other antiaggregatory substances.

8,11,14-Eicosatrienoic Acid↗
PubMed1990

Arachidonic acid metabolites do not mediate modulation of neurotransmitter release by adenosine in rat hippocampus or striatum.

The possible involvement of arachidonic acid metabolites as mediators of the modulation of neurotransmitter release by adenosine, acetylcholine, and GABA was examined in brain slices of rat hippocampus and striatum. The synaptic modulatory effects of these 3 agents on excitatory transmission in the CA1 region of hippocampus were completely unaffected by a phospholipase inhibitor (p-bromophenacyl bromide, BPB; 10-50 microM), a lipoxygenase inhibitor (nordihydroguaiaretic acid; 5-50 microM), the cyclooxygenase inhibitor indomethacin (10-20 microM), and a cyclooxygenase/lipoxygenase inhibitor (U53059; 5-10 microM). BPB was also found to be ineffective in altering the modulation of transmission by adenosine in the perforant path, and the adenosine inhibition of electrically stimulated release of endogenous dopamine from striatal slices. Arachidonic acid itself also had no effect on synaptic transmission. While these experiments do not rule out such a role for arachidonic acid or its metabolites in mammalian brain, they suggest that in a number of systems the inhibition of transmitter release must occur through an entirely independent mechanism.

Acetophenones↗
PubMed1979

Glass capillary gas chromatography with electron-capture detection. Separation of prostaglandins.

Glass capillary gas chromatography of the prostaglandins was performed on a system including an all-glass, solventless injector; thermostable methylphenyl-polysiloxane glass capillary columns; and a conventional electron-capture detector fitted with a make-up gas tee. The principal stable metabolites of prostaglandin endoperoxide were separated as perfluorinated derivatives in 35 min. Detection limits equal or exceed those obtained for packed column separations and electron capture detection. Prostaglandin endoperoxide metabolic profiles from mammalian cell cultures were obtained using this system. These profiling studies are not possible with other chromatographic methods because of inferior resolution and sensitivity.

Animals↗