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L Orning

Publications and source records attributed to L Orning.

At least 37 records · Page 2Linked 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

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↗
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↗
PubMed1990

Mechanism-based inactivation of leukotriene A4 hydrolase during leukotriene B4 formation by human erythrocytes.

Evidence is presented in support of a mechanism-based (suicide) inactivation of leukotriene A4 hydrolyase in intact human erythrocytes by leukotriene A4 and leukotriene A4 methyl ester. Loss of enzymatic activity, accompanying leukotriene B4 formation, was proportional to the substrate concentration. Inactivation was directly related to the amount of leukotriene B4 formation: for several, different experimental protocols 50% loss of hydrolase activity corresponded with formation of 10.3 +/- 2.1 microM leukotriene B4. The time course of inactivation was pseudo-first order and obeyed saturation kinetics. Apparent inactivation (KI) and first-order rate (ki) constants for leukotriene A4 were 28 microM and 0.35 min-1, respectively. Leukotriene A4 methyl ester was also a site-directed inactivator with a similar KI = 25 microM and a ki = 0.1 min-1. For single incubations substrate instability limited the extent of inactivation to 50% of the initial enzyme activity. Following multiple, consecutive incubations with leukotriene A4 this increased and approached 80-90%; however, a residual activity of 10-20% suggested that a pool of enzyme was not susceptible to inactivation. Recovery of enzymatic activity, following inactivation, was negligible in intact erythrocytes and isolated enzyme. A single radiolabeled protein, corresponding to leukotriene A4 hydrolase, was detected by electrophoretic analysis of the incubation between [3H]leukotriene A4 and erythrocytes, or partially purified enzyme. Incorporation of [3H]leukotriene A4 methyl ester into enzyme was linearly related to its inactivation: 191 +/- 5 pmol incorporated corresponded to 10% loss of activity. Results conform to criteria for a mechanism-based inactivation, in which leukotriene A4 participates in two parallel processes, one leading to leukotriene B4 formation, the other to "suicide" inactivation of leukotriene A4 hydrolase in intact erythrocytes. The specific, rather than indiscriminate nature of this process has implications for the regulation of cellular leukotriene B4 formation. It may also afford a basis to monitor transcellular biosynthesis of leukotriene B4 in vivo.

Epoxide Hydrolases↗
PubMed1989

Distribution and metabolism of leukotriene C4 after cisternal injection in guinea pigs.

Following cisternal injection of [3H8]LTC4 into guinea pigs, leukotriene metabolites were identified in the brain, cerebellum, perilymph, blood, liver and kidneys. LTC4 was metabolized into LTD4 and LTE4 in the cerebrospinal fluid and LTE4 was transported into the blood for general circulation and uptake into the liver and kidneys. The excretion of LTE4 from CNS to blood seemed to be the rate-limiting step in the elimination of leukotrienes from the body. Leukotrienes were also transported into the perilymph. The conversion of LTC4 into LTD4 and LTE4 was lower in perilymph as compared to the cerebrospinal fluid, suggesting a rate limiting function of the cochlear aqueduct that can be defined as a cerebrospinal fluid-labyrinth barrier.

Animals↗
PubMed1988

In vivo formation of omega-oxidized metabolites of leukotriene C4 in the rat.

[3H8]Leukotriene C4 was administered to germfree rats and to conventional rats having a bile duct cannula. Several radioactive metabolites were isolated. Two polar biliary metabolites from conventional rats were identified as N-acetyl-omega-carboxy-leukotriene E4 and N-acetyl-omega-hydroxy-leukotriene E4. A polar fecal metabolite from germfree rats was found to be N-acetyl-omega-carboxy-leukotriene E4. Chemical identities were established using UV spectroscopy and cochromatographies with authentic compounds in several HPLC systems. The fecal metabolite was further characterized by reductive desulfurization followed by gas-liquid-radiochromatography. The yield of the two biliary metabolites was 5% of the administered tritium after three hours and the yield of fecal N-acetyl-omega-carboxy-leukotriene E4 was 3.5% after three days.

Animals↗
PubMed1987

Omega-oxidation of cysteine-containing leukotrienes by rat-liver microsomes. Isolation and characterization of omega-hydroxy and omega-carboxy metabolites of leukotriene E4 and N-acetylleukotriene E4.

Leukotriene E4 was metabolized to two polar products by rat liver microsomes. These products were characterized by physico-chemical and chemical techniques. The chemical structures, (5S, 6R)-5,20-dihydroxy-6S-cysteinyl-7,9-trans-11,14-cis-icosatetraenoic acid (omega-hydroxy-leukotriene E4) and (5S, 6R)-5-hydroxy-6S-cysteinyl-7,9-trans-11,14-cis-icosatetraen-1,20-d ioic acid (omega-carboxy-leukotriene E4) suggested that leukotriene E4 was transformed by an omega-hydroxylase and omega-hydroxyleukotriene E dehydrogenase in sequence. N-Acetyl-leukotriene E4 was also transformed by these enzymes, but at a rate six times lower than leukotriene E4. The products formed from N-acetylleukotriene E4 were characterized as being N-acetyl-omega-hydroxy-leukotriene E4 and N-acetyl-omega-carboxy-leukotriene E4. Other substrates were 11-trans-leukotriene E4 and N-acetyl-11-trans-leukotriene E4. In contrast, leukotrienes C4 and D4 were not converted into omega-oxidized metabolites. The leukotriene E omega-hydroxylase reaction required NADPH and molecular oxygen as cofactors, and was most rapidly catalyzed by liver microsomes. Liver cytosol, fortified with NAD+, converted omega-hydroxyleukotriene E4 and N-acetyl-omega-hydroxy-leukotriene E4 into omega-carboxy metabolites. Microsomes contained at least 18 times less omega-hydroxy-leukotriene E dehydrogenase activity than did cytosol. Liver microsomes supplemented with acetyl-coenzyme A converted omega-hydroxy and omega-carboxy-leukotriene E4 into the corresponding N-acetyl derivatives. The novel enzyme, leukotriene E omega-hydroxylase, which is described here is distinct from a previously described leukotriene B omega-hydroxylase based on substrate competition and kinetic data.

Acetyltransferases↗
PubMed1987

Omega-hydroxylation of N-acetylleukotriene E4 by rat liver microsomes.

Previous investigations have demonstrated metabolism of leukotriene (LT) C4 in vivo involving transformations of the tripeptide, but not the fatty acid part, yielding N-acetyl LTE4 as a main biliary metabolite in the rat. In addition, several polar metabolites were detected in the same studies. The present report describes the characterization of a metabolite of N-acetyl LTE4 formed during incubations with rat liver microsomes. The structure, 5,20-dihydroxy-6-s-(2-acetamido-3-thiopropionyl)-7,9-trans-11, 14-cis-eicosa-tetraenoic acid, of this metabolite showed that it is formed by hydroxylation of the fatty acid part. Preliminary evidence indicates that it is one of several polar metabolites formed in vivo.

Animals↗
PubMed1987

Endogenous leukotriene D4 formation during anaphylactic shock in the guinea pig.

Experiments on the metabolism and excretion of i.v. administered selectively labeled [3H8]leukotriene C4 in bile duct-cannulated guinea pigs indicated predominantly biliary excretion of tritium. The major leukotriene metabolite in bile was identified as leukotriene D4. By monitoring leukotriene excretion radioimmunochromatographically, it was shown that guinea pigs suffering from anaphylactic shock produce leukotriene D4 endogenously. Immunological challenge of animals sensitized to ovalbumin was accompanied by an increase of biliary leukotriene D4 concentrations from 10 +/- 1 to 86 +/- 10 nM (mean +/- SEM, n = 5, P less than 0.001). When considering that bile flow was decreased to about half after challenge, the excretion rate of leukotriene D4 in bile increased from 0.88 +/- 0.16 before to 3.18 +/- 0.38 pmol X min-1 X kg-1 after challenge (mean +/- SEM, n = 5, P less than 0.002). It is concluded that systemic anaphylaxis in the guinea pig is associated with endogenous generation of leukotriene C4 (up to 1 nmol/kg during a 30-min period after the challenge.

Anaphylaxis↗
PubMed1986

In vivo metabolism of leukotriene C4 in germ-free and conventional rats. Fecal excretion of N-acetylleukotriene E4.

[5,6,8,9,11,12,14,15-3H8]Leukotriene C4 was subcutaneously injected into rats. Substantial amounts of the administered radioactivity were excreted in feces of germ-free and conventional animals during a 72-h period (78 and 64%, respectively). Analyses of fecal extracts by high performance liquid chromatography showed eight radioactive components for each type of animal. One metabolite amounted to 4.6% of the injected radioactivity in germ-free and 0.6% in conventional rats. Its chemical structure, 5-hydoxy-6-S-(2-acetamido-3-thiopropionyl)-7,9-trans-11,14-c is-eicosatetraenoi c acid (N-acetylleukotriene E4) was determined by ultraviolet spectroscopy, fast atom bombardment mass spectrometry, chemical and enzymatic transformations, and confirmed by chemical synthesis. Another metabolite (2.7% of the administered radioactivity in germ-free and 0.5% in conventional rats) was characterized as the 11-trans isomer of the former metabolite. The pathway of formation of these compounds appears to be analogous to the pathway of mercapturic acid biosynthesis.

Animals↗
PubMed1986

Metabolism and excretion of cysteinyl-leukotrienes.

In vitro and in vivo experiments have demonstrated that a major pathway of metabolism of the glutathione containing leukotrienes involves modifications of the tripeptide substituent. The metabolic alterations are initiated by elimination of the N-terminal gamma-glutamyl residue, catalyzed by the enzyme gamma-glutamyl transferase. This reaction is followed by hydrolysis of the remaining peptide bond resulting in elimination of the C-terminal glycine residue. The enzyme catalyzing the latter reaction is a membrane bound dipeptidase which occurs in kidney and other tissues. The product formed by these reactions, leukotriene E4, has been tentatively identified as a urinary metabolite in man following intravenous administration of leukotriene C4. In rats, two major fecal metabolites of leukotriene C4 were characterized as having the structures N-acetyl leukotriene E4 and N-acetyl 11-trans leukotriene E4. These compounds are formed from leukotriene E4 and 11-trans leukotriene E4 in reactions with acetyl coenzyme A. A membrane bound enzyme, present in liver, kidney and other tissues, catalyzes these reactions.

Animals↗
PubMed1985

In vivo metabolism of leukotriene C4 in man: urinary excretion of leukotriene E4.

Five - 20 nmoles of [5,6,8,9,11,12,14,15-3H8]leukotriene C4 was injected into three male volunteers. Forty-eight percent of the administered 3H was recovered from urine and 8% from feces, within a 72 hr period. Of the total urinary radioactivity 44% was excreted during the first hour after injection. This activity was mainly found in one compound, designated "I". The radioactivity excreted into urine later than one hour after injection, consisted partly of Compound I and two additional components, and partly of polar, non-volatile material. Compound I was identified as leukotriene E4 by UV-spectroscopy and cochromatographies in three high performance liquid chromatography systems with synthetic reference compounds. A total of 13% of administered radioactivity was excreted in urine as leukotriene E4.

Adult↗
PubMed1985

Leukotriene C4 formation catalyzed by three distinct forms of human cytosolic glutathione transferase.

The ability of three distinct types of human cytosolic glutathione transferase to catalyze the formation of leukotriene C4 from glutathione and leukotriene A4 has been demonstrated. The near-neutral transferase (mu) was the most efficient enzyme with Vmax= 180 nmol X min-1 X mg-1 and Km= 160 microM. The Vmax and Km values for the basic (alpha-epsilon) and the acidic (pi) transferases were 66 and 24 nmol X min-1 X mg-1 and 130 and 190 microM, respectively. The synthetic methyl ester derivative of leukotriene A4 was somewhat more active as a substrate for all the three forms of the enzyme.

Arachidonic Acids↗
PubMed1985

Metabolism of leukotrienes.

The in vitro metabolism of leukotriene B4 is initiated by omega-hydroxylation. This reaction is followed by oxidation of the omega-hydroxyl group to a carboxyl group. In vivo extensive beta-oxidation occurs and the main excreted products after administration of leukotriene B4 are water and carbon dioxide. Experiments performed in vitro and in vivo have demonstrated that a major pathway of metabolism of the glutathione containing leukotrienes involves modifications of the tripeptide substituent. The metabolic alterations are initiated by enzymatic elimination of the N-terminal gamma-glutamyl residue, catalyzed by the enzyme gamma-glutamyl transferase. This reaction is followed by hydrolysis of the remaining peptide bond resulting in elimination of the C-terminal glycine residue. The enzyme catalyzing the latter reaction is a membrane bound dipeptidase which occurs in kidney and other tissues. The product formed by these reactions, leukotriene E4, has been tentatively identified as a urinary metabolite in man following intravenous administration of leukotriene C4. In rats, the two major fecal metabolities of leukotriene C4 were characterized as being N-acetyl leukotriene E4 and N-acetyl 11-trans leukotriene E4. These compounds are formed in reactions between leukotriene E4 or 11-trans leukotriene E4 and acetyl coenzyme A. The reactions are catalyzed by a membrane bound enzyme present in liver, kidney and other tissues.

Animals↗
PubMed1984

Transformation of leukotriene A4 methyl ester to leukotriene C4 monomethyl ester by cytosolic rat glutathione transferases.

Six major basic cytosolic glutathione transferases from rat liver catalyzed the conversion of leukotriene A4 methyl ester to the corresponding leukotriene C4 monomethyl ester. Glutathione transferase 4-4, the most active among these enzymes, had a Vmax of 615 nmol X min-1 X mg protein-1 at 30 degrees C in the presence of 5 mM glutathione. It was followed in efficiency by transferase 3-4 which had a Vmax of 160 nmol X min-1 X mg-1 under the same conditions. Transferases 1-1, 1-2, 2-2 and 3-3 had at least 30 times lower Vmax values than transferase 4-4.

Animals↗