Mutation of tyrosine 383 in leukotriene A4 hydrolase allows conversion of leukotriene A4 into 5S,6S-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid. Implications for the epoxide hydrolase mechanism

Leukotriene A4 hydrolase is a bifunctional zinc metalloenzyme that catalyzes the final step in the biosynthesis of the proinflammatory mediator leukotriene B4. In previous studies with site-directed mutagenesis on mouse leukotriene A4 hydrolase, we have identified Tyr-383 as a catalytic amino acid involved in the peptidase reaction. Further characterization of the mutants in position 383 revealed that [Y383H], [Y383F], and [Y383Q] leukotriene A4 hydrolases catalyzed hydrolysis of leukotriene A4 into a novel enzymatic metabolite. From analysis by high performance liquid chromatography, gas chromatography/mass spectrometry of material generated in the presence of H216O or H218O, steric analysis of the hydroxyl groups, treatment with soybean lipoxygenase, and comparison with a synthetic standard, the novel metabolite was assigned the structure 5S, 6S-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid (5S,6S-DHETE). The kinetic parameters for the formation of 5S,6S-DHETE and leukotriene B4 were found to be similar. Also, both activities were susceptible to suicide inactivation and were equally sensitive to inhibition by bestatin. Moreover, from the stereochemical configuration of the vicinal diol, it could be inferred that 5S, 6S-DHETE is formed via an SN1 mechanism involving a carbocation intermediate, which in turn indicates that enzymatic hydrolysis of leukotriene A4 into leukotriene B4 follows the same mechanism. Inasmuch as soluble epoxide hydrolase utilizes leukotriene A4 as substrate to produce 5S,6R-DHETE, our results also suggest a functional relationship between leukotriene A4 hydrolase and xenobiotic epoxide hydrolases.     

Laxity and flexibility of the ankle following reconstruction with the Chrisman-Snook procedure

Anandamide (arachidonylethanolamide) is an endogenous ligand for cannabinoid receptors, and exerts various cannabimimetic activities. Since cannabinoids and anandamide were pharmacologically active with the eye, we examined metabolism of anandamide in a variety of porcine ocular tissues. In the presence of ethanolamine, [14C]arachidonic acid was converted to [14C]anandamide by a homogenate of retina, choroid, iris, optic nerve and lacrimal gland with a specific enzyme activity of 1.9-4.2 nmol min-1 mg-1 protein at 37 degrees C. On the other hand, [14C]anandamide was hydrolysed to [14C]arachidonic acid by a homogenate of each tissue with a specific enzyme activity of 1.2-3.5 nmol min-1 mg-1 protein. Thus, both activities of anandamide synthase and hydrolase were found in these ocular tissues. As for the subcellular distribution, the two enzyme activities were mostly recovered in particulate fractions rather than the cytosol. With the retina microsome palmitic acid was converted to its ethanolamide at a lower rate than arachidonic acid, and palmitoylethanolamide was less active than anandamide as a substrate for the hydrolase.     

Characterization of the aminopeptidase activity of epidermal leukotriene A4 hydrolase against the opioid dynorphin fragment 1-7

Leukotriene A4 hydrolase is a bifunctional cytosolic enzyme, which both hydrolyses leukotriene A4 (LTA4) into leukotriene B4 (LTB4) and exerts aminopeptidase activity against opioid peptides. In the present study we have investigated whether the peptides angiotensin I and II, bradykinin, kallidine, histamine, dynorphin fragment 1-7 and substance P can act as substrates for epidermal and neutrophil LTA4 hydrolase. Among the tested substrates, dynorphin fragment 1-7 was found to be the best substrate for the enzyme. The aminopeptidase activity of epidermal and neutrophil LTA4 hydrolase against dynorphin fragment 1-7 was further characterized. The enzyme was purified from human epidermis and human neutrophils by anion exchange chromatography (Q-Sepharose) and affinity chromatography on a column with the LTA4 hydrolase inhibitor bestatin coupled to AH-Sepharose. The incubation of the dynorphin fragment 1-7 with LTA4 hydrolase resulted in the formation of tyrosine. The presence of the N-terminal amino acid tyrosine is essential for the interaction of opioids with their receptors, and this finding indicates that the LTA4 hydrolase can inactivate dynorphin fragment 1-7. After the two purification steps no other aminopeptidases acting at the N-terminal tyrosine of dynorphin fragment 1-7 was present in the preparation. This was demonstrated by the abolishment of the degradation at the N-terminal end of dynorphin fragment 1-7 when preincubating the enzyme preparation with LTA4 before the incubation with the dynorphin fragment 1-7. The abolishment of the aminopeptidase activity shows that activation of the hydrolase part of the enzyme, with conversion of LTA4 into the potent proinflammatory compound LTB4, results in an inhibition of the aminopeptidase activity of the enzyme. As a result, the catabolism of dynorphin fragment 1-7 and probably of other opioid peptides is inhibited, resulting in sustained biological effects of these opioids. This phenomenon may be important for the maintenance of inflammation in skin conditions, such as psoriasis and atopic dermatitis, in which LTB4 is formed.     

Regulation of leukotriene A4 hydrolase activity in endothelial cells by phosphorylation

Endothelial cells contain leukotriene (LT) A4 hydrolase (LTA-H) as detected by Northern and Western blotting, but several studies have been unable to detect the activity of this enzyme. Since LTA-H could play a key role in determining what biologically active lipids are generated by activated endothelium during the inflammatory process, we studied possible mechanisms by which this enzyme may be regulated. We find that LTA-H is phosphorylated under basal conditions in human endothelial cells and in this state does not exhibit epoxide hydrolase activity (i.e. conversion of LTA4 to LTB4). LTA-H purified from endothelial cells is efficiently dephosphorylated by incubation with protein phosphatase-1 in the presence of an LTA-H peptide substrate and not at all in the absence of substrate. Under conditions that lead to dephosphorylation, protein phosphatase-1 activates the epoxide hydrolase activity of LTA-H. Using peptide mapping and site-directed mutagenesis, we have identified serine 415 as the site of phosphorylation of LTA-H by a kinase found in endothelial cell cytosol. In parallel, we have studied a human lung carcinoma cell line that expresses active LTA-H. Although these cells have cytosolic kinases that phosphorylate recombinant LTA-H, they do not target serine 415 and thus do not inhibit LTA-H activity. We believe that LTA-H is regulated in intact cells by a kinase/phosphatase cycle and further that the kinase in endothelial cells specifically recognizes and phosphorylates a regulatory site in the LTA-H.     

Molecular cloning and functional expression of a Caenorhabditis elegans aminopeptidase structurally related to mammalian leukotriene A4 hydrolases

In a search of the Caenorhabditis elegans DNA data base, an expressed sequence tag of 327 base pairs (termed cm01c7) with strong homology to the human leukotriene A4 (LTA4) hydrolase was found. The use of cm01c7 as a probe, together with conventional hybridization screening and anchored polymerase chain reaction techniques resulted in the cloning of the full-length 2.1 kilobase pair C. elegans LTA4 hydrolase-like homologue, termed aminopeptidase-1 (AP-1). The AP-1 cDNA was expressed transiently as an epitope-tagged recombinant protein in COS-7 mammalian cells, purified using an anti-epitope antibody affinity resin, and tested for LTA4 hydrolase and aminopeptidase activities. Despite the strong homology between the human LTA4 hydrolase and C. elegans AP-1(63% similarity and 45% identity at the amino acid level), reverse-phase high pressure liquid chromatography and radioimmunoassay for LTB4 production revealed the inability of the C. elegans AP-1 to use LTA4 as a substrate. In contrast, the C. elegans AP-1 was an efficient aminopeptidase, as demonstrated by its ability to hydrolyze a variety of amino acid p-nitroanilide derivatives. The aminopeptidase activity of C. elegans AP-1 resembled that of the human LTA4 hydrolase/aminopeptidase enzyme with a preference for arginyl-p-nitroanilide as a substrate. Hydrolysis of the amide bond of arginyl-p-nitroanilide was inhibited by bestatin with an IC50 of 2.6 +/- 1.2 microM. The bifunctionality of the mammalian LTA4 hydrolase is still poorly understood, as the physiological substrate for its aminopeptidase activity is yet to be discovered. Our results support the idea that the enzyme originally functioned as an aminopeptidase in lower metazoa and then developed LTA4 hydrolase activity in more evolved organisms.     

The bifunctional enzyme leukotriene-A4 hydrolase is an arginine aminopeptidase of high efficiency and specificity

Leukotriene-A4 hydrolase (EC 3.3.2.6) cleaved the NH2-terminal amino acid from several tripeptides, typified by arginyl-glycyl-aspartic acid, arginyl-glycyl-glycine, and arginyl-histidyl-phenylalanine, with catalytic efficiencies (kcat/Km) > or = 1 x 10(6) M-1 s-1. This exceeds by 10-fold the kcat/Km for its lipid substrate leukotriene A4. Catalytic efficiency declined for dipeptides which had kcat/Km ratios 10-100-fold lower than tripeptides. Tetrapeptides and pentapeptides were even poorer substrates with catalytic efficiencies below 10(3) M-1 s-1. The enzyme preferentially hydrolyzed tripeptide substrates and single amino acid p-nitroanilides with L-arginine at the NH2 terminus. Peptides with proline at the second position were not hydrolyzed, suggesting a requirement for an N-hydrogen at the peptide bond cleaved. Peptides with a blocked NH2 terminus were not hydrolyzed. The specificity constant (kcat/Km) was optimal at pH 7.2 with pK values at 6.8 and 7.9; binding was maximal at pH 8.0. Serum albumins activated the peptidase, increasing tripeptide affinities (Km) by 3-10-fold and specificities (kcat/Km) by 4-13-fold. Two known inhibitors of arginine peptidases, arphamenine A and B, inhibited hydrolysis of L-arginine p-nitroanilide with dissociation constants = 2.0 and 2.5 microM, respectively. Although the primary role of LTA4 hydrolase is widely regarded as the conversion of the lipid substrate leukotriene A4 into the inflammatory lipid mediator leukotriene B4, our data are the first showing that tripeptides are "better" substrates. This is compatible with a biological role for the peptidase activity of the enzyme and may be relevant to the distribution of the enzyme in organs like the ileum, liver, lung, and brain. We present a model which accommodates the available data on the interaction of substrates and inhibitors with the enzyme. This model can account for overlap in the active site for hydrolysis of leukotriene A4 and peptide or p-nitroanilide substrates.     

Visualization of a covalent intermediate between microsomal epoxide hydrolase, but not cholesterol epoxide hydrolase, and their substrates

Mammalian soluble and microsomal epoxide hydrolases have been proposed to belong to the family of alpha/beta-hydrolase-fold enzymes. These enzymes hydrolyse their substrates by a catalytic triad, with the first step of the enzymatic reaction being the formation of a covalent enzyme-substrate ester. In the present paper, we describe the direct visualization of the ester formation between rat microsomal epoxide hydrolase and its substrate. Microsomal epoxide hydrolase was precipitated with acetone after brief incubation with [1-(14)C]epoxystearic acid. After denaturing SDS gel electrophoresis the protein-bound radioactivity was detected by fluorography. Pure epoxide hydrolase and crude microsomes showed a single radioactive signal of the expected molecular mass that could be suppressed by inclusion of the competitive inhibitor 1,1,1-trichloropropene oxide in the incubation mixture. In a similar manner, 4-fluorochalcone-oxide-sensitive binding of epoxystearic acid to rat soluble epoxide hydrolase could be demonstrated in rat liver cytosol. Under similar conditions, no covalent binding of [26-(14)C]cholesterol-5alpha,6alpha-epoxide to microsomal proteins or solubilized fractions tenfold enriched in cholesterol epoxide hydrolase activity could be observed. Our data provide definitive proof for the formation of an enzyme-substrate-ester intermediate formed in the course of epoxide hydrolysis by microsomal epoxide hydrolase, show no formation of a covalent intermediate between cholesterol epoxide hydrolase and its substrate under the same conditions as those under which an intermediate was shown for both microsomal and soluble epoxide hydrolases and therefore indicate that the cholesterol epoxide hydrolase apparently does not act by a similar mechanism and is probably not structurally related to microsomal and soluble epoxide hydrolases.     

Site-directed mutagenesis of human leukotriene C4 synthase

The functional characteristics of leukotriene C4 synthase (LTC4S), which specifically conjugates leukotriene A4 with GSH, were assessed by mutagenic analysis. Human LTC4S and the 5-lipoxygenase-activating protein share substantial amino acid identity and predicted secondary structure. The mutation of Arg-51 of LTC4S to Thr or Ile abolishes the enzyme function, whereas the mutation of Arg-51 to His or Lys provides a fully active recombinant protein. The mutations Y59F, Y97F, Y93F, N55A, V49F, and A52S increase the Km of the recombinant microsomal enzyme for GSH. The mutation Y93F also markedly reduces enzyme function and increases the optimum for pH-dependent activity. The deletion of the third hydrophobic domain with the carboxyl terminus abolishes the enzyme activity, and function is restored by the substitution of the third hydrophobic domain and carboxyl terminus of 5-lipoxygenase-activating protein for that of LTC4S. Mutations of C56S and C82V alone or together and the deletion of Lys-2 and Asp-3 of LTC4S do not alter enzyme function. The direct linkage of two LTC4S monomers by a 12-amino acid bridge provides an active dimer, and the same bridging of inactive R51I with a wild-type monomer creates an active pseudo-dimer with function similar to that of the wild-type enzyme. These results suggest that in the catalytic function of LTC4S, Arg-51 probably opens the epoxide ring and Tyr-93 provides the thiolate anion of GSH. Furthermore, the monomer has independent conjugation activity, and dimerization of LTC4S maintains the proper protein structure.     

Driving the system: long-term-care coordination in Manitoba, Canada

We examined the enzymatic activity of leukotriene (LT) A4 hydrolase, which catalyzes the conversion of LTA4 to LTB4, in peripheral leukocytes of patients with atopic dermatitis. The patients were divided into three categories (severe, moderate and mild) on the basis of clinical severity. The LTA4 hydrolase activities in the supernatant fraction of peripheral blood polymorphonuclear leukocytes (PMN) were significantly higher in preparations of cells from severe atopic dermatitis patients (123.94 +/- 16.61 pmol/10(6) cells per min) than in those from moderate (49.03 +/- 9.43 pmol/ 10(6) cells per min; P < 0.01) and mild (28.75 +/- 11.42 pmol/10(6) cells per min; P < 0.01) atopic dermatitis patients and normal controls (15.14 +/- 1.74 pmol/10(6) cells per min; P < 0.01). LTA4 hydrolase activities were also higher in peripheral blood mononuclear cells (PBMC) from severe atopic dermatitis patients (27.81 +/- 8.28 pmol/10(6) cells per min) than in those from moderate (11.31 +/- 2.11 pmol/10(6) cells per min; P < 0.05) and mild (6.16 +/- 2.62 pmol/10(6) cells per min; P < 0.05) atopic dermatitis patients and normal controls (11.17 +/- 0.83 pmol/10(6) cells per min; P < 0.05). LTA4 hydrolase activities in PMN were reduced after improvement of the disease in eight patients with severe or moderate atopic dermatitis. These results suggest that LTA4 hydrolase, which synthesizes LTB4, plays a significant role in the pathogenesis and development of atopic dermatitis.     

Mechanism of action and cloning of epoxide hydrolase from the cabbage looper, Trichoplusia ni

The majority of the JH III epoxide hydrolase activity in last stadium day 3 (gate 1) wandering Trichoplusia ni was membrane bound with approximately 9% of the activity found in the cytosol. Both the microsomal and cytosolic JH epoxide hydrolases were stable, retaining 30% of their original activity after incubation at 4 degrees C for 15 days. 18O-labeled water underwent enzyme catalyzed regioselective addition to the least substituted C10 position of JH III. In multiple turnover reactions with JH epoxide hydrolase in 97.9% 18O-labeled water, only 91.3% 18O incorporation was observed. This is consistent with an SN2 reaction likely involving a carboxylate in the active site of JH epoxide hydrolase. The DNA amplification cloning of a fragment of a putative T. ni epoxide hydrolase is reported. The deduced amino acid sequence shares 67% similarity to the rat microsomal epoxide hydrolase.     

Limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis DCL14 belongs to a novel class of epoxide hydrolases

An epoxide hydrolase from Rhodococcus erythropolis DCL14 catalyzes the hydrolysis of limonene-1,2-epoxide to limonene-1,2-diol. The enzyme is induced when R. erythropolis is grown on monoterpenes, reflecting its role in the limonene degradation pathway of this microorganism. Limonene-1,2-epoxide hydrolase was purified to homogeneity. It is a monomeric cytoplasmic enzyme of 17 kDa, and its N-terminal amino acid sequence was determined. No cofactor was required for activity of this colorless enzyme. Maximal enzyme activity was measured at pH 7 and 50 degrees C. None of the tested inhibitors or metal ions inhibited limonene-1,2-epoxide hydrolase activity. Limonene-1,2-epoxide hydrolase has a narrow substrate range. Of the compounds tested, only limonene-1,2-epoxide, 1-methylcyclohexene oxide, cyclohexene oxide, and indene oxide were substrates. This report shows that limonene-1,2-epoxide hydrolase belongs to a new class of epoxide hydrolases based on (i) its low molecular mass, (ii) the absence of any significant homology between the partial amino acid sequence of limonene-1,2-epoxide hydrolase and amino acid sequences of known epoxide hydrolases, (iii) its pH profile, and (iv) the inability of 2-bromo-4'-nitroacetophenone, diethylpyrocarbonate, 4-fluorochalcone oxide, and 1, 10-phenanthroline to inhibit limonene-1,2-epoxide hydrolase activity.     

Formation of a novel enzymatic metabolite of leukotriene A4 in tissues of Xenopus laevis

Leukotriene-A4, hydrolase catalyzes the final step in the biosynthesis of the potent proinflammatory mediator leukotriene B4. Previously, leukotriene-A4 hydrolase has been characterized from human, mouse and rat sources, i.e. only from mammalian species. In the present investigation, expression of leukotriene-A4, hydrolase was studied in organs of Xenopus laevis. Enzyme activity was found in all nine organs tested with the highest levels in the intestine and the reproductive organs, i.e. oocytes and testes, previously unrecognized rich sources of the enzyme. No immunoreactive leukotriene-A4 hydrolase was detected in Western blots of 10000Xg supernatants of X. laevis organ homogenates, using a polyclonal antiserum raised against human leukotriene-A4 hydrolase. Likewise, Northern blot analysis of liver total RNA did not detect Xenopus leukotriene-A4 hydrolase mRNA using a human CDNA probe. These results indicate significant structural differences between the human and toad enzymes. Incubations of 10000Xg supernatants of organ homogenates with leukotriene A4 revealed the formation of a novel metabolite, denoted compound X. Conversion of leukotriene A4 into compound X was due to an enzymatic activity as judged by its protein dependence, heat sensitivity, and resistance to ultrafiltration, and this activity appeared to be linked, directly or indirectly,, to leukotriene A4 hydrolase. From data obtained by ultraviolet spectrophotometry, gas chromatography coupled to mass spectrometry, ultraviolet-induced isomerization, and comparison with a synthetic standard, compound X was assigned the structure 5S,12R-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid. Finally, compound X was found to exhibit contractile activity in guinea-pig lung parenchyma, apparently elicited via a leukotriene B receptor.     

Enzyme kinetic modelling as a tool to analyse the behaviour of cytochrome P450 catalysed reactions: application to amitriptyline N-demethylation

1. To determine kinetic parameters (Vmax, K(m)) for cytochrome P450 (CYP) mediated metabolic pathways, nonlinear least squares regression is commonly used to fit a model equation (e.g., Michaelis Menten [MM]) to sets of data points (reaction velocity vs substrate concentration). This method can also be utilized to determine the parameters for more complex mechanisms involving allosteric or multi-enzyme systems. Akaike's Information Criterion (AIC), or an estimation of improvement of fit as successive parameters are introduced in the model (F-test), can be used to determine whether application of more complex models is helpful. To evaluate these approaches, we have examined the complex enzyme kinetics of amitriptyline (AMI) N-demethylation in vitro by human liver microsomes. 2. For a 15-point nortriptyline (NT) formation rate vs substrate (AMI) concentration curve, a two enzyme model, consisting of one enzyme with MM kinetics (Vmax = 1.2 nmol min-1 mg-1, K(m) = 24 microM) together with a sigmoidal component (described by an equation equivalent to the Hill equation for cooperative substrate binding; Vmax = 2.1 nmol min-1 mg-1, K' = 70 microM; Hill exponent n = 2.34), was favoured according to AIC and the F-test. 3. Data generated by incubating AMI under the same conditions but in the presence of 10 microM ketoconazole (KET), a CYP3A3/4 inhibitor, were consistent with a single enzyme model with substrate inhibition (Vmax = 0.74 nmol min-1 mg-1, K(m) = 186 microM, K1 = 0.0028 microM-1). 4. Sulphaphenazole (SPA), a CYP2C9 inhibitor, decreased the rate of NT formation in a concentration dependent manner, whereas a polyclonal rat liver CYP2C11 antibody, inhibitory for S-mephenytoin 4'-hydroxylation in humans, had no important effect on this reaction. 5. Incubation of AMI with 50 microM SPA resulted in a curve consistent with a two enzyme model, one with MM kinetics (Vmax = 0.72 nmol min-1 mg-1, K(m) = 54 microM) the other with 'Hill-kinetics' (Vmax = 2.1 nmol min-1 mg-1, K' = 195 microM; n = 2.38). 6. A fourth data-set was generated by incubating AMI with 10 microM KET and 50 microM SPA. The proposed model of best fit describes two activities, one obeying MM-kinetics (Vmax = 0.048 nmol min-1 mg-1, K(m) = 7 microM) and the other obeying MM kinetics but with substrate inhibition (Vmax = 0.8 nmol min-1 mg-1, K(m) = 443 microM, K1 = 0.0041 microM-1). 7. The combination of kinetic modelling tools and biological data has permitted the discrimination of at least three CYP enzymes involved in AMI N-demethylation. Two are identified as CYP3A3/4 and CYP2C9, although further work in several more livers is required to confirm the participation of the latter.     

Clinical spectrum of nonmenstrual toxic shock syndrome (TSS): comparison with menstrual TSS by multivariate discriminant analyses

1. The present experiments were undertaken in order to characterize further the apparently irreversible inhibition of the contraction of depolarized rat aorta caused by lacidipine, a 1,4-dihydropyridine calcium antagonist. 2. We studied the effect of lacidipine on contraction evoked by 100 mM KCl solution in rat aorta, treated by N omega-nitro-L-arginine (0.1 mM), an inhibitor of nitric oxide (NO) synthesis. We compared the effect of prolonged depolarization on lacidipine and (+)-isradipine inhibition and the reversal of this inhibition after washout in the absence of dihydropyridines. Assuming that the onset of lacidipine-evoked inhibition was a pseudo-first order association kinetics, we estimated the dissociation rate constant (k-1 = 0.031 min-1), the association rate constant (k1 = 2.70 x 10(8) M-1 min-1) and the dissociation constant (KD = k-1/k1 = 115 pM) which was close to the IC50 value in steady-state conditions (160 pM). 3. The inhibitory effects of lacidipine and (+)-isradipine on rat aorta contraction were reversibly enhanced after preincubation with the drug in a 40 mM KCl-solution. Washout with drug-free 40 mM K(+)-depolarizing solution reversed inhibition in the (+)-isradipine-treated preparations, but not in the lacidipine-treated ones. 4. Radioligand binding studies were performed with [3H]-lacidipine and [3H]-isradipine in microsomes from rat aorta and rat ileum. Both ligands bound to a homogeneous population of binding sites (for[3H]-lacidipine: KD = 23 +/- 2.6 pM, Bmax = 380 +/- 21 fmol mg-1 protein in membranes from aorta; KD =23 +/- 3.1 pM, Bmax = 790 +/- 60 fmol mg-1 protein in membranes from ileum; for [3H]-isradipine:KD = 140 +/- 46 pM, Bmax = 350 +/- 64 fmol mg-1 protein in membrane from aorta; KD = 68 +/- 14 pM,Bmax = 760 +/- 75 fmol mg-1 protein in membranes from ileum). After isotopic dilution, [3H]-lacidipine and [3H]-isradipine dissociated according to a monoexponential kinetics. In membranes from ileum, the calculated dissociation rate constants (kappa_ 1) were 0.0257 min-1 and 0.0595 min-1, for [3H]-lacidipine and[3H]-isradipine, respectively.5. The non specific binding of [3H]-lacidipine and [3H]-isradipine, was measured in intact rat aorta preparations incubated under the conditions of the functional experiments, in the presence of nifedipine(1 microM). After incubation with [3H]-lacidipine 77.6 +/- 1.9 pM for 2 h the concentration of drug in the tissue was 15.15 +/- 1.18 fmol mg-1 w.wt. and still amounted to 7.24 +/- 0.61 fmol mg-1 w.wt. after 3.5 h washout in drug-free solution. After incubation with [3H]-isradipine 47.2 +/- 0.4 pM for 2 h it was 2.26 +/-0.07 fmol mg-1 w.wt. and was undetectable after 3.5 h washout in a drug-free solution.6. It is concluded that lacidipine interacts reversibly with dihydropyridine binding sites and that the apparent irreversible inhibition of contraction in depolarized preparations could be related to a nonspecific binding in a tissue compartment different from the plasma membrane.     

Kinetic mechanism of rabbit muscle glycogen synthase I

The kinetic mechanism of rabbit muscle glycogen synthase I was investigated by determining isotope-exchange rates at chemical equilibrium between uridine diphosphoglucose (UDPG) and glycogen and between UDPG and uridine 5'-diphosphate (UDP). The rates were followed simultaneously by use of UDPG labeled with 14C in the glucose moiety and with 3H in the uracil group. They were found to be independent of the concentrations of glycogen and the UDPG-UDP pair, averaging 6 X 10(-9) mol min-1 mg-1, with a ratio of UDPG-glycogen exchange to UDPG-UDP exchange of 0.85-0.95. The conclusion is that glycogen synthase has a rapid equilibrium random bi bi mechanism. The previously reported slow activation of glycogen-free synthase in the presence of glycogen was examined kinetically. The activation rate appears to be independent of glycogen concentration over a wide range, while the maximum activation is related to the third or fourth root of the glycogen concentration. This suggest that the slow bimolecular reaction mechanism proposed for human polymorphonuclear leucocyte glycogen synthase I [S?lling, H., & Esmann, V. (1977) Eur. J. Biochem. 81, 129] does not apply to rabbit muscle synthase I. The rate of exchange of glycogen molecules in the complex between glycogen and rabbit muscle synthase I under conditions where the enzyme is catalytically active was estimated by a novel method. The enzyme-glycogen complex was treated with [glucose-14C]UDPG and glycogen of different molecular weight. The distribution of isotope between the two forms of glycogen was determined after their separation by agarose gel chromatography. A rate constant of 0.3 min-1 was estimated for the exchange. It can be calculated, on the basis of the specific activity of the enzyme (20 mumol min-1 mg-1) and its action pattern, that hundreds of individual chains in the glycogen molecule must be available to the enzyme during the average lifetime of the complex. A mechanism is proposed for this process.     

Reactions catalyzed by tetrahydrobiopterin-free nitric oxide synthase

Murine macrophage nitric oxide synthase (NOS) was expressed in E. coli and purified in the presence (holoNOS) or absence (H4B-free NOS) of (6R)-tetrahydro-L-biopterin (H4B). Isolation of active enzyme required the coexpression of calmodulin. Recombinant holoNOS displayed similar spectral characteristics and activity as the enzyme isolated from murine macrophages. H4B-free NOS exhibited a Soret band at approximately 420 nm and, by analytical gel filtration, consisted of a mixture of monomers and dimers. H4B-free NOS catalyzed the oxidation of NG-hydroxy-L-arginine (NHA) with either hydrogen peroxide (H2O2) or NADPH and O2 as substrates. No product formation from arginine was observed under either condition. The amino acid products of NHA oxidation in both the H2O2 and NADPH/O2 reactions were determined to be citrulline and Ndelta-cyanoornithine (CN-orn). Nitrite and nitrate were also formed. Chemiluminescent analysis did not detect the formation of nitric oxide (*NO) in the NADPH/O2 reaction. The initial inorganic product of the NADPH/O2 reaction is proposed to be the nitroxyl anion (NO-) based on the formation of a ferrous nitrosyl complex using the heme domain of soluble guanylate cyclase as a trap, and the formation of a ferrous nitrosyl complex of H4B-free NOS during turnover of NHA and NADPH. NO- is unstable and, under the conditions of the reaction, is oxidized to nitrite and nitrate. At 25 degreesC, the H2O2-supported reaction had a specific activity of 120 +/- 14 nmol min-1 mg-1 and the NADPH-supported reaction had a specific activity of 31 +/- 6 nmol min-1 mg-1 with a KM,app for NHA of 129 +/- 9 microM. HoloNOS catalyzed the H2O2-supported reaction with a specific activity of 815 +/- 30 nmol min-1 mg-1 and the NADPH-dependent reaction to produce *NO and citrulline at 171 +/- 20 nmol min-1 mg-1 with a KM, app for NHA in the NADPH reaction of 36.9 +/- 0.3 microM.     

Primary structure and catalytic mechanism of the epoxide hydrolase from Agrobacterium radiobacter AD1

The epoxide hydrolase gene from Agrobacterium radiobacter AD1, a bacterium that is able to grow on epichlorohydrin as the sole carbon source, was cloned by means of the polymerase chain reaction with two degenerate primers based on the N-terminal and C-terminal sequences of the enzyme. The epoxide hydrolase gene coded for a protein of 294 amino acids with a molecular mass of 34 kDa. An identical epoxide hydrolase gene was cloned from chromosomal DNA of the closely related strain A. radiobacter CFZ11. The recombinant epoxide hydrolase was expressed up to 40% of the total cellular protein content in Escherichia coli BL21(DE3) and the purified enzyme had a kcat of 21 s-1 with epichlorohydrin. Amino acid sequence similarity of the epoxide hydrolase with eukaryotic epoxide hydrolases, haloalkane dehalogenase from Xanthobacter autotrophicus GJ10, and bromoperoxidase A2 from Streptomyces aureofaciens indicated that it belonged to the alpha/beta-hydrolase fold family. This conclusion was supported by secondary structure predictions and analysis of the secondary structure with circular dichroism spectroscopy. The catalytic triad residues of epoxide hydrolase are proposed to be Asp107, His275, and Asp246. Replacement of these residues to Ala/Glu, Arg/Gln, and Ala, respectively, resulted in a dramatic loss of activity for epichlorohydrin. The reaction mechanism of epoxide hydrolase proceeds via a covalently bound ester intermediate, as was shown by single turnover experiments with the His275 --> Arg mutant of epoxide hydrolase in which the ester intermediate could be trapped.     

Pharmacokinetics of acyclovir in the cat

To investigate the detoxification of bromobenzene-induced hepatic lipid peroxidation by Oenanthe javanica DC, the hepatic lipid peroxide level and the activities of enzymes responsible for production and removal of epoxide were studied. The level of lipid peroxide elevated by bromobenzene was significantly reduced by the methanol extract (250 mg/kg) and persicarin (5 mg/kg). The methanol extract and persicarin administered daily over 4 weeks before intoxication with bromobenzene did not affect the activities of aminopyrine N-demethylase, aniline hydroxylase, and glutathione S-transferase. Epoxide hydrolase activity was decreased significantly by bromobenzene, which was restored to the control level by pretreatment with persicarin. However, the identical pretreatment with isorhamnetin and hyperoside did not change the enzyme activity or lipid peroxide level. The results suggest that the reduction of bromobenzene-induced hepatic lipid peroxidation by O. javanica under our experimental conditions is effected through enhancing the activity of epoxide hydrolase, an enzyme removing bromobenzene epoxide. In addition, the bioactive component of this plant responsible for the detoxification of bromobenzene, at least in part, is thought to be persicarin.