Nuclear localization of leukotriene A4 hydrolase in type II alveolar epithelial cells in normal and fibrotic lung

Leukotriene A4 (LTA4) hydrolase catalyzes the final step in leukotriene B4 (LTB4) synthesis. In addition to its role in LTB4 synthesis, the enzyme possesses aminopeptidase activity. In this study, we sought to define the subcellular distribution of LTA4 hydrolase in alveolar epithelial cells, which lack 5-lipoxygenase and do not synthesize LTA4. Immunohistochemical staining localized LTA4 hydrolase in the nucleus of type II but not type I alveolar epithelial cells of normal mouse, human, and rat lungs. Nuclear localization of LTA4 hydrolase was also demonstrated in proliferating type II-like A549 cells. The apparent redistribution of LTA4 hydrolase from the nucleus to the cytoplasm during type II-to-type I cell differentiation in vivo was recapitulated in vitro. Surprisingly, this change in localization of LTA4 hydrolase did not affect the capacity of isolated cells to convert LTA4 to LTB4. However, proliferation of A549 cells was inhibited by the aminopeptidase inhibitor bestatin. Nuclear accumulation of LTA4 hydrolase was also conspicuous in epithelial cells during alveolar repair following bleomycin-induced acute lung injury in mice, as well as in hyperplastic type II cells associated with fibrotic lung tissues from patients with idiopathic pulmonary fibrosis. These results show for the first time that LTA4 hydrolase can be accumulated in the nucleus of type II alveolar epithelial cells and that redistribution of the enzyme to the cytoplasm occurs with differentiation to the type I phenotype. Furthermore, the aminopeptidase activity of LTA4 hydrolase within the nucleus may play a role in promoting epithelial cell growth.     

Purification and characterization of leukotriene A4 epoxide hydrolase from dog lung

Leukotriene A4 epoxide hydrolase from dog lung, a soluble enzyme catalyzing the hydrolysis of leukotriene A4 (LTA4) to leukotriene B4 (LTB4) was partially purified by anion exchange HPLC. The enzymatic reaction obeys Michaelis- Menten kinetics. The apparent Km ranged between 15 and 25 microM and the enzyme exhibited an optimum activity at pH 7.8. An improved assay for the epoxide hydrolase has been developed using bovine serum albumin and EDTA to increase the conversion of LTA4 to LTB4. This method was used to produce 700 mg of LTB4 from LTA4 methyl ester. The partial by purified enzyme was found to be uncompetitively inhibited by divalent cations. Ca+2, Mn+2, Fe+2, Zn+2 and Cu+2 were found to have inhibitor constants (Ki) of 89 mM, 3.4 mM, 1.1 mM, 0.57 mM, and 28 microM respectively Eicosapentaenoic acid was shown to be a competitive inhibitor of this enzyme with a Ki of 200 microM. From these inhibition studies, it can be theorized that the epoxide hydrolase has at least one hydrophobic and one hydrophilic binding site.     

Bestatin inhibits covalent coupling of [3H]LTA4 to human leukocyte LTA4 hydrolase.

The covalent coupling of [3H]LTA4 to human leukocyte LTA4 hydrolase is inhibited in a concentration-dependent fashion by pre-incubation with bestatin. This inhibition correlated with the concentration-dependent inhibition by bestatin of LTB4 and LTB5 synthesis by LTA4 hydrolase. Epibestatin, a diastereomer of bestatin, neither inhibited LTB4 or LTB5 production by LTA4 hydrolase nor prevented the covalent coupling of [3H]LTA4 to the enzyme. In contrast, captopril inhibited both LTB4 synthesis by LTA4 hydrolase and covalent coupling of [3H]LTA4 to the enzyme.     

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.     

Leukotriene A4 hydrolase, insights into the molecular evolution by homology modeling and mutational analysis of enzyme from Saccharomyces cerevisiae

Mammalian leukotriene A4 (LTA4) hydrolase is a bifunctional zinc metalloenzyme possessing an Arg/Ala aminopeptidase and an epoxide hydrolase activity, which converts LTA4 into the chemoattractant LTB4. We have previously cloned an LTA4 hydrolase from Saccharomyces cerevisiae with a primitive epoxide hydrolase activity and a Leu aminopeptidase activity, which is stimulated by LTA4. Here we used a modeled structure of S. cerevisiae LTA4 hydrolase, mutational analysis, and binding studies to show that Glu-316 and Arg-627 are critical for catalysis, allowing us to a propose a mechanism for the epoxide hydrolase activity. Guided by the structure, we engineered S. cerevisiae LTA4 hydrolase to attain catalytic properties resembling those of human LTA4 hydrolase. Thus, six consecutive point mutations gradually introduced a novel Arg aminopeptidase activity and caused the specific Ala and Pro aminopeptidase activities to increase 24 and 63 times, respectively. In contrast to the wild type enzyme, the hexuple mutant was inhibited by LTA4 for all tested substrates and to the same extent as for the human enzyme. In addition, these mutations improved binding of LTA4 and increased the relative formation of LTB4, whereas the turnover of this substrate was only weakly affected. Our results suggest that during evolution, the active site of an ancestral eukaryotic zinc aminopeptidase has been reshaped to accommodate lipid substrates while using already existing catalytic residues for a novel, gradually evolving, epoxide hydrolase activity. Moreover, the unique ability to catalyze LTB4 synthesis appears to be the result of multiple and subtle structural rearrangements at the catalytic center rather than a limited set of specific amino acid substitutions.     

Crystal structure of human leukotriene A(4) hydrolase, a bifunctional enzyme in inflammation

Leukotriene (LT) A(4) hydrolase/aminopeptidase (LTA4H) is a bifunctional zinc enzyme that catalyzes the biosynthesis of LTB4, a potent lipid chemoattractant involved in inflammation, immune responses, host defense against infection, and PAF-induced shock. The high resolution crystal structure of LTA4H in complex with the competitive inhibitor bestatin reveals a protein folded into three domains that together create a deep cleft harboring the catalytic Zn(2+) site. A bent and narrow pocket, shaped to accommodate the substrate LTA(4), constitutes a highly confined binding region that can be targeted in the design of specific anti-inflammatory agents. Moreover, the structure of the catalytic domain is very similar to that of thermolysin and provides detailed insight into mechanisms of catalysis, in particular the chemical strategy for the unique epoxide hydrolase reaction that generates LTB(4).     

Leukotriene A4 hydrolase, a bifunctional enzyme. Distinction of leukotriene A4 hydrolase and aminopeptidase activities by site-directed mutagenesis at Glu-297.

We previously obtained evidence for intrinsic aminopeptidase activity for leukotriene (LT)A4 hydrolase, an enzyme characterized to specifically catalyse the hydrolysis of LTA4 to LTB4, a chemotactic compound. From a sequence homology search between LTA4 hydrolase and several aminopeptidases, it became clear that they share a putative active site for known aminopeptidases and a zinc binding domain. Thus, Glu-297 of LTA4 hydrolase is a candidate for the active site of its aminopeptidase activity, while His-296, His-300 and Glu-319 appear to constitute a zinc binding site. To determine whether or not this putative active site is also essential to LTA4 hydrolase activity, site-directed mutagenesis experiments were carried out. Glu-297 was mutated into 4 different amino acids. The mutant E297Q (Glu changed to Gln) conserved LTA4 hydrolase activity but showed little aminopeptidase activity. Other mutants at Glu-297 (E297A, E297D and E297K) showed markedly reduced amounts of both activities. It is thus proposed that either a glutamic or glutamine moiety at 297 is required for full LTA4 hydrolase activity, while the free carboxylic acid of glutamic acid is essential for aminopeptidase.     

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.     

The slow, tight binding of bestatin and amastatin to aminopeptidases

Bestatin reversibly inhibits Aeromonas aminopeptidase (EC 3.4.11.10) in a process that is remarkable for its unusual degree of time dependence. The binding of bestatin by both Aeromonas aminopeptidase and cytosolic leucine aminopeptidase (EC 3.4.11.1) is slow and tight, with Ki values (determined from rate constants) of 1.8 X 10(-8) and 5.8 X 10(-10) M, respectively. In contrast, microsomal aminopeptidase (EC 3.4.11.2) binds bestatin in a rapidly reversible process with a Ki value of 1.4 X 10(-6) M. Kinetic analysis of the slow inhibition observed is facilitated by the use of a variety of experimental treatments, primarily measurements made during pre-equilibrium; however, careful selection of conditions permits use also of steady state observations. When titrated with bestatin, 1 mol of cytosolic leucine aminopeptidase (containing 6 g atoms each of zinc and manganese) is rendered 80% inactive by 1 mol of inhibitor, thus suggesting that enzymatic activity depends on one active site/hexamer; titration of Aeromonas aminopeptidase by bestatin reveals a 1:1 stoichiometry. Amastatin inhibits all three aminopeptidases through the mechanism of slow, tight binding with Ki values ranging from 3.0 X 10(-8) to 2.5 X 10(-10) M. This behavior of microsomal aminopeptidase contrasts sharply with its rapidly reversible inhibition by bestatin. The slow, tight binding observed with five of the six aminopeptidase-inhibitor pairs investigated suggests the formation of a transition state analog complex between the enzyme and inhibitor. Physical evidence consistent with this possibility was provided by the observation that both bestatin and amastatin perturb the absorption spectrum of cobalt Aeromonas aminopeptidase.     

Impairment of IGF-I gene splicing and MGF expression associated with muscle wasting

An aminopeptidase was purified from bovine skeletal muscle by ammonium sulfate fractionation and by successive chromatographies of DEAE-cellulose, Sehacryl S-200, phenyl-sepharose CL-4B, hydroxyapatite and Hi-Trap chelating HP columns. The aminopeptidase was purified about 14-fold over the crude extract with a yield of 1.0% activity. The molecular mass of the enzyme was found to be 58 kDa on SDS-PAGE. The enzyme activity was enhanced by the addition of some anions, such as Cl(-), NO(3)(-) and SCN(-), which is the most unique property of this enzyme. While, the activity was strongly inhibited by bestatin, PMSF and puromycin, suggesting that it was a serine protease. In addition, this enzyme was identical with leukotriene (LT) A4 hydrolase, converting LTA4 to LTB4.     

Identification of aminopeptidase M as an enkephalin-inactivating enzyme in rat cerebral membranes

Two membrane-bound enkephalin-hydrolyzing aminopeptidase activities were partially purified from rat brain membranes. The first, which represents 90% of the total activity, was highly sensitive to both puromycin (Ki = 1 microM) and bestatin (Ki = 0.5 microM). The second was inhibited much more by bestatin (Ki = 4 microM) than by puromycin (Ki = 100 microM). The latter puromycin-insensitive aminopeptidase was found to resemble aminopeptidase M purified from rat kidney brush border membranes. Both displayed the same purification pattern and the same kinetic constants of substrates and inhibitors, and both were similarly inactivated by metal chelating agents. Moreover, antibodies raised in rabbits against rat kidney aminopeptidase M inhibited the aminopeptidase activities of both kidney and brain puromycin-insensitive enzymes at similar dilutions, while the brain puromycin-sensitive aminopeptidase activity was not affected. Thus, aminopeptidase M (EC 3.4.11.2) was found to occur in brain, and the role of this enzyme in inactivating endogenous enkephalins released from their neuronal stores is suggested.     

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.     

Determination of the contribution of cysteinyl leukotrienes and leukotriene B4 in acute inflammatory responses using 5-lipoxygenase- and leukotriene A4 hydrolase-deficient mice

Arachidonic acid metabolism by 5-lipoxygenase leads to production of the potent inflammatory mediators, leukotriene (LT) B4 and the cysteinyl LT. Relative synthesis of these subclasses of LT, each with different proinflammatory properties, depends on the expression and subsequent activity of LTA4 hydrolase and LTC4 synthase, respectively. LTA4 hydrolase differs from other proteins required for LT synthesis because it is expressed ubiquitously. Also, in vitro studies indicate that it possesses an aminopeptidase activity. Introduction of cysteinyl LT and LTB4 into animals has shown LTB4 is a potent chemoattractant, while the cysteinyl LT alter vascular permeability and smooth muscle tone. It has been impossible to determine the relative contributions of these two classes of LT to inflammatory responses in vivo or to define possible synergy resulting from the synthesis of both classes of mediators. To address this question, we have generated LTA4 hydrolase-deficient mice. These mice develop normally and are healthy. Using these animals, we show that LTA4 hydrolase is required for the production of LTB4 in an in vivo inflammatory response. We show that LTB4 is responsible for the characteristic influx of neutrophils accompanying topical arachidonic acid and that it contributes to the vascular changes seen in this model. In contrast, LTB4 influences only the cellular component of zymosan A-induced peritonitis. Furthermore, LTA4 hydrolase-deficient mice are resistant to platelet-activating factor, identifying LTB4 as one mediator of the physiological changes seen in systemic shock. We do not identify an in vivo role for the aminopeptidase activity of LTA4 hydrolase.     

Leukotriene A5 is a substrate and an inhibitor of rat and human neutrophil LTA4 hydrolase

The epoxide 5(S) trans-5,6 oxido, 7,9 trans-11,14,17 cis eicosatetraenoic acid (leukotriene A5) was chemically synthesized and demonstrated to be both a substrate and an inhibitor of partially purified rat and human LTA4 hydrolase. Both rat and human LTA4 hydrolase utilized leukotriene A5 less effectively as a substrate than leukotriene A4. Incubation of leukotriene A5 (10 microM) or leukotriene A4 (10 microM) with rat neutrophils demonstrated formation of 123 pmol LTB5/min/10(7) cells and 408 pmol LTB4/min/10(7) cells respectively. Purified rat neutrophil LTA4 hydrolase incubated with 100 microM leukotriene A5 produced 22 nmol LTB5/min/mg protein and when incubated with 100 microM leukotriene A4 produced 50 nmol LTB4/min/mg protein. Human neutrophil LTA4 hydrolase incubated with 100 microM leukotriene A5 produced 24 nmol LTB5/min/mg protein and when incubated with 100 microM leukotriene A4 produced 52 nmol LTB4/min/mg protein. Leukotriene A5 was an inhibitor of the formation of leukotriene B4 from leukotriene A4 by both the rat and human neutrophil LTA4 hydrolase. Excess leukotriene A5 prevented covalent coupling of [3H] leukotriene A4 to LTA4 hydrolase suggesting inhibition may involve covalent coupling of leukotriene A5 to the LTA4 hydrolase.     

Monkey brain arylamidase. II. Further characterization and studies on mode of hydrolysis of physiologically active peptides.

A large-scale purification of monkey brain arylamidase was carried out. Amino acid analyses indicate that the enzyme is rich in acidic amino acids and is poor in cystine. The amino terminal residue was determined to be alanine by dansylation. The enzyme was activated by sulfhydryl compounds. Dithiothreitol was more effective than beta-mercaptoethanol. Bestatin competitively inhibited the enzyme activity and the Ki value was calculated to be 2.5 x 10(-7) M, which was of the same order as that of puromycin. The inhibitions by puromycin and bestatin were reversible. The enzyme hydrolyzed di-, tri-, and oligopeptides including physiologically active peptides. Of physiologically active peptides, enkephalins and Met-Lys-bradykinin, which possess a neutral amino acid at the N-terminal position, were more rapidly hydrolyzed by the enzyme. Peptides such as LH-RH and TRH, which possess a pyrrolidonecarboxylyl group at the N-terminal position, and substance P and bradykinin, which possess a proline residue adjacent to the N-terminal residue, were not hydrolyzed by the enzyme. The Km values for various peptides indicate that the enzyme has higher affinity for oligopeptides than di- and tripeptides. The aminopeptidase activity of the enzyme was also competitively inhibited by puromycin and bestatin. Analyses of the hydrolysis products of various peptides by the dansylation method indicate that the enzyme has both kinin-converting activity and angiotensinase activity.     

Bestatin inhibition of human tissue carnosinase, a non-specific cytosolic dipeptidase

Bestatin is a dipeptide containing a unique beta-amino acid. It is usually referred to as an aminopeptidase inhibitor. Current interest has focused on the immunostimulating activity of bestatin and several clinical trials have demonstrated that it is an effective adjunct to radiation or chemotherapy in the treatment of certain types of cancer. We found that bestatin was much more effective against human tissue carnosinase than against aminopeptidases. Inhibition was competitive, with a Ki of 0.5nM. Carnosinase did not hydrolyse bestatin and the enzyme-inhibitor complex formed rapidly. A hog kidney dipeptidase similar to human tissue carnosinase was equally sensitive to this inhibitor. Bestatin has a backbone structure identical to that of carnosine; however, our results indicate that the inhibitory activity of this compound is primarily attributable to the side chains of the beta-amino-acid moiety. Human tissue carnosinase is a non-specific dipeptidase, actively hydrolysing many dipeptides, including prolinase substrates. Inhibition of this cytosolic enzyme is probably at least partially responsible for the intracellular accumulation of dipeptides which occurs following the in vivo administration of bestatin.     

Use of azidobestatin as a photoaffinity label to identify the active site peptide of leucine aminopeptidase.

Aminopeptidases catalyze the hydrolysis of amino acid residues from the amino terminus of peptide substrates. They are found in most cells and tissues, and their activity has been implicated in myriad fundamental biochemical and physiological processes. Nevertheless, little is known about the structure of the aminopeptidase active sites. Beef lens leucine aminopeptidase (blLAP) can be considered prototypical of many enzymes in this family of peptidases. Bestatin, [(2S,3R)-(3-amino-2-hydroxy-4-phenyl-butanoyl)-L-leucine] is a nonhydrolyzable substrate analogue of a peptide, PheLeu, which is rapidly cleaved by blLAP. Bestatin incorporates elements of the putative tetrahedral intermediate, and this results in a greater than 10(5)-fold enhancement of binding relative to analogous peptides. Bestatin is the most tightly bound inhibitor of many aminopeptidases. Bestatin was successively converted to nitrobestatin, p-aminobestatin, [3H]-p-aminobestatin, and finally [3H]-p-azidobestatin (pAB). Like bestatin, pAB is a slow binding inhibitor of LAP (Ki*, the dissociation constant for the final complex, = approximately 4 x 10(-9); Ki, the dissociation constant for the initial collision complex, = approximately 10(-8). The t1/2 for binding of 2 x 10(-8) M and 8 x 10(-8) M bestatin are approximately 60 min and approximately 38 min, respectively. pAB, nitrobestatin, bestatin, and physiological peptides appear to bind in the same site, the first three with similar avidity. In the dark, pAB and bestatin protect low concentrations of the enzyme against inactivation upon extensive dialysis. The t1/2 for photoactivation of pAB is approximately 3 s. Irradiation of blLAP for such short periods of time resulted in insignificant change in activity. blLAP which was placed in 254-nm light in the presence of pAB was inactivated significantly. Treatment of photolabeled blLAP with trypsin produces only two peptides. Autoradiography and scintillation counting indicate that the active site is in the peptide which includes residues 138-487. Treatment of the same blLAP with hydroxylamine produces two different peptides, with the active site in the peptide 323-487. This indicates that the active site is in the carboxyl-terminal one-third of the protomer. It is likely that this photoaffinity label will be useful in identifying active sites in other aminopeptidases as well.     

Surface aminopeptidase activity of human lymphocytes. I. Biochemical and biologic properties of intact cells

Surface aminopeptidase activity in intact lymphocytes was studied and was shown to have the following properties when alanine-p-nitroanilide was used as substrate: 1) The activity was surface associated and not secreted as determined by extracellular location of product and the effect of proteases and diazotized sulfanilic acid on enzyme activity. 2) The enzyme activity was shown to have a pH optimum of 7.4 to 8.0. 3) Enzyme activity was shown to be inhibited by amastatin, bestatin, and 1,10 phenanthroline. Inhibition by amastatin consisted of a high-affinity component (Ki = 3.5 x 10(-6) M) which accounted for approximately 20% of the total activity and a low-affinity component (Ki = 3.5 x 10(-5) M) which accounted for the remainder suggesting that two forms of aminopeptidase exist. Only a single component of inhibition was seen with bestatin (Ki = 3.5 x 10(-6) M) and 1,10 phenanthroline (Ki = 2.0 x 10(-4) M) which accounted for 80 and 90% of the total enzyme activity, respectively. Unlike the competitive inhibitors bestatin and amastatin, inhibition by 1,10 phenanthroline was shown to be non-competitive. Finally, surface aminopeptidase activity essentially doubled in the presence of PHA (10 micrograms/ml) or Con A (10 micrograms/ml), at 72 h. This enhancing effect was shown to be dose dependent, time dependent, and mitogen dependent and correlated with the cellular state of activation as determined by [3H]TdR incorporation.     

1-[4-[3-[4-[bis(4-fluorophenyl)hydroxymethyl]-1- piperidinyl]propoxy]-3-methoxyphenyl]ethanone(AHR-5333): a selective human blood neutrophil 5-lipoxygenase inhibitor

In this study we report the in vitro inhibition of leukotriene synthesis in calcium ionophore (A23187)-stimulated, intact human blood neutrophils by AHR-5333. The results showed that AHR-5333 inhibits 5-HETE, LTB4 and LTC4 synthesis with IC50 values of 13.9, 13.7 and 6.9 microM, respectively. Further examination of the effect of AHR-5333 on individual reactions of the 5-lipoxygenase pathway (i.e. conversion of LTA4 to LTB4, LTA4 to LTC4, and arachidonic acid to 5-HETE) showed that this agent was not inhibitory to LTA4 epoxyhydrolase and glutathione-S-transferase activity in neutrophil homogenates. However, conversion of arachidonic acid (30 microM) to 5-HETE was half maximally inhibited by 20 microM AHR-5333 in the cell-free system. The inhibition of LTB4 and LTC4 formation in intact neutrophils by AHR-5333 appears to be entirely due to a selective inhibition of 5-lipoxygenase activity and an impaired formation of LTA4, which serves as substrate for LTA4 epoxyhydrolase and glutathione-S-transferase. AHR-5333 did not affect the transformation of exogenous arachidonic acid to thromboxane B2, HHT and 12-HETE in preparations of washed human platelets, indicating that this agent has no effect on platelet prostaglandin H synthase, thromboxane synthase and 12-lipoxygenase activity. The lack of inhibitory activity of AHR-5333 on prostaglandin H synthase activity was confirmed with microsomal preparations of sheep vesicular glands.     

Leukotriene A4 hydrolase is a zinc-containing aminopeptidase

A comparison of amino acid sequences revealed that leukotriene A4 (LTA4) hydrolase is homologous to various types of aminopeptidases. Consistently with the finding, the purified LTA4 hydrolases from both human and guinea pig sources contained equimolar zinc ion, as determined by atomic absorption spectrometry. The enzyme had a significant amount of aminopeptidase activity toward synthetic peptide substrates. Both LTA4 hydrolase and aminopeptidase activities were inhibited by o-phenanthroline, p-chloromercuribenzoic acid, and Leu-thiol with similar IC50 values. Co-purification as well as co-immunoprecipitation of both enzyme activities with an affinity-purified antibody against LTA4 hydrolase strongly suggest that the two enzyme activities reside in a single protein.