Photoaffinity labeling and purification of ZG-16p, a high-affinity dihydropyridine binding protein of rat pancreatic zymogen granule membranes that regulates a K(+)-selective conductance.
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In rat pancreatic zymogen granules (ZG), an ATP-sensitive K(+) conductance and a Cl(-) conductance have been characterized that are inversely regulated by an approximately 65-kDa multidrug resistance P-glycoprotein (mdr1) gene product. In search of a label for purification of this protein, we found that the dihydropyridine derivative (-)-[(3)H]BZDC-DHP, a recently developed high-affinity ligand for Mdr1, binds with similar affinity to ZG membranes (ZGM) (K(d) = 6.2 nM). Binding was inhibited by nanomolar concentrations of the L-type Ca(2+) channel blockers azidopine and verapamil and by micromolar concentrations of the K(+) channel blockers glibenclamide and quinidine. Inhibition by glibenclamide was noncompetitive. The Mdr1 modulators cyclosporin A and vinblastine did not inhibit binding, which is different from Mdr1. In addition, only (+/-)-BZDC-DHP, azidopine, and verapamil selectively inhibited the K(+) conductance in ZGs, whereas the Cl(-) conductance was not affected. In photoaffinity labeling experiments, (-)-[(3)H]BZDC-DHP surprisingly specifically and selectively labeled a approximately 19-kDa protein in ZGM with a pharmacological profile identical with the high-affinity binding site but did not label a 65-kDa protein. The 19-kDa protein was purified by ion exchange chromatography and SDS-polyacrylamide gel electrophoresis and sequenced. The sequence obtained corresponds to ZG-16p, a recently cloned ZG protein with no apparent homology to Mdr1. The identity of the 19-kDa protein was confirmed by immunoprecipitation of (-)-[(3)H]BZDC-DHP-labeled ZGM with an anti-ZG-16p antibody. Furthermore, it is shown that ZG-16p is associated with the ZGM. We propose that ZG-16p, as part of the submembranous granule matrix, regulates the ATP-sensitive K(+) conductance of ZGs. (+info)
Probing subunit interactions in alpha class rat liver glutathione S-transferase with the photoaffinity label glutathionyl S-[4-(succinimidyl)benzophenone].
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Glutathionyl S-[4-(succinimidyl)benzophenone] (GS-Succ-BP), an analogue of the product of glutathione and electrophilic substrate, acts as a photoaffinity label of dimeric rat liver glutathione S-transferase (GST), isoenzyme 1-1. A time-dependent loss of enzyme activity is observed upon irradiation of the enzyme with long wavelength UV light in the presence of the reagent. The initial rate of inactivation exhibits nonlinear dependence on the concentration of the reagent, characterized by an apparent dissociation constant of the enzyme-reagent complex (K(R)) of 99 +/- 2 microM and k(max) of 0.082 +/- 0.005 min(-1). Protection against this inactivation is provided by the electrophilic substrate (ethacrynic acid), electrophilic substrate analogue (dinitrophenol), and product analogues (S-hexylglutathione and p-nitrobenzylglutathione) but not by steroids (Delta(5)-androstene-3,17-dione and 17beta-estradiol-3, 17-disulfate). These results suggest that GS-Succ-BP binds and reacts with the enzyme within the xenobiotic substrate binding site, and this reaction site is distinct from the substrate and nonsubstrate steroid binding sites of the enzyme. About 1 mol of reagent is incorporated into 1 mol of enzyme dimer when the enzyme is completely inactivated. Met-208 is the only amino acid target of the reagent, and modification of this residue in one enzyme subunit of the GST 1-1 dimer completely abolishes the enzyme activity of both subunits. In order to evaluate the role of subunit interactions in the Alpha class glutathione S-transferases, inactive GS-Succ-BP-modified GST 1-1 was mixed with unlabeled, active GST 2-2. The enzyme subunits were dissociated in dilute trifluoroacetic acid and then renatured at pH 7.8 and separated by chromatofocusing into GST 1-1, 1-2, and 2-2. The specific activities of the heterodimer toward several substrates indicate that the loss of catalytic activity in the unmodified subunit of the modified GST 1-1 is the indirect result of the interaction between the two enzyme subunits and that this subunit interaction is absent in the heterodimer GST 1-2. (+info)
LTA(4)-derived 5-oxo-eicosatetraenoic acid: pH-dependent formation and interaction with the LTB(4) receptor of human polymorphonuclear leukocytes.
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5-oxo-(7E,9E,11Z,14Z)-eicosatetraenoic acid (5-oxo-ETE) has been identified as a non-enzymatic hydrolysis product of leukotriene A(4) (LTA(4)) in addition to 5,12-dihydroxy-(6E,8E,10E, 14Z)-eicosatetraenoic acids (5,12-diHETEs) and 5,6-dihydroxy-(7E,9E, 11Z,14Z)-eicosatetraenoic acids (5,6-diHETEs). The amount of 5-oxo-ETE detected in the mixture of the hydrolysis products of LTA(4) was found to be pH-dependent. After incubation of LTA(4) in aqueous medium, the ratio of 5-oxo-ETE to 5,12-diHETE was 1:6 at pH 7.5, and 1:1 at pH 9.5. 5-Oxo-ETE was isolated from the alkaline hydrolysis products of LTA(4) in order to evaluate its effects on human polymorphonuclear (PMN) leukocytes. 5-Oxo-ETE induced a rapid and dose-dependent mobilization of calcium in PMN leukocytes with an EC(50) of 250 nM, as compared to values of 3.5 nM for leukotriene B(4) (LTB(4)500 nM for 5(S)-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE). Pretreatment of the cells with LTB(4) totally abolished the calcium response induced by 5-oxo-ETE. In contrast, the preincubation with 5-oxo-ETE did not affect the calcium mobilization induced by LTB(4). The calcium response induced by 5-oxo-ETE was totally inhibited by the specific LTB(4) receptor antagonist LY223982. These data demonstrate that 5-oxo-ETE can induce calcium mobilization in PMN leukocyte via the LTB(4) receptor in contrast to the closely related analog 5-oxo-(6E,8Z,11Z, 14Z)-eicosatetraenoic acid which is known to activate human neutrophils by a mechanism independent of the receptor for LTB(4). (+info)
Catechol-O-methyltransferase inhibition attenuates levodopa toxicity in mesencephalic dopamine neurons.
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Inhibition of catechol-O-methyltransferase (COMT; EC 2.1.1.6) is a new therapeutic strategy in the treatment of Parkinson's disease. However, nothing is known about the effects of COMT inhibition on levodopa (L-dopa)-induced toxicity in dopamine (DA) neurons. Therefore we evaluated the effects of the selective COMT inhibitors Ro 41-0960, OR-486, and tolcapone alone and in combination with L-dopa in primary mesencephalic cultures from rat. Neither COMT inhibitor affected the growth of tyrosine hydroxylase immunoreactive (THir) cells with concentrations up to 10 microM when studied alone. However, Ro 41-0960 reduced the L-dopa-induced THir cell loss after 24 h in a dose-dependent manner, shifting the TD(50) value from 21 microM in the absence to 71 microM in the presence of 1 microM Ro 41-0960 (P <.01) without affecting survival of non-DA neurons. OR-486 and the clinically used COMT inhibitor tolcapone showed similar effects. In contrast, toxicity induced by D-dopa was not altered by COMT inhibitors. Furthermore, the primary metabolite of L-dopa formed by COMT, 3-O-methyldopa, and the methyl group donor S-adenosyl-L-methionine used by COMT did not alter THir neuron survival and L-dopa-induced toxicity, respectively, with concentrations up to 100 microM. These data demonstrate that COMT inhibition attenuates L-dopa toxicity toward DA neurons in vitro, but probably not by preventing 3-O-methyldopa production or cellular S-adenosyl-L-methionine depletion. (+info)
The estrogen receptor relative binding affinities of 188 natural and xenochemicals: structural diversity of ligands.
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We have utilized a validated (standardized) estrogen receptor (ER) competitive-binding assay to determine the ER affinity for a large, structurally diverse group of chemicals. Uteri from ovariectomized Sprague-Dawley rats were the ER source for the competitive-binding assay. Initially, test chemicals were screened at high concentrations to determine whether a chemical competed with [3H]-estradiol for the ER. Test chemicals that exhibited affinity for the ER in the first tier were subsequently assayed using a wide range of concentrations to characterize the binding curve and to determine each chemical's IC50 and relative binding affinity (RBA) values. Overall, we assayed 188 chemicals, covering a 1 x 10(6)-fold range of RBAs from several different chemical or use categories, including steroidal estrogens, synthetic estrogens, antiestrogens, other miscellaneous steroids, alkylphenols, diphenyl derivatives, organochlorines, pesticides, alkylhydroxybenzoate preservatives (parabens), phthalates, benzophenone compounds, and a number of other miscellaneous chemicals. Of the 188 chemicals tested, 100 bound to the ER while 88 were non-binders. Included in the 100 chemicals that bound to the ER were 4-benzyloxyphenol, 2,4-dihydroxybenzophenone, and 2,2'-methylenebis(4-chlorophenol), compounds that have not been shown previously to bind the ER. It was also evident that certain structural features, such as an overall ring structure, were important for ER binding. The current study provides the most structurally diverse ER RBA data set with the widest range of RBA values published to date. (+info)
Selective action of a CCK-B/gastrin receptor antagonist, S-0509, on pentagastrin-, peptone meal- and beer-stimulated gastric acid secretion in dogs.
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BACKGROUND: The pharmacological effects of a novel CCK-B/gastrin receptor antagonist, S-0509, on gastric acid secretion in dogs remain unknown. AIM: To evaluate the antisecretory effects of S-0509 on gastric acid secretion and to compare such effects with famotidine or atropine in dogs stimulated with various gastric stimulants. METHODS: Ten beagle dogs with a denervated Heidenhain pouch and three beagle dogs with an innervated gastric fistula were used. Gastric acid secretion was stimulated by either continuous intravenous administration of pentagastrin, carbachol or histamine, or oral administration of a peptone meal or beer. RESULTS: In the Heidenhain pouch model, both intravenously administered and orally administered S-0509 significantly inhibited the gastric acid secretion stimulated by pentagastrin, peptone meal and beer. Nonetheless, the drug had little or no effect on carbachol-stimulated or histamine-stimulated acid secretion. Famotidine extensively inhibited all gastric acid secretion stimulated by the above stimulants in a non-selective manner. Atropine also significantly inhibited the acid secretion stimulated by pentagastrin, peptone meal, beer or carbachol, but was not able to inhibit stimulation due to histamine. Oral administration of peptone meal or beer significantly increased the plasma gastrin level. Similarly to the Heidenhain pouch model, even in the gastric fistula (GF) model, S-0509 significantly inhibited pentagastrin-stimulated gastric acid secretion, yet the drug had no effect on carbachol-stimulated secretion. CONCLUSIONS: S-0509 is a selective CCK-B/gastrin receptor antagonist in dogs that inhibits gastric acid secretion stimulated by pentagastrin and gastrin-releasing substances, but does not inhibit histamine-stimulated and carbachol-stimulated acid secretion. (+info)
The specificity of glucuronidation of entacapone and tolcapone by recombinant human UDP-glucuronosyltransferases.
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The COMT inhibitors entacapone and tolcapone are rapidly metabolized in vivo, mainly by glucuronidation. In this work, the main UGT isoforms responsible for their glucuronidation in vitro were characterized by using a subset of representative cloned and expressed human UGT isoforms. Entacapone in particular was seen to be an exceptionally good substrate for UGT1A9 with an even higher reaction velocity value at 500 microM substrate concentration compared with that of the commonly used substrate, propofol (1.3 and 0.78 nmol min(-1) mg(-1), respectively). Neither entacapone nor tolcapone was glucuronidated by UGT1A6. Tolcapone was not detectably glucuronidated by UGT1A1, and the rate of glucuronidation of entacapone was also low by this isoform. However, UGT1A1 was the only UGT capable of catalyzing the formation of two glucuronides of the catecholic entacapone. Both COMT inhibitors were glucuronidated at low rates by the representative members of the UGT2B family, UGT2B7 and UGT2B15. Michaelis-Menten parameters were determined for entacapone and tolcapone using recombinant human UGT isoforms and human liver microsomes to compare the kinetic properties of the two COMT inhibitors. The kinetic data illustrates that UGT1A9 exhibited a much greater rate of glucuronidation and a far lower K(m) value for both entacapone and tolcapone than UGT2B15 and UGT2B7 whose contribution is minor by comparison. Entacapone showed a 3 to 4 times higher V(max) value and a 4 to 6 times lower K(m) value compared with those of tolcapone both in UGT1A9 cell lysates and in human liver microsomes. (+info)
Site-directed mutagenesis of squalene-hopene cyclase: altered substrate specificity and product distribution.
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BACKGROUND: Two regions of squalene-hopene cyclase (SHC) were examined to define roles for motifs posited to be responsible for initiation and termination of the enzyme-catalyzed polyolefinic cyclizations. Specifically, we first examined the triple mutant of the DDTAVV motif, a region deeply buried in the catalytic cavity and thought to be responsible for the initiation of squalene cyclization. Next, four mutants were prepared for Glu45, a residue close to the substrate entrance channel proposed to be involved in the termination of the cyclization of squalene. RESULTS: The DDTAVV motif in SHC was changed to DCTAEA, the corresponding conserved region of eukaryotic oxidosqualene cyclase (OSC), by the triple mutation of D377C/V380E/V381A; selected single mutants were also examined. The triple mutant showed no detectable cyclization of squalene, but effectively cyclized 2,3-oxidosqualene to give mono- and pentacyclic triterpene products. Of the Glu45 mutants, E45A and E45D showed reduced activity, E45Q showed slightly increased activity, and E45K was inactive. A normal yield of pentacyclic products was produced, but the ratio of hopene 2 to hopanol 3 was significantly changed in the less active mutants. CONCLUSIONS: Initiation and substrate selectivity may be determined by the interaction of the DDTAVV motif with the isopropylidene of squalene (for SHC) and of the DCTAEA motif with the epoxide of oxidosqualene (for OSC). This is the first report of a substrate switch determined by a central catalytic motif in a triterpenoid cyclase. At the termination of cyclization, the product ratio may be largely controlled by Glu45 at the entrance channel to the active site. (+info)