2-naphthylacetic Acid Research Articles

A Role for Threonine 302 in the Mechanism-Based Inactivation of P450 2B4 by 2-Ethynylnaphthalene

2-Ethynylnaphthalene (2EN) is a mechanism-based inactivator of P450 2B4 that covalently modifies an amino acid in the peptide Glu273-Met314 with a 2-naphthylacetyl group [Robertset al.(1994)Biochemistry33, 3766–3771]. Truncated 2B4 lacking amino acids 2–27, 2B4 (Δ2–27), was expressed inEscherichia coli,purified, and found to catalyze the oxidation of 2EN to 2-naphthylacetic acid (2NA). The metabolism of 2EN resulted in the inactivation and covalent modification of the protein moiety as we have previously reported with P450 2B4 purified from the livers of phenobarbital-induced rabbits. The rate constants of inactivation of theO-deethylation activity of 7-ethoxy-4-trifluoromethylcoumarin (EFC) were 0.15 ± 0.01 and 0.20 ± 0.05 min−1for the protein purified from rabbit liver and 2B4 (Δ2–27), respectively. A protein in which threonine 302 was replaced with alanine, P450 2B4 (Δ2–27, T302A), was inactivated by 2EN with a much slower rate constant (0.05 ± 0.01 min−1) and formed 1.8-fold more 2NA as compared to P450 2B4 (Δ2–27) over a 10-min incubation. When the formation of 2NA was supported by cumene hydroperoxide, 2B4 (Δ2–27, T302A) formed 30% less product than 2B4 (Δ2–27) over a 5-min incubation. After incubation with [3H]2EN and NADPH, P450 2B4 (Δ2–27) had significant radioactivity associated with the P450 in an NADPH-dependent manner when the incubation mixture was analyzed by SDS–PAGE followed by autoradiography and 10-fold more radioactivity associated with the P450 as compared to P450 2B4 (Δ2–27, T302A) when analyzed by reverse-phase HPLC. Thus, threonine 302 is not required by 2B4 for oxidation of 2EN or EFC but appears to play an important role in the inactivation of P450 2B4 by 2EN and the covalent labeling of the P450 protein by 2EN.

Micellar liquid chromatography of plant growth regulators detected by derivative fluorometry

A micellar liquid chromatographic method with fluorimetric detection was developed for the determination of the plant growth regulators indol 3-yl acetic acid (IAA), 2-(1-naphthyl) acetic acid (1-NAA), indol 3-yl propionic acid (IPA), 2-(2-naphthyl) acetic acid (2-NAA), indol 3-yl butyric acid (IBA), 2-(1-naphthyl) acetamide (1-Namide) and indol 3-yl acetic acid ethyl ester (IAA ethyl ester). Extraction of plant hormones was performed with methanol, followed by purification with ethyl acetate, solid-phase extraction and again partitioning against diethyl ether. Sodium dodecylsulphate (10 m M) is used as mobile phase to elute the compounds in a maximum run-time of 23 min. Partially or unresolved peaks are separated by calculation of the 1st derivative chromatogram. Detection limits are between 0.30 μg g −1 and 1.10 μg g −1.

Solution Conformations of a Biantennary Glycopeptide and a Series of Its Exoglycosidase Products from Sequential Trimming of Sugar Residues

Linkages between sugar residues in branched oligosaccharides exhibit various degrees of flexibility. This flexibility, together with other forces, determines the overall solution conformation of oligosaccharides. We used the method of time-resolved resonance energy transfer to study the solution conformations of a biantennary glycopeptide and its partially trimmed products by exoglycosidases. The N-terminal of the glycopeptide was labeled with 2-naphthyl acetic acid as a fluorescent donor. Either terminal sugar residue, Gal6', on the branch bearing 6-linked Man (antenna 6'), or Neu5Ac on the branch bearing 3-linked Man (antenna 6) was labeled with 5-dimethylaminophthalene-1-sulfonyl as an acceptor. The distance and distance distributions between the terminals were measured. In the intact biantennary glycopeptide, the donor-acceptor distance distribution of antenna 6' is bimodal with a majority of the population in the extended conformation and that of antenna 6 in one very broad population. The Neu5Ac on antenna 6 is oriented toward the N-terminal at low temperature and adopts a more extended form at high temperature. The removal of individual sugar residues along one of the two antenna in the biantennary oligosaccharide has a small effect on the distance distribution of the remaining antenna for both antennae 6 and 6'. Together with previous studies of the triantennary glycopeptides (Rice, K. G., Wu, P. G., Brand, L., and Lee, Y.C. (1993) Biochemistry 32, 7264-7270), our results suggest that both steric hindrance and inter-residue hydrogen bonding are very important in the folding pattern in oligosaccharide structures.

A New Approach to Assay Endo-Type Carbohydrases: Bifluorescent-Labeled Substrates for Glycoamidases and Ceramide Glycanases

Glycoamidases and ceramide glycanases are important "endo-type" enzymes for structural elucidations of glycoconjugates as well as for construction of neoglycoconjugates. The assay methods currently available for these enzymes are tedious and do not permit continual assay of the enzyme activities. We modified a desialylated biantennary glycopeptide with 2-naphthylacetic acid at the N-terminus and at the nonreducing terminal galactosyl residues with mono-N-dansylethylenediamine, via a specific oxidation of the C-6 hydroxyl group with galactose oxidase. In such a substrate, the naphthyl fluorescence (λem = 335 nm) is quenched due to absorption of its emitted light by the dansyl group, which in turn results in emission of fluorescence (λex = 520 nm) by the latter. However, when the link between the two fluorophores is severed by glycoamidase (PNGase), the energy transfer ceases to occur. Consequently the emission of the dansyl fluorescence and the quenching of naphthyl fluorescence diminish or disappear. Likewise, the energy transfer between the fluorophores in an alkyl lactoside containing a dansyl group at the terminal position of aglycon and a 2-naphthylmethyl group on the galactosyl residue is also eliminated by the glycosidic cleavage by a ceramide glycanase from American leech, Macrobdella decora, resulting in enhancement of the naphthyl emission and decrease in the dansyl emission. The substrates presented here permit continuous fluorescent monitoring of the enzymatic reaction. This allows precise analyses of enzyme kinetics not possible with the conventional assay methods for the endo-type enzymes which usually require separation of reaction products.

A Method for the Determination of UDP-Glucuronosyltransferase Activity toward Arylcarboxylic Acids

A fast and reliable method for the assay of UDP-glucuronosyltransferase (UGT) activity toward aglycones containing a carboxylic acid function is described. The procedure involves incubation with UDP-[U- 14C]-glucuronic acid, the common substrate for the reaction, solid-phase separation of the radiolabeled acylglucuronides and unreacted cofactor, and quantification by liquid scintillation counting. The technique was validated for each of the seven substrates tested by reversed-phase HPLC, and was then applied successfully to the determination of optimal conditions for the activation of the carboxylic acid-UGT, and the estimation of kinetic constants for the glucuronidation of clofibric acid, 2-naphthylacetic acid, naproxen, and 4,4,4-triphenyl-butanoic acid in rat liver microsomes. From the results obtained, we believe that this is an assay which, with only minor modification, could be applied to a wide range of carboxylic acid substrates for which, until now, specific and sensitive assays have been largely unavailable.

Mechanism-based inactivation of cytochrome P450 2B1 by 2-ethynylnaphthalene: identification of an active-site peptide.

The 7-ethoxycoumarin O-deethylase activity of rat liver cytochrome P450 2B1 reconstituted with NADPH-cytochrome P450 reductase and lipid was inactivated by 2-ethynylnaphthalene (2EN) in a time- and NADPH-dependent manner, and the loss of activity followed pseudo-first-order kinetics. The extrapolated KI and kinactivation were 0.08 microM and 0.83 min-1, respectively. The loss of 7-ethoxycoumarin O-deethylation activity displayed a number of characteristics consistent with mechanism-based inactivation, including irreversibility, saturability, protection by an alternate substrate, and the lack of an effect of exogenous nucleophiles on the inactivation. The inactivation was not accompanied by a concomitant loss of spectrally detectable cytochrome P450. HPLC analysis showed that [3H]2EN was irreversibly bound to the protein moiety of cytochrome P450 and the stoichiometry of inactivation was approximately 1.3 mol of 2EN bound per mole of cytochrome P450. Liquid chromatographic and GC-MS analyses of the organic extracts from these incubations showed that the major metabolite was 2-naphthylacetic acid, and a partition ratio of 4-5 mol of acid produced per mole of cytochrome P450 2B1 inactivated was determined. A radiolabeled peptide, approximately 6.5 kDa when analyzed by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was isolated by HPLC from a tryptic digest of the [3H]2EN-inactivated cytochrome P450 and NADPH-cytochrome P450 reductase. Sequence data were obtained after cyanogen bromide cleavage of this amino-terminally blocked peptide. These results in conjunction with the results from the cleavage of the intact [3H]2EN-inactivated cytochrome P450 by cyanogen bromide and separation of the peptides either by HPLC or by Tricine-SDS-PAGE followed by transfer of the peptides to a poly(vinylidene difluoride) membrane and sequencing of the labeled peptides from both experiments, led to the identification of a 2EN-modified active-site peptide with the sequence ISLLSLFFAGTETSSTTLRYGFLLM. This corresponds to positions 290-314 in cytochrome P450 2B1. Sequence alignments of cytochrome P450 2B1 with cytochrome P450 2B1 with cytochrome P450 101 predict that this region might correspond to helix I of the bacterial protein [Poulos, T.L. (1988) Pharm. Res. 5, 67-75] that contains a highly conserved threonine residue involved in oxygen binding.

Control of Apoplast pH in Corn Coleoptile Segments. II: The Effects of Various Auxins and Auxin Analogues

Using abraded Zea mays L. coleoptile segments, proton extrusion has been measured in the presence of growth promoting auxins and their inactive structural analogues. Prior to application, the segments spontaneously acidified the external medium to pH 4.8 within 7h. Indole-3-acetic acid (IAA) concentration dependently decreases external pH to 4.2, fusicoccin (FC) to below 3.5. Whereas the IAA-effect spontaneously recovers and can be repeated, the FC-effect is final and irreversible. The growth promoting auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), indole-3-butyric acid (IBA) and 1-naphthylacetic acid (1-NAA) all stimulate proton extrusion similar to IAA. The growth inactive structural analogues 2,3-dichlorophenoxyacetic acid (2,3-D) and 2-naphthylacetic acid (2-NAA) also acidify the external medium, but quantitatively to a lesser extent. p-chlorophenoxyisobutyric acid (PCIB) does not inhibit the IAA response. No effects were obtained with deactivated H+-ATPase (anoxia) and in the presence of benzoic acid.

Inelastic electron tunnelling spectra of some derivatives of naphthalene

Abstract Inelastic electron tunnelling spectra of 1- and 2-naphthylacetic acid, 1- and 2-naphthoic acid, 1- and 2-naphthylacetate and 1-naphthylacetamide were measured at 4.2 K in the range 100–4000 cm−1. The molecules were transferred onto the alumina surface by the vacuum deposition method. The spectra showed intensive and sharp peaks originating from aromatic rings. However, the spectra from naphthylacetates were of poorer quality than those from the other compounds and hence their adsorption orientation was concluded to be more parallel with respect to the surface of alumina. The adsorption of the naphthylacetates was possibly followed by dissociation of the molecules. The I, U characteristics were also measured, and it was observed that the naphthoic acids yielded the most nonlinear curves.

Studies on the metabolism of arylacetic acids. 7. The influence of varying dose size upon the conjugation pattern of 2-naphthylacetic acid in the guinea pig, mouse and hamster.

1. The influence of dose size upon the metabolic conjugation of 2-naphthylacetic acid with various amino acids and glucuronic acid has been studied in the guinea pig, mouse and hamster. 2. Guinea pigs conjugated 2-naphthylacetic acid with glycine and glucuronic acid. 3. Mice conjugated 2-naphthylacetic acid with glycine, taurine and glucuronic acid. Taurine conjugation had the highest capacity, and both this and the glycine mechanism were saturated at doses above 100 mg/kg. 4. Hamsters utilized glutamine, glycine, taurine and glucuronic acid for the conjugation of 2-naphthylacetic acid. No conjugation pathway was saturated by doses up to 200 mg/kg. 5. The thus-far unique ability of 2-naphthylacetic acid to evoke multiple amino acid conjugations, using the taurine and glutamine mechanisms hitherto unknown in these species, appears to be due to its affinity for previously unrecognized enzyme systems, rather than to saturation of 'normal' pathways revealing novel routes at high doses.

Head-to-Head Polymers XXXV. Head-to-Head Poly(2-vinylnaphthalene)

Head-to-head poly(2-vinylnaphthalene) was prepared in five steps from 2-naphthylacetic acid. The methyl ester was brominated with N-bromosuccinimide and 2(2-naphthyl)2-bromoacetate treated with a copper/zinc couple, which gave dimethyl 2,3-di(2-naphthyl)succinate in moderate yield. This compound was reduced with lithium aluminum hydride to 2,3-di(2-naphthyl)butanediol-1,4 and the hydroxyl groups replaced by bromine. 2,3-Di(2-naphthyl)-1,4-dibromobutane was subjected to the Yamamoto coupling polycondensation; head-to-head poly(2-vinylnaphthalene) was obtained in relatively low yield and of modest molecular weight.

Liquid chromatographic assay for the measurement of glucuronidation of arylcarboxylic acids using uridine diphospho-[U- 14C]glucuronic acid

A general method for the assay of UDP-glucuronosyltransferase activity towards arylcarboxylic acids (clofibric acid, 1- and 2-naphthylacetic acid) using UDP-[U- 14C]glucuronic acid in liver microsomes is described. The 14C-labelled glucuronide was separated by high-performance liquid chromatography, identified by hydrolysis by β-glucuronidase, characterized by laser desorption mass spectrometry and quantified by scintillation counting. The coefficient of variation of the enzyme activity for the inter-assay repeatability was below 4.5%. As little as 2.5 nmol of the arylcarboxylic acid glucuronides could be detected and precisely quantified. The method was applied to the determination of the apparent kinetic constants for glucuronidation of the acids. Clofibric acid was the best substrate for UDP-glucuronosyltransferase ( V max/ K M, the ratio of the maximum initial velocity and the Michaelis-Menten constant, is 12.3). The two isomers, 1- and 2-naphthylacetic acids, were transformed at a similar rate. However, they exhibited different enzymatic affinities, as the K M values were 1.0 m M and 5.6 m M for 1- and 2-naphthylacetic acid, respectively. This indicates that the spatial organization of the substrates played a critical role in this acyl glucuronoconjugation.

Auxin-binding protein from coleoptile membranes of corn (Zea mays L.). I. Purification by immunological methods and characterization.

The purification of a putative auxin receptor is one possibility to elucidate the first event in the mechanism of auxin action. By affinity chromatography of membrane proteins on 2-OH-3,5-diiodobenzoic acid-Sepharose and gel filtration on Ultrogel a fraction enriched in auxin-binding protein (ABP) was obtained and used for rabbit immunization. From the immunoglobulin G (IgG) fraction of the antisera IgGs against proteins not binding auxin (nonABP) could be obtained which were used to eliminate the nonABP from the eluates of the 2-OH-3,5-diiodobenzoic acid-Sepharose. The remainder fraction was further purified and concentrated on IgG-Sepharose which retained the ABP that could be eluted without loss of binding activity. A 600-fold purification with a yield of 42% was achieved. The ABP could be identified as the site I "receptor" described by Dohrmann et al. (Dohrmann, U., Hertel, R., and Kowalik, H. (1978) Planta (Berl.) 140, 97-106). It is shown that the competitors tested reduce [14C]1-naphthylacetic acid-(NAA) binding in the following order of effectiveness: NAA greater than 2-naphthylacetic acid greater than 1-phenylacetic acid greater than 2,3,5-triiodobenzoic acid greater than 3-indolylacetic acid greater than 2,4-dichlorophenoxyacetic acid. The ABP has a sharp binding optimum at pH 5.5, and the KD was calculated to be 5.7 X 10(-8) M to [14C]NAA. The binding activity of the ABP linearly decreased with increasing temperature but could partially be restored upon chilling in the presence of auxin. The ABP seems to be a 40-kDa dimer in its native form without disulfide bonds between its monomers.

The utilization of exogenous taurine for the conjugation of xenobiotic acids in the ferret.

Although the occurrence of the taurine conjugation mechanism for various xenobiotic acids is well established, nothing is known of the source of the taurine used for this conjugation. [14C]Taurine was administered alone and in combination with 2-naphthylacetic acid or clofibric acid (both of which are known to form taurine conjugates) to to ferrets, and the 0--24 h urine collected. Of the dose of [14C]taurine, 26% was recovered in the urine in 24 h and the only 14C-containing material present was unchanged taurine. When either 2-naphthylacetic acid or clofibric acid was co-administered with [14C]taurine, 21 and 17%, respectively, of the 14C dose was recovered in the 0--24 h urine. In both cases, two 14C compounds were present--unchanged taurine (minor) and the taurine conjugate of the acid in question (major). Comparison of these results with those previously obtained with 14C-labelled 2-naphthylacetic and clofibric acids, shows that the taurine used for their conjugation is derived from a pool freely accessible to exogenous taurine. The results are discussed in terms of the availability for metabolic utilization of taurine in the animal body, and of the use of co-administration of [14C]taurine with a xenobiotic acid for the identification of taurine conjugates.

Metabolic conjugation of some carboxylic acids in the horse.

1. 14C-Labelled benzoic acid, salicylic acid and 2-naphthylacetic acid were administered orally to horses, and urinary metabolites investigated by chromatographic and mass spectral techniques. 2. [14C]Benzoic acid (5 mg/kg) was eliminated rapidly in the urine, and quantitatively recovered in 24 h. The major urinary metabolite was hippuric acid (95% of dose) with much smaller amounts of benzoic acid, benzoyl glucuronide and 3-hydroxy-3-phenylpropionic acid. Administration of [ring-D5]benzoic acid together with [14C]benzoic acid to a pony permitted the mass spectral determination of metabolites of the exogenous benzoic acid metabolites in the presence of the same endogenous compounds. 3. [14C]Salicylic acid (35 mg/kg) was eliminated rapidly in the urine, 98% of the 14C dose being excreted in 24 h. The major excretion product was unchanged salicylate (94% of dose). Gentisic acid, salicyluric acid and the ester and ether glucuronides of salicylic acid were very minor metabolites. 4. 2-Naphthyl[14C]acetic acid (2 mg/kg) was excreted very slowly in the urine, with 53 and 77% of the 14C dose being recovered in six days. 2-Naphthylacetylglycine was the major metabolite (26 and 38% dose) and in addition, the glucuronic acid and taurine conjugates were excreted together with unchanged 2-naphthylacetic acid. 5. The study has shown that the horse can utilize glycine, taurine and glucuronic acid for conjugation of xenobiotic carboxylic acids, and that the relative extents of these pathways are governed by the structure of the carboxylic acid.

Amino Acid Conjugation Responses to 2-Naphthylacetic Acid in Rodents

Amino Acid Conjugation Responses to 2-Naphthylacetic Acid in Rodents

Studies on the metabolism of arylacetic acids. 5. The metabolic fate of 2-naphthylacetic acid in the rat, rabbit and ferret.

1. The fate of [14C]-2-naphthylacetic acid has been studied in rat, ferret and rabbit and the findings compared with those for the 1-isomer.2. 2-Naphthylacetic acid was conjugated in all three species with glycine, glutamine, taurine and glucuronic acid.3. In rat, but not rabbit, 2-naphthylacetic acid is eliminated in the bile (50% dose) and undergoes entergohepatic circulation.4. The pattern of metabolism and elimination of 2-naphthylacetic acid in rat is dose-dependent. Small doses (5 mg/kg) are excreted in urine and bile mainly as the glucuronic acid conjugate, while larger doses (250 mg/kg) are eliminated in the bile mainly as amino acid conjugates.5. Metabolic conjugation of 1- and 2-naphthylacetic acids differs in the three species studied. 1-Naphthylacetic acid is conjugated largely with glucuronic acid and amino acid conjugation is minimal, whereas the converse is true of the 2-isomer. These differences in conjugation have been correlated with structural differences between the two acids.6. 2-Naphthylacetic acid is the first carboxylic acid found to undergo simultaneous amino acid conjugation with glycine, glutamine and taurine in non-primate species. 2-Naphthylacetic acid may be a useful probe for exploring the species occurrence of the various conjugations.

Thermochemical properties of naphthalene compounds III. Enthalpies of combustion and formation of the 1- and 2-naphthylacetic acids and 1- and 2-naphthyl acetates

The energies of combustion of 1-naphthylacetic and 2-naphthylacetic acids and 1-naphthyl and 2-naphthyl acetates have been determined using a static bomb calorimeter. The enthalpies of formation in the crystalline state at 298.15 K have been derived for these four naphthalene compounds. The values obtained for − ΔH f o(c)/kcal th mol −1 are: 1-naphthylacetic acid, 85.85 ± 0.42; 2-naphthylacetic acid, 88.90 ± 0.45; 1-naphthyl acetate, 72.89 ± 0.46; 2-naphthyl acetate, 73.99 ± 0.46.

Structure activity relationship among 6-substituted-2-naphthylacetic acids. A new series of nonsteroidal anti-inflammatory agents.

Chemical modification of 2-naphthylacetic acid has led to (+)-2-(6-methoxy-2-naphthyl)-propionic acid, naproxen. Maximal biological activity in the naph-thylpropionic acid series is associated with the S absoiuti configuration. In experimental animals naproxen shows anti-inflammatory, analgetic and antipyretic activity o: llxphenylbutazone, and 7 and 22xacetylsalicylic acid re spectively. It is noteworthy that highly active compound! were obtained without resorting to nitrogenous substitu cnts.

15H-cyclohepta(2,1-a:4,5-a')dinaphthalen-15-one. The condensation of naphthalene-1,2-dicarboxylic anhydride with 2-naphthylacetic acid.

The condensation of naphthalene-1,2-dicarboxylic anhydride with 2-naphthylacetic acid gave the two structurally isomeric (2-naphthylmethylene)naphtho[1,2-c]furanones (VII) and (VIII) in the ratio 7 : 1. 2-[2-(2-Naphthyl)-ethyl]-1-naphthoic acid (IX), formed by reduction of the 3-substituted 1-ketone (VII) has been converted into 15H-cyclohepta[2,1-a:4,5-a′]dinaphthalen-15-one (IV).

Condensation of 2-benzoylbenzoic with 2-naphthylacetic acid and synthesis of benzo[a]pentaphene

Condensation of 2-benzoylbenzoic with 2-naphthylacetic acid and synthesis of benzo[a]pentaphene