Meta Hydroxyl Research Articles

Site-Directed Mutagenesis of α-Adrenergic Receptors

The application of in vitro site-directed mutagenesis has led to the identification of conserved amino acids that play important roles in receptor structure and function. Precise amino acid substitutions can be obtained and then correlated with changes in receptor phenotype. Here, we describe several techniques commonly employed to Introduce site-specific mutations. The benefits and potential drawbacks of each method are discussed. Site-directed mutagenesis of the human α2A-adrenergic receptor (α2AAR) has been successfully employed to identify conserved amino acids involved in agonist binding and receptor activation. Aspartate residues in the second and intracellular side of the third transmembrane domain of the α2AAR are implicated in receptor/G-protein interactions. Since these aspartate residues are highly conserved among all G-protein-coupled receptors, and elimination of these residues has been shown to abolish the ability of other receptors in this class to activate their respective intracellular signaling pathways, It seems likely that these residues are critical for agonist-induced conformational changes that underlie receptor/G-protein interactions. In contrast to the role played by the conserved residues mentioned above, a conserved aspartate residue situated near the extracellular side of the third transmembrane domain plays a pivotal role in adrenergic ligand binding. Genetic analysis of the fifth transmembrane domain of the α2AAR suggests that a conserved serine residue in this region participates in hydrogen binding to the meta-hydroxyl group of catecholamines. These findings point to the utility of site-directed mutagenesis in identifying structure-function relationships among G-protein-coupled receptors.

Hydroquinone degradation via reductive dehydroxylation of gentisyl-CoA by a strictly anaerobic fermenting bacterium

Anaerobic degradation of hydroquinone was studied with the fermenting bacterium strain HQGö1. The rate of hydroquinone degradation by dense cell suspensions was dramatically accelerated by addition of NaHCO3. During fermentation of hydroquinone in the presence of 14C-Na2CO3 benzoate was formed as a labelled product, indicating an initial ortho-carboxylation of hydroquinone to gentisate. Gentisate was activated to the corresponding CoA-ester in a CoA ligase reaction at a specific activity of 0.15 μmol x min−1 x mg protein−1. Gentisyl-CoA was reduced to benzoyl-CoA with reduced methyl viologen as electron donor by simultaneous reductive elimination of both the ortho and meta hydroxyl group. The specific activity of this novel gentisyl-CoA reductase was 17 nmol x min−1 x mg protein−1. Further degradation to acetate was catalyzed by enzymes which occur also in other bacteria degrading aromatic compounds via benzoyl-CoA.

Hydroquinone degradation via reductive dehydroxylation of gentisyl-CoA by a strictly anaerobic fermenting bacterium

Anaerobic degradation of hydroquinone was studied with the fermenting bacterium strain HQGö1. The rate of hydroquinone degradation by dense cell suspensions was dramatically accelerated by addition of NaHCO3. During fermentation of hydroquinone in the presence of 14C-Na2CO3 benzoate was formed as a labelled product, indicating an initial ortho-carboxylation of hydroquinone to gentisate. Gentisate was activated to the corresponding CoA-ester in a CoA ligase reaction at a specific activity of 0.15 μmol x min−1 x mg protein−1. Gentisyl-CoA was reduced to benzoyl-CoA with reduced methyl viologen as electron donor by simultaneous reductive elimination of both the ortho and meta hydroxyl group. The specific activity of this novel gentisyl-CoA reductase was 17 nmol x min−1 x mg protein−1. Further degradation to acetate was catalyzed by enzymes which occur also in other bacteria degrading aromatic compounds via benzoyl-CoA.

Cytotoxic effects of biphenyl and hydroxybiphenyls on isolated rat hepatocytes

The cytotoxic effects of biphenyl (BP) and its hydroxylated derivatives, o-phenylphenol (OPP), m-phenylphenol (MPP), p-phenylphenol (PPP), 2-biphenylyl glycidyl ether (OPP-epoxide), phenyl-hydroquinone (PHQ), o, o′-biphenol ( o, o′-BPol) and p, p′-biphenol ( p, p′-BPol), were investigated in freshly isolated rat hepatocytes. OPP, MPP and PPP, at concentration of 0.75 mM, resulted in the loss of intracellular ATP, glutathione (GSH) and protein thiols, causing cell death. OPP-epoxide and BP were less toxic than the OPP isomers. MPP or PPP compared with OPP caused serious impairments in oxidative phosphorylation in mitochondria isolated from rat liver. PHQ (0.75 mM) caused a rapid loss of intracellular ATP which preceded the onset of cell death. PHQ was more toxic than o, o′-BPol or p, p′-BPol. PHQ dissolved in Krebs-Henseleit buffer without hepatocytes was rapidly converted to its corresponding quinone, phenyl-benzoquinone. The cytotoxicity produced by PHQ depends on the rate of formation of reactive intermediates. These results indicate that the addition of a hydroxyl group to the aromatic ring of BP enhances BP-induced cytotoxicity and that the mitochondria are a common target of the OPP isomers and other BP derivatives. In addition, the para- or meta-hydroxyl groups rather than the ortho-hydroxyl group increase the toxicity. The cytotoxicity produced by PHQ depends on the rate of formation of reactive intermediate(s) such as phenyl-benzoquinone.

The hydroxylation step in the biosynthetic pathway leading from norcoclaurine to reticuline

A phenolase was purified to homogeneity (488-fold) from cell suspension cultures of Berberis stolonifera. This enzyme in the presence of O 2 and ascorbate introduces a meta-hydroxyl group into tyrosine, tyramine, coclaurine, and N-methylcoclaurine. No other hydroxylating activity directed towards N-methylcoclaurine could be found in this tissue. The enzyme ( M r 60 × 10 3) is probably a dimer with two identical subunits, it has a pH optimum at 6.0 and a temperature optimum in the range of 20–30°. Evidence is presented that this enzyme catalyses two separate reactions in the formation of reticuline, namely the hydroxylation of tyrosine (tyramine) to DOPA (dopamine) and the 3′-hydroxylation of N-methylcoclaurine to 3′-hydroxy- N-methylcoclaurine, the penultimate intermediate to reticuline.

Identification of Two Serine Residues Involved in Agonist Activation of the β-Adrenergic Receptor

Pharmacophore mapping of the ligand binding domain of the β-adrenergic receptor has revealed specific molecular interactions which are important for agonist and antagonist binding to the receptor. Previous site-directed mutagenesis experiments have demonstrated that the binding of amine agonists and antagonists to the receptor involves an interaction between the amine group of the ligand and the carboxylate side chain of Asp113 in the third hydrophobic domain of the receptor (Strader, C. D., Sigal, I. S., Candelore, M. R., Rands, E., Hill, W. S., and Dixon, R. A. F. (1988) J. Biol. Chem. 263, 10267–10271). We have now identified 2 serine residues, at positions 204 and 207 in the fifth hydrophobic domain of the β-adrenergic receptor, which are critical for agonist binding and activation of the receptor. These serine residues are conserved with G-protein-coupled receptors which bind catecholamine agonists, but not with receptors whose endogenous ligands do not have the catechol moiety. Removal of the hydroxyl side chain from either Ser204 or Ser207 by substitution of the serine residue with an alanine attenuates the activity of catecholamine agonists at the receptor. The effects of these mutations on agonist activity are mimicked selectively by the removal of the catechol hydroxyl moieties from the aromatic ring of the agonist. The data suggest that the interaction of catecholamine agonists with the β-adrenergic receptor involves two hydrogen bonds, one between the hydroxyl side chain of Ser204 and the meta-hydroxyl group of the ligand and a second between the hydroxyl side chain of Ser207 and the para-hydroxyl group of the ligand.

Neuropharmacology and Stereochemistry of Dopamine Receptor Agonist and Antagonist Enantiomeric Pairs

Neuropharmacological evaluation of the R and S isomers of 11-hydroxy-N-n-propylnoraporphine (11-OH-NPa) supports the impression that the 11-OH group in aporphines (analogous to the meta hydroxyl of dopamine, DA) is sufficient to confer high affinity and activity at DA receptors. As in the case of the catechol congeners, (R)-apomorphine (APO) and (R)-N-n-propylnorapomorphine (NPA), (R)-11-OH-NPa is a potent DA agonist while, like (S)-NPA, (S)-11-OH-NPa is a DA antagonist. Thus, (R) and (S)-11-OH-NPa are an additional pair of compounds in which one enantiomer is a DA agonist and the other an antagonist. Other analogous pairs are the enantiomers of 3-(3-hydroxyphenyl)-N-n-propylpiperidine (3-PPP), and cis-1-methyl-5-hydroxy-2-(di-n-propylamino)tetralin (5-OH-MDAT). All contain a meta hydroxyphenyl, an N-n-propyl, and a phenethylamine moiety which can be superimposed in a consistent way to discriminate the DA agonists from the antagonists, with the key feature in this discrimination being the direction of the ammonium hydrogen. An energy penalty must be incurred by 3-PPP to assume the required conformations and it may account for the relatively low potency of the 3-PPP enantiomers. This analysis supports the view that rigid analogs of flexible compounds when "frozen" in their biologically active conformation exhibit higher affinity interactions with the receptor.

Pharmacological characterization of octopamine-sensitive adenylate cyclase in the flight muscle of Locusta migratoria L.

Octopamine stimulates (20-30-fold) the adenylate cyclase activity in a broken cell preparation of locust flight muscle. This stimulation is mediated by one receptor with an apparent affinity of 3.3 X 10(-6) M. The hydroxyl group in the para position on phenylethanolamine was absolutely necessary to obtain an agonist whereas the meta hydroxyl group or the presence of a catechol almost suppressed the activity. Similar pharmacological profiles were obtained on other locust muscles, i.e. mandibular and extensor tibiae muscles. The affinities of a large series of agonists and antagonists for the octopamine receptor coupled with an adenylate cyclase were compared to those reported for octopamine 1, 2A and 2B receptors based on physiological studies in the locust extensor tibiae muscle. It appears that the octopamine receptor coupled with adenylate cyclase is likely to be identical with the octopamine 2A receptor mediating the increase in the amplitude of slow motoneuron twitch tension. The results of the pharmacological experiments are discussed, in particular concerning the possible relationship between the octopamine receptor and alpha 1- and alpha 2-adrenoceptors.

A study of the biologically active conformers for prodine opiates and their derivatives

AbstractConformational energy calculations using the empirical MM2 (molecular mechanics II) and semiempirical quantum mechanical PCILO (perturbative configuration interaction using localized orbitals) methods are reported for various prodine derivatives. These include 3‐demethylprodine, α‐prodine, β‐prodine, the α‐2‐methyl derivative, α‐promedol, the γ‐2,3‐dimethyl derivative, and γ‐isopromedol. The results are consistent with all of the compounds activating the opiate receptor in a phenyl equatorial conformation with optimum activity resulting from a particular orientation of the phenyl and propionoxyl groups. In disagreement with previous limited experimental data, α‐promedol is found to prefer a phenyl equatorial conformer. It is confirmed that, of the two mirror image phenyl equatorial conformers that are preferred for 3‐demethylprodine, the more active prodines antipodes consistently prefer the one in which the phenyl orientation is the opposite (mirror image) of that found in morphine and in the preferred conformer of the morphine‐like (+)‐phenylmorphan. This is a possible molecular basis for the nonmorphine‐like effects that occur with the introduction of a phenyl meta hydroxyl into some prodine derivatives. It is also suggested that the less active prodine antipodes, which have a morphine‐like phenyl orientation, may act in a morphine‐like manner at opiate receptors.

Effects of exogenous L-dopa on the metabolism of methionine and S-adenosylmethionine in the brain.

The catecholamines dopamine, norepinephrine, and epinephrine are metabolized by oxidative deamination and by O-methylation. This latter pathway is catalyzed by the enzyme catechol-O-methyl-transferase (1); it utilizes S-adenosyl-methionine (SAMe) as the methyl donor (Fig. 1), and its products are, for the most part, methylated on the metahydroxyl group. The catecholamines themselves are formed within only a few specific tissues in the body (21). These include a relatively small number of brain neurons, postganglionic sympathetic neurons, adrenomedullary chromaffin cells, and interneurons within sympathetic ganglia. The catechol-amines can be O-methylated within their cells of origin, within synapses, or, after secretion into the blood stream, within the liver, kidneys, and other organs.KeywordsMethionine AdenosyltransferaseMethionine ConcentrationPostganglionic Sympathetic NeuronHomocysteine MethyltransferaseMethyl Group MetabolismThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

P-O-methylation of protocatechuic acid, a metabolite of epinephrine and norepinephrine, in perfused rat liver.

O-methylation by catechol-O-methyltransferase (COMT; EC 2.1.1.6) is an important pathway for biological degradation of catecholamines. In the healthy organism this enzyme is known to catalyse the methylation of the meta hydroxyl group of catecholamines and their metabolites.

Purification and properties of an o-dihydricphenol meta- O-methyltransferase from cell suspension cultures of parsley and its relation to flavonoid biosynthesis

1. 1. An O-methyltransferase directly related to flavone glycoside biosynthesis has been isolated from cell suspension cultures of parsley. The enzyme has been purified 82-fold by MnCl 2 and (NH 4) 2SO 4 precipitation and chromatography on DEAE-cellulose, Sephadex G-100 and hydroxylapatite. The enzyme catalyses the transfer of the methyl group of S-adenosyl- l-methionine to the meta position of o-dihydric phenols. 2. 2. Highest enzyme activities were obtained after illumination of the cells for 24 h with high intensities of white light from fluorescent lamps. 3. 3. The enzyme has a pH optimum around pH 9.7. It requires Mg 2+ and is not inhibited by p-chloromercuribenzoate or iodoacetamide. A molecular weight of about 48 000 was estimated from the elution volume after chromatography on a Sephadex G-100 column. 4. 4. Only o-dihydric phenols can function as substrates for the enzyme and only the meta-hydroxyl group is methylated. Luteolin (5,7,3′,4′-tetrahydroxyflavone) and its 7- O-glucoside with apparent K m values of 4.6·10 −5 and 3.1·10 −5 M, respectively, are the best substrates tested, whereas eriodictyol (5,7,3′,4′-tetrahydroxyflavanone) ( K m = 1.2·10 −3M) and caffeic acid ( K m = 1.6·10 −3M) had much lower affinity for the enzyme. With luteolin as substrate the apparent K m value for S-adenosyl- l-methionine is 1.5·10 −4 M. 5. 5. Cell cultures also contain enzymatic activity for the synthesis of S-adenosyl- l-methionine from l-methionine and ATP. In contrast to the O-methyltransferase the activity of this enzyme is not influenced by illumination of the cultures.

O-methylation of simple phenols in the rat.

Eleven simple mono‐, di‐ and thihydric phenols were given by stomach tube at a dose of 100 mg/kg body weight. The subsequent 48‐hour urines were hydrolyzed, extracted and analyzed by thin‐layer and gas chromatography and by infrared spectrophotometry. The mean extent of direct O‐methylation was 6.9% with catechol, 6.0% with 4‐methylcatechol, 8.9% with 4‐ethylcatechol and 6.2% with pyrogallol. The preferred site of methylation was the middle hydroxyl group of pyrogallol and the meta hydroxyl group of 4‐methylcatechol. Methoxyl groups were introduced ortho to the hydroxyl group of the monohydric phenols 4‐ethyl‐phenol, p‐cresol and phenol, but this occurred to a very small extent. No O‐methylated metabolites were detected following the administration of resorcinol, hydroquinone, hydroxyquinol and phloroglucinol. There was no correlation between the extent of O‐methylation of the simple phenols and their potencies as convulsant agents. Formation of anisoles and O‐methylation of more than one hydroxyl group were not observed with any of the compounds. The rats dosed with hydroxyquinol but not those given pyrogallol or phloroglucinol excreted small amounts of resorcinol. Metabolites produced by oxidation and unknown metabolites were also observed.

Clinical evaluation of aramine, a new sympathomimetic amine.

Aramine ® hydrogen tartrate (levo-1-[ m -hydroxyphenyl]-2-amino-1-propanol d -hydrogen tartrate) 1 is a new sympathomimetic amine designed for active, prolonged constriction of blood vessels. As a compound for nasal medication, it fulfills the requirements of being isotonic, slightly acid (pH 6.0), buffered, an effective vasoconstrictor without vasodilatory action, and without local or systemic toxicity. 2 Controlled laboratory experiments revealed that aramine ® has no deleterious effect (gross or microscopic) upon rabbit nasal mucosa or conjunctiva after 45 daily instillations of 0.5% aramine ® solution. The ciliary activity of normal rabbit trachea is not affected by immersion in aramine ® for one to two minutes. After two to four minutes, there may be mild inhibition of ciliary motility. 3 Aramine ® usually causes no increase in pulse rate or cardiac arrhythmias when given in less than maximal pressor doses, a characteristic of sympathomimetic amines with a metahydroxyl group on the aromatic

SPECTROCHEMICAL STUDIES OF HYDROXYAZO-COMPOUNDS. PART III

Abstract 1. Tautomeric transpositions may take place in these hydroxyazo-compounds by the addition of a potassium hydroxide solution, with an accompanying change in colour. 2. We can respectively assign A(azo)-, R(red)-, and B(blue)-forms to these tautomers. (Sometimes we recognise more readily the mixture of these forms.) 3. Tautoinerism is due to the labile hydrogen atoms in hydroxyl-groups which take the ortho- or para-position with respect to the azo-group. 4. The hydrogen atom which is in the meta hydroxyl-group with regard to the azo-group does not only move by itself, but moreover hinders the transposition of the other hydrogen atoms. 5. When there are many labile hydrogen atoms, mutual hindrance may occur. 6. B-form is sometimes produced only when there is a para nitroradical with regard to the azo-group. 7. Nitro-groups which take the ortho- or meta-position with respect to the azo-group do not change into the isonitro-structure. 8. The nitro-group has considerable bathochromic influence upon these hydroxyazo-compounds. 9. Generally only one absorption band exists in the solutions, of these substances, but there are two bands when methyl-group is included.