Henri B. Weems

Immunoprecipitation of high-affinity, guanine nucleotide-sensitive, solubilized μ-opioid receptors from rat brain : Coimmunoprecipitation of the G proteins Gαo, Gαi1, and Gαi3

Abstract: Antibodies directed against the C‐terminal and the N‐terminal regions of the μ‐opioid receptor were generated to identify the G proteins that coimmunoprecipitate with the μ receptor. Two fusion proteins were constructed: One contained the 50 C‐terminal amino acids of the μ receptor, and the other contained 61 amino acids near the N terminus of the receptor. Antisera directed against both fusion proteins were capable of immunoprecipitating ∼70% of solubilized rat brain μ receptors as determined by [3H][D‐Ala2,N‐Me‐Phe4,Gly‐ol5]‐enkephalin ([3H]DAMGO) saturation binding. The material immunoprecipitated with both of the antisera was recognized as a broad band with a molecular mass between 60 and 75 kDa when screened in a western blot. Guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS) had an EC50 of 0.4 nM in diminishing [3H]DAMGO binding to the immunoprecipitated pellet. The ratio of G proteins to μ receptors in the immunoprecipitated material was 1:1. When the material immunoprecipitated with affinity‐purified antibody was screened for the presence of G protein α subunits, it was determined that Gαo, Gαi1, Gαi3, and to a lesser extent Gαi2, but not Gαs or Gαq/11, were coimmunoprecipitated with the μ receptor. Inclusion of GTPγS during the immunoprecipitation process abolished the coimmunoprecipitation of G proteins.

Resolution of optical isomers by chiral high-performance liquid chromatography: Separation of dihydrodiols and tetrahydrodiols of benzo[a]pyrene and benz[a]anthracene

Abstract Enantiomers of several underivatized dihydrodiols and tetrahydrodiols of benzo[ a ]pyrene and benz[ a ]anthracene have been resolved with high-performance liquid chromatography using a commercially available column packed with a d -3,5-dinitrobenzoylphenylglycine derivative of aminopropyl silica gel. Separation of optical isomers was confirmed by ultraviolet-visible absorption and circular dichroism spectral analyses. This simple method has been applied to the determination of optical purity of two dihydrodiol metabolites formed from the in vitro incubation of 11-methylbenz[ a ]anthracene by rat liver microsomes.

Direct resolution of mono- and diol enantiomers of unsubstituted and methyl-substituted benz[a]anthracene and benzo[a]pyrene by high-performance liquid chromatography with a chiral stationary phase

The direct resolution of 86 structurally related monomethyl, mono-ol, and trans- and cis-diol enantiomers of unsubstituted and methyl-substituted benz[a]anthracene and benzo[a]pyrene was investigated by high-performance liquid chromatography with a commercially available column, packed with an (R)-N-(3,5-dinitrobenzoyl)phenylglycine, ionically bonded to gamma-aminopropylsilanized silica. The results indicate that structural factors, such as conformation, presence of a methyl substituent, molecular size and shape, and ring saturation all contribute to chiral interactions between the chiral stationary phase and the solutes. Detailed chiral recognition mechanisms can not yet be established, due to complex structural factors that influence enantiomeric resolutions and the lack of data on the absolute configurations of the resolved enantiomers. Nevertheless, the chromatographic method can be applied to the determination of enantiomeric purity of mono- and diol metabolites of polycyclic aromatic hydrocarbons. The absolute configurations of a limited number of resolved enantiomers have been established.

Solubilization of High-Affinity, Guanine Nucleotide-Sensitive μ-Opioid Receptors from Rat Brain Membranes†

Abstract: High‐affinity μ‐opioid receptors have been solubilized from rat brain membranes. In most experiments, rats were treated for 14 days with naltrexone to increase the density of opioid receptors in brain membranes. Occupancy of the membrane‐associated receptors with morphine during solubilization in the detergent 3‐[(3‐cholamidopropyl)dimethyl]‐1‐propane sulfonate appeared to stabilize the μ‐opioid receptor. After removal of free morphine by Sephadex G50 chromatography and adjustment of the 3‐[(3‐cholamidopropyl)dimethyl]‐1‐propane sulfonate concentration to 3 mM, the solubilized opioid receptor bound [3H][d‐Ala2,N‐Me‐Phe4,Gly‐ol5]‐enkephalin ([3H]DAMGO), a μ‐selective opioid agonist, with high affinity (KD = 1.90 ± 0.93 nM; Bmax = 629 ± 162 fmol/mg of protein). Of the membrane‐associated [3H]‐DAMGO binding sites, 29 ± 7% were recovered in the solubilized fraction. Specific [3H]DAMGO binding was completely abolished in the presence of 10 µM guanosine 5′‐O‐(3‐thiotriphosphate). The solubilized receptor also bound [3H]diprenorphine, a nonselective opioid antagonist, with high affinity (KD = 1.4 ± 0.39 nM, Bmax = 920 ± 154 fmol/mg of protein). Guanosine 5′‐O‐(3‐thiotriphosphate) did not diminish [3H]diprenorphine binding. DAMGO at concentrations between 1 nM and 1 µM competed with [3H]diprenorphine for the solubilized binding sites; in contrast, [d‐Pen2,d‐Pen5]‐enkephalin, a δ‐selective opioid agonist, and U50488H, a κ‐selective opioid agonist, failed to compete with [3H]diprenorphine for the solubilized binding sites at concentrations of <1 µM. In the absence of guanine nucleotides, the DAMGO displacement curve for [3H]diprenorphine binding sites better fit a two‐site than a one‐site model with KDhigh = 2.17 ± 1.5 nM, Bmax = 648 ± 110 fmol/mg of protein and KDlow = 468 ± 63 nM, Bmax = 253 ± 84 fmol/mg of protein. In the presence of 10 µM guanosine 5′‐O‐(3‐thiotriphosphate), the DAMGO displacement curve better fit a one‐ than a two‐site model with KD = 815 ± 33 nM, Bmax = 965 ± 124 fmol/mg of protein.

Direct separation of non-K-region mono-ol and diol enantiomers of phenanthrene, benz[a]anthracene, and chrysene by high-performance liquid chromatography with chiral stationary phases.

The direct separation of 26 bay region and non-bay region mono-ol and diol enantiomers of phenanthrene, benz[a]anthracene, and chrysene was compared by high-performance liquid chromatography on commercially available columns, packed with gamma-aminopropylsilanized silica to which either (R)-N-(3,5-dinitrobenzoyl)phenylglycine(R-DNBPG) or (S)-N-(3,5-dinitrobenzoyl)leucine(S-DNBL) was either ionically or covalently bonded. In general, enantiomers of bay region mono-ols and diols are more efficiently resolved than those of non-bay region derivatives. Elution orders of enantiomers on either chiral stationary phase are the same, regardless of whether the chiral stationary phase is ionically or covalently bonded. Except for the enantiomers of 4-hydroxy-4-methyl-1,2,3,4-tetrahydrobenz[a]anthracene, 1,2,3,4-tetrahydrobenz[a]anthracene trans-1,2-diol, and benz[a]anthracene trans-1,2-dihydrodiol, elution orders of resolved enantiomers on R-DNBPG are reversed on S-DNBL. The enantiomers are generally more efficiently resolved on R-DNBPG than on S-DNBL. With the exception of the elution order of the enantiomeric 4-hydroxy-1,2,3,4-tetrahydrochrysene, the results of this study are consistent with the chiral recognition mechanisms proposed by Pirkle and co-workers, who developed the chiral stationary phases used in this study.

Enzymatic formation of an 8,9-diol from 8-methylbenz[a]anthracene

Summary An optically active 8,9-diol has been identified as a rat liver microsomal metabolite of the carcinogenic 8-methylbenz[a]anthracene. This is the first example indicating that the presence of a methyl group in a polycyclic aromatic hydrocarbon does not sterically block the enzymatic formation of a diol at the methyl-substituted double bond.

Resolution of epoxide enantiomers of polycyclic aromatic hydrocarbons by chiral stationary-phase high-performance liquid chromatography☆

Enantiomers of nine K-region and one non-K-region epoxides of polycyclic aromatic hydrocarbons have been resolved by high-performance liquid chromatography with chiral stationary phases either ionically or covalently bonded to gamma-aminopropylsilanized silica. Resolution of enantiomers was confirmed by ultraviolet-visible absorption, circular dichroism, and mass spectral analyses. This method has been applied to the determination of optical purity and absolute configuration of the K-region epoxides formed in the metabolism of 1-methylbenz[a]anthracene, 7-methylbenz[a]anthracene, and 12-methylbenz[a]anthracene by rat liver microsomes.

Resolution and absolute configuration of 7,12-dimethylbenz[a]anthracene 5,6-epoxide enantiomers

The enantiomers of 7,12-dimethylbenz[a]anthracene (DMBA) 5,6-epoxide were directly resolved by normal-phase high-performance liquid chromatography with an ionically bonded chiral stationary phase. The absolute configurations of the resolved enantiomers were determined by comparison of circular dichroism spectra of the methanolysis products formed from the epoxide enantiomers with that of a DMBA trans-5,6-dihydrodiol enantiomer of known absolute stereochemistry. DMBA 5R,6S-epoxide is hydrated by rat liver microsomal epoxide hydrolase predominantly (95%) to a 5S,6S-dihydrodiol. The results indicate that the 5S,6S-dihydrodiol formed from the metabolism of DMBA by microsomes prepared from the livers of 3-methylcholanthrene-treated rats is predominantly derived from a 5R,6S-epoxide intermediate.

Improved enantiomeric separation of dihydrodiols of polycyclic aromatic hydrocarbons on chiral stationary phases by derivatization to O-methyl ethers

K-region trans-dihydrodiol derivatives of phenanthrene, 1-methylphenanthrene, 4,5-methylenephenanthrene, pyrene, 1-bromopyrene, chrysene, benzo[c]phenanthrene, benz[a]anthracene, 1-, 4-, 6-, 7-, 11- and 12-methylbenz[a]anthracenes, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene, and benzo[a]pyrene, and non-K-region trans-3,4-dihydrodiols of benz[a]anthracene, chrysene, and 7,12-dimethylbenz[a]anthracene are converted to O-methyl ethers. Enantiomers of these O-methyl ethers are generally more efficiently separated on Pirkles chiral stationary phases than the enantiomers of underivatized dihydrodiols. O-Methyl ethers are substantially less polar than dihydrodiols, and O-methyl ethers are eluted with shorter retention times. Eluents of lower polarity can hence be used. This enhances chiral interactions between chiral stationary phase and solutes, allowing improved separation of enantiomers.

Stereoselective formation and hydration of benzo[c]phenanthrene 3,4-and 5,6-epoxide enantiomers by rat liver microsomal enzymes

The K-region 5,6-epoxides, formed in the metabolism of benzo[c]phenanthrene (BcPh) in the presence of an epoxide hydrolase inhibitor 3,3,3-trichloropropylene 1,2-oxide (TCPO) by liver microsomes from untreated, phenobarbital-treated, 3-methylcholanthrene-treated, and polychlorinated biphenyls (Aroclor 1254)-treated rats of the Sprague-Dawley and the Long-Evans strains, were found by chiral stationary phase high-performance liquid chromatography analyses to be enriched (58-72%) in the 5S, 6R enantiomer. In the absence of TCPO, the metabolically formed BcPh trans-5,6-dihydrodiol was enriched (78-86%) in the 5S,6S enantiomer. The major enantiomer of the BcPh 3,4-epoxide metabolite was found to be enriched in the 3S,4R enantiomer which undergoes racemization under the experimental conditions. The major enantiomer of the 5,6-dihydrodiol metabolite was elucidated by the exciton chirality circular dichroism (CD) method to have a 5S,6S absolute stereochemistry. Absolute configurations of enantiomeric methoxylation products derived from each of the two BcPh 5,6-epoxide enantiomers. Optically pure BcPh 5S,6R-epoxide was enzymatically hydrated exclusively at the C6 position to form an optically pure BcPh 5S,6S-dihydrodiol. However, optically pure BcPh 5R,6S-epoxide was hydrated at both C5 and C6 positions to form a BcPh trans-5,6-dihydrodiol with a (5S,6S):(5R,6R) enantiomer ratio of 32:68.