Enantiomers of 2-[(Acylamino)ethyl]-1,4-benzodiazepines, potent ligands of kappa-opioid receptor: chiral chromatographic resolution, configurational assignment and biological activity.

Compounds 2a and 3a-e are racemic 2-[(acylamino)ethyl]-1,4-benzodiazepines, tifluadom analogs, with high affinity and selectivity towards the kappa-opioid receptor. We describe the enantiomeric separation of all compounds through liquid chromatography with chiral stationary phases, as well as the resolution of the enantiomers of the most interesting compounds, 2a and 3a, by the semipreparative column Chiralpak AD. The configuration of the resolved enantiomers was investigated: the comparative study of CD and (1)H NMR spectra shows that compounds (-)-2a and (-)-3a have the same absolute configuration of (+)-(S)-tifluadom. A study on the stereoselective interaction with opiate receptors is reported.

Stepwise modulation of neurokinin-3 and neurokinin-2 receptor affinity and selectivity in quinoline tachykinin receptor antagonists.

A stepwise chemical modification from human neurokinin-3 receptor (hNK-3R)-selective antagonists to potent and combined hNK-3R and hNK-2R antagonists using the same 2-phenylquinoline template is described. Docking studies with 3-D models of the hNK-3 and hNK-2 receptors were used to drive the chemical design and speed up the identification of potent and combined antagonsits at both receptors. (S)-(+)-N-(1-Cyclohexylethyl)-3-[(4-morpholin-4-yl)piperidin-1-yl]methyl-2-phenylquinoline-4-carboxamide (compound 25, SB-400238: hNK-3R binding affinity, K(i) = 0.8 nM; hNK-2R binding affinity, K(i) = 0.8 nM) emerged as the best example in this approach. Further studies led to the identification of (S)-(+)-N-(1,2,2-trimethylpropyl)-3-[(4-piperidin-1-yl)piperidin-1-yl]methyl-2-phenylquinoline-4-carboxamide (compound 28, SB-414240: hNK-3R binding affinity, K(i) = 193 nM; hNK-2R binding affinity, K(i) = 1.0 nM) as the first hNK-2R-selective antagonist belonging to the 2-phenylquinoline chemical class. Since some members of this chemical series showed a significant binding affinity for the human mu-opioid receptor (hMOR), docking studies were also conducted on a 3-D model of the hMOR, resulting in the identification of a viable chemical strategy to avoid any significant micro-opioid component. Compounds 25 and 28 are therefore suitable pharmacological tools in the tachykinin area to elucidate further the pathophysiological role of NK-3 and NK-2 receptors and the therapeutic potential of selective NK-2 (28) or combined NK-3 and NK-2 (25) receptor antagonists.

Recent advances in the development of dopamine D(3) receptor agonists and antagonists.

Advances in molecular cloning techniques have allowed the characterization of five subtypes (D(1)-D(5)) of dopamine (DA) receptors. The limbic location of the D(3) receptor has led to speculation about its possible role in schizophrenia and drug abuse. Since the D(3) receptor is localized in the limbic region rather than the striatum, antipsychotics with D(3) receptor selectivity could be devoid of extrapyramidal side effects commonly seen with D(2) receptor antagonists. Recent work in our laboratory revealed that the benz[e] indole cis-(+/-)-44b demonstrated high selectivity for the D(3) receptor. This compound exhibits a typical antipsychotic profile without the motor effects found in commonly used antipsychotic agents. This mini-review will give a brief introduction on D(3) receptors and a detailed description of selectively-acting D(3) agonists and antagonists which have recently appeared in the literature.

SPECT tracer [(123)I]IBZM has similar affinity to dopamine D2 and D3 receptors.

Emission tomography investigations of the pathophysiological involvement of the cerebral dopaminergic transmitter system in the living human brain relies heavily on a careful selection of the most suitable radioligand. In recent years, many clinical studies have employed [(123)I]IBZM in SPECT studies. The aim of the present study was to characterize the binding of IBZM to dopaminergic receptor subtypes as a means of elucidating which receptor subtypes are visualized and examined by [(123)I]IBZM. The affinity of IBZM for each of the major human dopamine receptors (D1, D2(short), D3, D4(4. 2), and D5 receptor) was determined by competitive radioligand binding assay using membranes prepared from clonal cell lines expressing the different subtypes. Radioligands with high affinity for the D1(A) and D5 receptors ([(3)H]SCH-23390), dopamine D2(short) and D4(4.2) receptors ([(3)H]Spiroperidol), and dopamine D3 receptor ([(3)H]7-OH-DPAT) were used to measure specific binding. Corresponding unlabeled displacing ligands for determination of nonspecific binding were employed. Assays were performed at 25 degrees C. These experiments show that for IBZM K(i) values were 1.6 nM for dopamine D2(s) receptors and 2.2 nM for dopamine D3 receptors. There was no binding of IBZM to D1(A), D5, or D4(4.2) receptors. In conclusion, when [(123)I]IBZM is used as SPECT tracer, the studies reflect dopaminergic D2 as well as D3 receptor binding.

Synthesis and antinociceptive activity of pyrrolidinylnaphthalenes.

In this paper the synthesis of the racemates (2R,3S/2S,3R)-1,2-dimethyl-3-[2-(6-substituted naphthyl)]-3-hydroxypyrrolidine 1b-d [(2R,3S/2S,3R)-1b-d] are reported. Compounds 1b-d were prepared by reaction of the racemic 1,2-dimethyl-3-pyrrolidone 2 with the lithiation product obtained from 2-bromo-6-substituted naphthalene 3b-d. Pharmacological properties of (2R,3S/2S,3R)-1a-d are also described. Analgesic activity was investigated by the hot plate test and binding affinities towards mu, delta and kappa opioid receptors were evaluated. A preliminary evaluation of the in vivo side-effects was also accomplished using the rota-rod test. Interesting antinociceptive activity was shown by all compounds and in particular by 1d, which is the most active compound, since it is six-fold more potent than morphine and has lower side effects on the locomotory activity.

Dopamine D1B receptor chimeras reveal modulation of partial agonist activity by carboxyl-terminal tail sequences.

NNC 01-0012, a second-generation benzazepine compound, pharmacologically differentiates multiple vertebrate D1 receptor subtypes (D1A, D1B, D1C, and D1D) and displays high selectivity and affinity for dopamine D1C receptors. Functionally, whereas NNC 01-0012 acts as a full or poor antagonist at D1C and D1A receptor-mediated cyclic AMP production, respectively, it exhibits partial agonist activity at the D1B receptor. To define some of the structural motifs that regulate the pharmacological and functional differentiation of vertebrate dopamine D1 receptors by NNC 01-0012, a series of receptor chimeras were constructed in which the divergent carboxyl-terminal (CT) receptor tails were replaced with the corresponding sequences of D1A, D1B, or D1C receptors. Substitution of the vertebrate D1B carboxyl-terminal-tail at position Tyr345 with carboxyl-terminal-tail sequences of the D1A receptor abolished the partial agonist activity of NNC 01-0012 without affecting dopamine-stimulated cyclic AMP accumulation. At vertebrate D1B/D1CcT-tail receptor mutants, however, the intrinsic activity of the partial agonist NNC 01-0012 (10 microM) was markedly enhanced (approximately 60% relative to 10 microM dopamine) with no concomitant alteration in the molecule's ligand binding affinity or constitutive activity of the chimeric receptor. Similar results were obtained with other benzazepines such as SKF-38393 and SCH-23390, which act as partial agonists at vertebrate D1B receptors. Substitution of D1A and D1C receptor carboxyl-terminal tails with sequences encoded by the D1B receptor carboxyl-terminal tail did not, however, produce receptors with functional characteristics significantly different from wild type. Taken together, these data clearly suggest that in addition to well-characterized domains and amino acid residues in the third cytoplasmic loop, partial agonist activity at the D1B receptor is modulated by sequence-specific motifs within the carboxyl-terminal tail, a region that may underlie the possible structural basis for functionally divergent roles of multiple dopamine D1-like receptors.

Functional differentiation of multiple dopamine D1-like receptors by NNC 01-0012.

Although members of the multiple vertebrate/mammalian dopamine D1 receptor gene family can be selectively classified on the basis of their molecular/phylogenetic, structural, and tissue distribution profiles, no subtype-specific discriminating agents have yet been identified that can functionally differentiate these receptors. To define distinct pharmacological/functional attributes of multiple D1-like receptors, we analyzed the ligand binding profiles, affinity, and functional activity of 12 novel NNC compounds at mammalian/vertebrate D1/D1A and D5/D1B, as well as vertebrate D1C/D1D, dopamine receptors transiently expressed in COS-7 cells. Of all the compounds tested, only NNC 01-0012 displayed preferential selectivity for vertebrate D1C receptors, inhibiting [3H]SCH-23390 binding with an estimated affinity (approximately 0.6 nM) 20-fold higher than either mammalian/vertebrate D1/D1A or D5/D1B receptors or the D1D receptor. Functionally, NNC 01-0012 is a potent antagonist at D1C receptors, inhibiting to basal levels dopamine (10 microM)-stimulated adenylyl cyclase activity. In contrast, NNC 01-0012 (10 microM) exhibits weak antagonist activity at D1A receptors, inhibiting only 60% of maximal cyclic AMP production by dopamine, while acting as a partial agonist at vertebrate D1B and D1D receptors, stimulating adenylyl cyclase activity by approximately 33% relative to the full agonist dopamine (10 microM), an effect that was blocked by the selective D1 receptor antagonist NNC 22-0010. These data clearly suggest that the benzazepine NNC 01-0012, despite lacking the N-methyl residue in the R3 position, is a selective and potent D1C receptor antagonist. Moreover, the differential signal transduction properties exhibited by NNC 01-0012 at these receptor subtypes provide further evidence, at least in vertebrates, for the classification of the D1C receptor as a distinct D1 receptor subtype.

Behavioral and neurochemical effects of the preferential dopamine D3 receptor agonist cis-8-OH-PBZI.

In the present study we investigated the in vivo pharmacological profile of the benz[e]indole cis-8-hydroxy-3-(n-propyl)],2,3a,4,5,9b-hexahydro-1H-benz[e]indole (cis-8-OH-PBZI), which has been described as a preferential dopamine D3 receptor agonist in vitro. The compound inhibited spontaneous locomotor activity in mice, an effect which was antagonized by the dopamine D3 receptor antagonist 5,6-dimethoxy-2-(di-u-propylamino) indan (U99194A). Moreover, cis-8-OH-PBZI inhibited conditioned avoidance responding in rats, a preclinical test indicative of antipsychotic efficacy, at doses which did not induce catalepsy. Doses of cis-8-OH-PBZI (6 and 12 mg/kg) that inhibited spontaneous locomotor activity in rats did not affect interstitial levels of dopamine and dihydroxyphenylacetic acid (DOPAC) in the nucleus accumbens or dorsolateral striatum. In contrast to the effect of the dopamine receptor agonist (+/-)-2-dipropylamino-7-hydroxy-1,2,3,4-tetrahydronaphythalene (7-OH-DPAT), cis-8-OH-PBZI did not induce locomotor activity in reserpinized mice. In conclusion, cis-8-OH-PBZI exhibits a pharmacological profile that suggests it has antipsychotic activity but lacks the motoric side effects often associated with antipsychotic medication. The data suggest a mechanism requiring the activation of postsynaptic dopamine D3 receptors and support the hypothesis that these receptors mediate inhibitory behavioral effects.

The preferential dopamine D3 receptor agonist cis-8-OH-PBZI induces limbic Fos expression in rat brain.

The affinity, selectivity and agonistic properties of a constrained dopaminergic compound, the benz[e]indole cis-8-hydroxy-3-(n-propyl)1,2,3a.4,5,9b-hexahydro-1H-benz[e]indole (cis-8-OH-PBZI), for the dopamine D3 receptor were evaluated in competition binding experiments with cloned human dopamine receptor subtypes and, to further extend its profile, in in vitro radioligand binding assays. The Ki value measured for competition binding of this compound to the dopamine D3 receptor was 27.4+/-3.1 nM; this was 775-fold, 550-fold, 90-fold and 10-fold higher affinity than that measured at dopamine D1A, D5, D2s and D4 receptors, respectively. Evidence of dopamine receptor activation by cis-8-OH-PBZI was obtained by measuring dose-dependent increases in extracellular acidification rates and decreases in cAMP synthesis. In vivo, cis-8-OH-PBZI potently induced Fos protein immunoreactivity in the rat medial prefrontal cortex and shell region of the nucleus accumbens, but only marginally in the motor dorsolateral striatum, indicating a selective limbic site of action. In conclusion, the present data identify cis-8-OH-PBZI as having preference for the dopamine D3 receptor in vitro, and as having dopamine agonist activity and limbic sites of action in vivo.

Agonist-induced functional desensitization of the mu-opioid receptor is mediated by loss of membrane receptors rather than uncoupling from G protein.

The effects of acute exposure of the opioid peptide [D-Ala2,N-MePhe4, Gly-ol5]enkephalin (DAMGO) on the mu-opioid receptor were examined in Chinese hamster ovary (CHO) K-1 and baby hamster kidney stable transfectants. In the CHO cell line, acute 1-hr treatment with DAMGO decreased the density of receptors without affecting the affinity or proportion of agonist-detected sites and attenuated the ability of the agonist to inhibit forskolin-stimulated cAMP accumulation. In contrast, similar 1-hr treatment of baby hamster kidney cells did not affect receptor density or agonist ability to inhibit cAMP accumulation, but longer duration of agonist exposure resulted in a reduction in membrane receptor, identical to the CHO cells. These results suggested that for the mu-opioid receptor, alteration in receptor density was the major determinant for the observed agonist-induced desensitization. Consistent with this notion, the ratio of the DAMGO concentration yielding half-maximal occupation of the mu receptor to that yielding half-maximal functional response was < 1. This suggests the necessity for a high mu receptor occupancy rate for maximal functional response, so that any loss of cell surface opioid-binding sites was a critical determinant in reducing the maximal response. This hypothesis was further supported by the observation that irreversible inactivation of fixed proportions of opioid-binding sites with beta-chlorn-altrexamine demonstrated that there were few spare receptors, which is in contrast to what has been reported for other G protein-coupled receptors, including the delta-opioid receptor. Taken together, these data suggest that the opioid agonist DAMGO has a high affinity for the mu receptor but must occupy > 70% of the available receptors to generate the maximal second messenger-linked response.

Behavioural and anticonvulsant effects of Ca2+ channel toxins in DBA/2 mice.

This study investigated the behavioural and anticonvulsant effects of voltage-sensitive calcium channel blockers in DBA/2 mice. Omega-Conotoxin MVIIC (0.1, 0.3 micrograms ICV/mouse) and omega-agatoxin IVA (0.1, 0.3, 1 micrograms ICV), which act predominantly at P- and/or Q-type calcium channels, prevented clonic and tonic sound-induced seizures in this animal model of reflex epilepsy (ED50 values with 95% confidence limits for protection against clonic sound-induced seizures were 0.09 (0.04-0.36) micrograms ICV and 0.09 (0.05-0.15) micrograms ICV respectively and against tonic seizures 0.07 (0.03-0.16) micrograms ICV and 0.08 (0.04-0.13) micrograms ICV, respectively). The N-type calcium channel antagonists omega-conotoxin GVIA and omega-conotoxin MVIIA were also tested in this model. Omega-Conotoxin GVIA was anticonvulsant in DBA/2 mice, but only at high doses (3 micrograms ICV prevented tonic seizures in 60% of the animals; 10 micrograms ICV prevented clonic seizures in 60% and tonic seizures in 90% of the animals), whereas omega-conotoxin MVIIA did not inhibit sound-induced seizures in doses up to 10 micrograms ICV. Both omega-conotoxin GVIA and omega-conotoxin MVIIA induced an intense shaking syndrome in doses as low as 0.1 microgram ICV, whereas omega-conotoxin MVIIC and omega-agatoxin IVA did not produce shaking at any of the doses examined. Finally, omega-conotoxin GI (0.01-1 microgram ICV) and alpha-conotoxin SI (0.3-30 micrograms ICV), which both act at acetylcholine nicotinic receptors, were not anticonvulsant and did not induce shaking in DBA/2 mice. These results confirm that blockers of N- and P-/Q-type calcium channels produce different behavioural responses in animals. The anticonvulsant effects of omega-conotoxin MVIIC and omega-agatoxin IVA in DBA/2 mice are consistent with reports that P- and/or Q-type calcium channel blockers inhibit the release of excitatory amino acids and are worthy of further exploration.

The cloning and chromosomal mapping of two novel human opioid-somatostatin-like receptor genes, GPR7 and GPR8, expressed in discrete areas of the brain.

Following the cloning of the opioid receptors mu, kappa, and delta, we conducted a search for related receptors. Using oligonucleotides based on the opioid and also the structurally related somatostatin receptors, we amplified genomic DNA using the polymerase chain reaction and isolated fragments of novel G protein-coupled receptor genes. Two of these gene fragments designated clones 12 and 11 were used to isolate the full-length genes. The intronless coding sequences of these genes, named GPR7 and GPR8, shared 70% identity with each other, and each shared significant similarity with the sequences encoding transmembrane regions of the opioid and somatostatin receptors. GPR7 was mapped to chromosome 10q11.2-q21.1 and GPR8 to chromosome 20q13.3. Northern blot analysis using human mRNA demonstrated expression of GPR7 mainly in cerebellum and frontal cortex, while GPR8 was located mainly in the frontal cortex. In situ hybridization revealed expression of GPR7 in the human pituitary. A partial sequence of the mouse orthologue of GPR7 was obtained, and in situ hybridization demonstrated expression in discrete nuclei of brain, namely suprachiasmatic, arcuate, and ventromedial nuclei of hypothalamus. A stable cell line expressing the GPR7 gene was created, but expression levels of the receptor were low. The available pharmacology indicated binding to several opioid drugs such as bremazocine, levorphanol, and beta-FNA, but not to the opioid receptor subtype-selective mu, delta, or kappa agonists.

F11 neuroblastoma x DRG neuron hybrid cells express inhibitory mu- and delta-opioid receptors which increase voltage-dependent K+ currents upon activation.

The F11 cell line is a fusion product of cells of mouse neuroblastoma cell line N18TG-2 with embryonic rat dorsal-root ganglion (DRG) neurons. Previous biochemical results suggest that they express mu- and delta-opioid receptors that are negatively coupled to adenylate cyclase. The present study provides direct agonist-binding and electrophysiologic evidence of mu and delta, but not kappa, receptor expression in F11 cells. Radioligand binding assays show that F11 cell membranes bind the mu- and delta-opioid receptor agonists, DAGO and DPDPE with Kd = 4.5 and 4.9 nM and Bmax = 111 and 195 fmol/mg, respectively. Tight-seal patch-clamp recordings of F11 cells after several days in a differentiating culture medium (low serum, cyclic AMP and nerve growth factor) showed that: (i) the outward K+ current during pulsed depolarization in most of these cells was increased by either DAGO or DPDPE, but none were responsive to both opioids or to the kappa-opioid receptor agonist, U-50,488H. The response was blocked by relevant receptor antagonists, naloxone, beta-funaltrexamine or naltrindole; (ii) cells without processes responded neither to DAGO nor to DPDPE; (iii) treatment with pertussis toxin blocked all opioid-induced increases in outward K+ current. The opioid-induced increase in voltage-dependent membrane K+ current in F11 cells resembles the inhibitory effect elicited by mu- and delta-opioid agonists in primary cultures of mouse DRG neurons.

High efficiency reconstitution of a phencyclidine/MK-801 receptor binding site solubilized from rat forebrain membranes.

Phencyclidine (PCP) receptors have been solubilized from rat forebrain membranes with the zwitterionic detergent 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate. Specific binding of the potent PCP receptor ligands [3H]thienyl-phencyclidine (TCP) and [3H]MK-801 was restored by incorporating extracted membrane protein into lipid vesicles prepared from a total brain lipid extract. A nearly quantitative recovery of solubilized receptor activity was achieved; this was dependent upon both the concentration of detergent used during membrane solubilization and the concentration of added lipid used during the reconstitution. The single, saturable, binding site measured for both [3H]TCP and [3H]MK-801 in solubilized and reconstituted preparations exhibited properties similar to those of the high affinity PCP binding site labeled by these ligands in brain membranes. The ability of ligands selective for this site (MK-801, TCP, and dexoxadrol) to competitively displace specific [3H]TCP binding was retained after solubilization and reconstitution, although IC50 values measured for these ligands were shifted to higher concentrations. Levoxadrol and haloperidol were ineffective at displacing the radioligand binding in both membrane and vesicle preparations. The additive and dose-dependent ability of glutamate and glycine to enhance [3H]TCP binding to the solubilized/reconstituted receptor further suggests that a direct interaction with the N-methyl-D-aspartate receptor/ion channel complex has been preserved in the vesicle preparations. The photoaffinity labeling of two polypeptides (Mr 98,000 and 59,000) by azido-[3H]PCP was demonstrated in the vesicle preparations; this was largely prevented by competitive displacement of the radioligand with PCP before photolysis. These results establish both an essential lipid dependency and polypeptide composition for the high affinity, haloperidol-insensitive, PCP receptor in brain.

Characterization of active and latent forms of the membrane-associated sn-glycerol-3-phosphate acyltransferase of Escherichia coli.

The intrinsically active, sn-glycerol-3-phosphate acyltransferase present in membranes prepared from both wild type Escherichia coli and from strains which overproduce the enzyme can be kinetically distinguished from a latent enzyme species which is unmasked by solubilization and reconstitution. Both membrane-associated and solubilized/reconstituted enzyme preparations exhibited cooperativity with respect to sn-glycerol-3-phosphate and palmitoyl-coenzyme A substrates; positive cooperativity in membranes toward palmitoyl-coenzyme A (napp = 4) and negative cooperativity toward sn-glycerol-3-phosphate (napp = 0.75) were significantly altered upon solubilization and reconstitution. Since the degree of alteration increased with the amount of sn-glycerol-3-P acyltransferase present in the membranes, a detergent-dissociable homooligomerization of the sn-glycerol-3-phosphate acyltransferase was considered as an underlying mechanism. This possibility was investigated by changing the protein-to-Triton X-100 ratio of homogeneous enzyme prior to reconstitution and then analyzing the subsequent migration of samples on a Sephacryl S-300 sizing column. The elution positions were consistent with monomeric and dimeric polypeptide bound to micelles of Triton X-100. Hill coefficients for monomeric, reconstituted enzyme preparations were comparable to those obtained for the active, membrane-associated sn-glycerol-3-phosphate acyltransferase. The reduced cooperativity of dimeric, reconstituted enzyme preparations correlated closely to the Hill coefficient values obtained for latent, solubilized/reconstituted sn-glycerol-3-phosphate acyltransferase from membranes of Escherichia coli which overproduce the enzyme. The physiological significance of these findings is discussed.

Glycine-like modulation of N-methyl-D-aspartate receptors by a monoclonal antibody that enhances long-term potentiation.

We have identified a monoclonal antibody, B6B21, that significantly elevates long-term potentiation when applied to CA1 pyramidal cell apical dendrites in rat hippocampal slices and characterized its binding to N-methyl-D-aspartate-receptor complexes using extensively washed hippocampal membranes. Five micrograms of affinity-purified B6B21 per 100 micrograms of membranes gave a two- to threefold elevation in N-[1-(2-thienyl)cyclohexyl]-3,4-[3H]piperidine ([3H]TCP) binding. When [3H]TCP binding was stimulated by the combined addition of maximal concentrations of glutamate, glycine, and magnesium, B6B21 no longer stimulated [3H]TCP binding. Like glycine, B6B21 enhanced the effect of N-methyl-D-aspartate and glutamate in stimulating [3H]TCP binding. Moreover, B6B21 reversed 7-chlorokynurenic acid inhibition of [3H]TCP binding, but it had no effect on the inhibition of [3H]TCP binding by D-(-)-2-amino-5-phosphonovaleric acid. B6B21 increased the rate of association and dissociation of [3H]TCP, but had no effect on equilibrium binding. Glutamate, but not glycine, however, increased B6B21-enhancement of [3H]TCP association and dissociation. B6B21 binding at strychnine-insensitive glycine sites was confirmed by direct measurement of [3H]glycine binding. These results suggest that B6B21 binds directly to N-methyl-D-aspartate receptors and displays properties similar to glycine.

Reconstitution of solubilized delta-opiate receptor binding sites in lipid vesicles.

Delta-opiate receptors have been solubilized with the non-ionic bile salt detergent digitonin from NG108-15 cell membranes and reconstituted into lipid vesicles. Specific opiate binding was restored to soluble receptor preparations after supplementation with a brain lipid extract, and dilution below the effective detergent concentration. Saturable and specific opiate binding was measured for both membrane and vesicle preparations; dissociation constants (Kd) obtained from saturation isotherms of [3H]bremazocine binding were 1.3 and 4.2 nM, respectively. Relative affinity (IC50) values of ligand binding measured for subtype-selective agonists confirmed that a delta-opiate binding site interaction was recovered in vesicle preparations. Changes in agonist binding affinity noted for these experiments were explained by dissociation of the GTP-binding protein Gi from the receptor in detergent. The recovery of solubilized opiate receptors was nearly quantitative, and strictly dependent upon the total brain lipid preparation used in the reconstitution. Ligand binding was incompletely recovered after substituting pure, vesicle-forming phospholipid preparations. [3H]Bremazocine binding was also reconstituted after lectin affinity chromatography of solubilized receptor preparations, using conditions which likely effect the removal of endogenous lipid cofactors. A photoaffinity cross-linking methodology was employed to verify recovery of the delta-opiate receptor after its solubilization from membranes and reconstitution. Two membrane-associated proteins (50 and 70 kDa) were covalently tagged with an azido analog of beta-endorphin(Leu5) in cell membranes and subsequently identified by immunoblotting with antisera directed against this opioid. Labeling of the 50-kDa polypeptide was prevented by coincubating assay samples with a relative excess of (D-Pen2,5)enkephalin. This opioid binding polypeptide was also present in solubilized/reconstituted receptor preparations.

Phospholipid dependence of homogeneous, reconstituted sn-glycerol-3-phosphate acyltransferase of Escherichia coli.

A novel mixed micelle assay for the sn-glycerol-3-phosphate acyltransferase of Escherichia coli was developed using the nonionic detergent octaethylenegly-coldodecyl ether. The assay permitted investigation of the phospholipid dependence of enzyme activity at phospholipid/detergent ratios of 5:1 (w/w) to 2:1 depending on the phospholipid employed. The higher ratio yielded maximal activity when E. coli phospholipids were used; the lower ratio was observed with cardiolipin(E. coli). Phosphatidylglycerol(E. coli) and phosphatidylethanolamine(E. coli) also restored enzyme activity. Activation by phosphatidylethanolamine(E. coli) was pH-dependent and relatively inefficient. The synthetic, disaturated (1,2-palmitoyl)phosphatidylglycerol reconstituted only 25% of the total enzyme activity as that observed with the monounsaturated (1-palmitoyl, 2-oleoyl) species. Full activation of enzyme was achieved with (1,2-dioleoyl)phosphatidylglycerol. Phosphatidylcholine and phosphatidic acid were unable to reconstitute enzyme activity. Chromatographic sizing of the sn-glycerol-3-phosphate acyltransferase, following reconstitution in cardiolipin(E. coli)/octaethyleneglycoldodecyl ether mixed micelles, suggested that the monomeric form of the enzyme was active.

Efficiency of reconstitution of the membrane-associated sn-glycerol 3-phosphate acyltransferase of Escherichia coli.

Membrane-associated enzymes are often solubilized with detergents, purified, and then reconstituted with phospholipid cofactors to regain function. Insofar as most purification and reconstitution procedures are not quantitative, the final reconstituted preparations could reflect a population of molecules ranging from fully functional to completely inactive. Quantitative studies on the efficiency of reconstitution of the Triton X-100-solubilized sn-glycerol 3-phosphate (glycerol-P) acyltransferase of Escherichia coli cytoplasmic membrane were undertaken at each step of purification. Physical recovery of the 83,000 Mr polypeptide was quantitated in polyacrylamide gels using membranes from cells labeled with [3H]leucine. The 83,000 Mr polypeptide in such gels was demonstrated to consist exclusively of the glycerol-P acyltransferase peptide by V8 peptide mapping. Comparison between physical recovery of 83,000 Mr polypeptide and reconstituted activity allowed the efficiency of reconstitution to be determined. Unexpectedly, disproportionalities occurred during the purification. However, the final purification of reconstituted enzyme activity matched that of the 83,000 Mr polypeptide. This method also allowed measurement of the specific activities of the glycerol-P acyltransferase in membranes from a wild type E. coli strain and from plasmid-containing strains which express the plsB gene product to different extents. The physical amounts of the 83,000 Mr polypeptide and glycerol-P acyltransferase activity measured in membranes were not strictly proportional. In strains where the amount of 83,000 Mr polypeptide was enhanced, a larger proportion of latent activity was observed following solubilization and reconstitution. The results establish the suitability of the reconstituted preparations of glycerol-P acyltransferase for detailed kinetic analysis and permit inferences pertaining to regulation.