Specific adsorbents for affinity chromatography of benzodiazepine binding proteins

The use of affinity chromatography as means of isolating/purifying proteins which have an affinity for benzodiazepine is described. Three such drugs are employed: chlorazepate, clonazepam and delorazepam. The results presented in this paper indicate that the proposed technique only works for chlorazepate and delorazepam. In fact these benzodiazepine-Sepharose derivatives are able to retain specifically proteins from human serum and rat kidney, lung, skeletal muscle and brain.     

Isolation of Putative Benzodiazepine Receptors from Rat Brain Membranes by Affinity Chromatography

Abstract: The benzodiazepine receptor from rat brain was solubilised and purified 5200-fold by affinity chromatography. The affinity column contained an immobilized benzodiazepine (delorazepam) and biospecific elution with 6 m m -chlorazepate was achieved. The purified receptor is apparently homogeneous in SDS-polyacrylamide gel electrophoresis. The native protein had a molecular weight of 240,000, and the subunit one of 60,000. The dissociation constant ( K _D) is 8 n m for [³H]diazepam. A correlation exists between the value of affinity obtained for benzodiazepine derivatives and their known pharmacological effectiveness.     

Human heat shock gene expression and the modulation of plasma membrane Na+, K+-ATPase activity

Benzodiazepine receptor solubilized from bovine cortical membranes was bound to a new benzodiazepine affinity column, the synthesis of which is described. Bio-specific elution with the benzodiazepine compound chlorazepate resulted in the elution of fractions highly enriched in specific binding for the GABA receptor agonist muscimol. Specific activity for [³H]muscimol binding was >1.3 nmol/mg protein. It is shown that [³H]flunitrazepam binding activity can be recovered by removal of chlorazepate from the purified fraction. These results strongly support a model which suggests that the 2 binding sites reside on the same physical entity.     

Isolation and cloning of a voltage-dependent anion channel-like Mr 36,000 polypeptide from mammalian brain.

A polypeptide of M(r) 36,000 (36 kDa) was isolated from detergent-solubilized membrane fractions of mammalian brain on a benzodiazepine affinity column utilized for the purification of the gamma-aminobutyric acid/benzodiazepine receptor protein, followed by preparative gel electrophoresis. Partial protein sequence for two fragments of the 36-kDa polypeptide allowed the isolation of cDNA clones from a rat hippocampal library. An open reading frame coding a sequence of 295 amino acid residues containing the two probe peptide sequences with minor differences, and a putative N-terminal signal peptide of 25 residues was found. Hydropathy index revealed no regions of alpha-helix suitable for membrane spanning, but several areas of alternating hydrophilic and hydrophobic residues consistent with beta-strands. The sequence of this brain protein was 24% identical to that of a yeast mitochondrial protein, the voltage-dependent anion channel (VDAC), and over 70% identical with the VDAC from human B lymphocytes. The gamma-aminobutyric acid type A (GABAA) receptor/36-kDa preparation purified on benzodiazepine affinity column has channel-forming activity in lipid bilayer membranes that is virtually identical to VDAC isolated from mitochondria of various sources, indicating that the 36-kDa protein is a new member of the VDAC family of proteins. An antiserum raised against the purified 36-kDa polypeptide was able to precipitate [3H]muscimol binding activity, indicating a tight association with the GABAA receptor protein in vitro and copurification on the benzodiazepine affinity column due to this association. Further studies are needed to determine whether such an association occurs in vivo.     

The role of benzodiazepine receptors in the induction of differentiation of HL-60 leukemia cells by benzodiazepines and purines

A series of benzodiazepines was evaluated for their capacity to induce the differentiation of HL-60 acute promyelocytic leukemia cells. Benzodiazepines were effective initiators of maturation in the concentration range of 50 to 150 microM. The possible involvement of benzodiazepine receptors in mediating the differentiation induced by these agents was investigated. The presence of high affinity, peripheral type benzodiazepine binding sites (KD = 7.3 nM, TB = 14.5 pmol/mg protein with Ro5-4864) was demonstrated in HL-60 membranes. The occupancy of peripheral type high affinity benzodiazepine receptors by various benzodiazepines showed some correlation (r = 0.76) with their differentiation-inducing capabilities, but binding potencies were 1,000-fold higher than the concentrations required to produce differentiation. A class of benzodiazepine receptors with lower binding affinity was also detected in HL-60 membranes (KD = 28.6 microM; TB = 199 pmol/mg protein with diazepam). A higher level of correlation (r = 0.88) was demonstrated between benzodiazepine occupancy of these lower affinity receptors and the capacity to induce maturation. Significantly, benzodiazepine concentrations needed for low affinity binding and induction of differentiation were the same (25-200 microM), suggesting that low affinity benzodiazepine receptors may be involved in the induction process. We have shown that the molecular form responsible for the induction of the differentiation of HL-60 cells to mature forms by 6-thioguanine (TGua) is the free base, TGua, itself [Ishiguro, Schwartz, and Sartorelli (1984) J. Cell. Physiol., 121:383-390]. Since hypoxanthine (Hyp) and inosine (Ino) have been identified as putative endogenous ligands for high affinity benzodiazepine receptors in brain tissue, the potential involvement of benzodiazepine receptors in the differentiation of HL-60 cells by the purines was investigated. Physiological purines such as Hyp and Ino were inactive in displacing the benzodiazepines from their high and low affinity binding sites in HL-60 membranes. In contrast, TGua caused inhibition of benzodiazepine binding to high and low affinity sites. The inhibition of Ro5-4864 binding to high affinity binding sites by TGua appeared to be due to the binding of TGua to membranes through the formation of a mixed disulfide between the 6-thiopurine and protein thiols, since the inhibition was reversed by the presence of 2-mercaptoethanol. The findings suggest a possible relationship between the occupancy of benzodiazepine receptors by TGua and the induction of leukemic cell differentiation.     

Postmortem Degradation Alters Fluorographic Labeling Patterns and Affinities of Benzodiazepine Binding Proteins

To investigate the effect of endogenous proteolysis on the molecular weights of the benzodiazepine binding proteins, brains of trout, chicken, and rat were removed immediately after death and stored at room temperature for various periods of time before they were frozen. Photoaffinity labeling of membranes with [3H]flunitrazepam, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, revealed proteolytic fragments of 47K in trout, chicken, and rat. The proteolysis set in rapidly after death. Seemingly in parallel with the degradation observed fluorographically, the affinity for [3H]flunitrazepam increased without systematic changes in receptor density. The degradation pattern was not identical to that of the photolabeled trypsinized benzodiazepine binding proteins. The endogenous proteolytic fragments were deglycosylated in two steps. In conclusion, proteolytic effects must be taken into account when interpreting labeling patterns and binding parameters.     

Microcalorimetric method to determine competitive binding. Action of a psychotropic drug (dipotassium chlorazepate) on L-tryptophan . human serum albumin complex.

A mathematical treatment and an original microcalorimetric method are developed to verify an eventual competitive binding between any two substances for the same macromolecule. To apply this method, a competitive binding of L-tryptophan and one benzodiazepin (dipotassium chlorazepate) for human serum albumin is perfectly demonstrated. The association constants and the enthalpy variations are equal to 14 000 +/- 2000 M-1 and --6.6 +/- 0.2 kcal/mol for human serum albumin . tryptophan complex and 13 000 +/- 1000 M-1 and --10.0 +/- 0.2 kcal/mol for human serum albumin . chlorazepate complex. In all cases the stoichiometry is equal to one. The binding of tryptophan to human serum albumin is partially stereospecific; the association constant and the enthalpy variation for D-tryptophan complex are equal, respectively, to 1000 +/- 200 M-1 and --2.6 +/- 0.3 kcal/mol.     

Irreversible Binding of [3H]Flunitrazepam to Different Proteins in Various Brain Regions

Irreversible photolabeling by [3H]flunitrazepam of four proteins with apparent molecular weights 51,000 (P51), 53,000 (P53), 55,000 (P55), and 59,000 (P59) was investigated in various rat brain regions by SDS-polyacrylamide gel electrophoresis, fluorography, and quantitative determination of radioactivity bound to proteins. On maximal labeling of these proteins, only 15-25% of [3H]flunitrazepam reversibly bound to membranes becomes irreversibly attached to proteins. Results presented indicate that for every [3H]flunitrazepam molecule irreversibly bound to membranes, three molecules dissociate from reversible benzodiazepine binding sites. This seems to indicate that these proteins are either closely associated or identical with reversible benzodiazepine binding sites, and supports the hypothesis that four benzodiazepine binding sites are associated with one benzodiazepine receptor. When irreversible labeling profiles of proteins P51, P53, P55, and P59 were compared in different brain regions, it was found that labeling of individual proteins varied independently, supporting previous evidence that these proteins are associated with distinct benzodiazepine receptors.     

Behavioural actions of Ro 5-4864: A peripheral-type benzodiazepine?

Ro 5-4864 is a 1,4 benzodiazepine lacking typical benzodiazepine behavioural actions, and which has very low affinity for the “classical” CNS benzodiazepine binding sites. However, Ro 5-4864 has very high affinity for the peripheral type of binding site in the periphery and in the brain. Evidence is reviewed that Ro 5-4864 is sedative, convulsant and anxiogenic in rodents. We also describe the effects of combining Ro 5-4864 treatment with benzodiazepines (e.g. diazepam, chlordiazepoxide) and with other drugs that modify the activity of benzodiazepines (Ro 15-1788, CGS 8216, picrotoxin, PK 11195, phenytoin). The binding sites that might be mediating these behavioural actions of Ro 5-4864 are discussed.     

Further Characterization of the Avian Benzodiazepine Receptor Subunits Including Phylo-and Ontogenetic Aspects

The two avian benzodiazepine binding proteins offer an opportunity for further studies concerning their regional variation and their phylo- and ontogenetic development. Accordingly, regional variation of the benzodiazepine binding proteins is investigated further in two reptiles and chicken using photoaffinity labeling with [3H]flunitrazepam followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Whereas regional heterogeneity is pronounced in chicken, it is not readily apparent in the two reptiles. The ontogeny of the benzodiazepine binding proteins in chicken forebrain and cerebellum is remarkably similar to that previously reported in rodents. The results are discussed in light of the possible existence of the gamma-aminobutyric acid/benzodiazepine receptor as an isoreceptor complex.     

3-Ethoxy-beta-carboline: a high affinity benzodiazepine receptor ligand with partial inverse agonist properties

3-Ethoxy-beta-carboline binds with high affinity to benzodiazepine receptors in the central nervous system (Ki approximately equal to 10.1, 15.3, and 25.3 nM in rat cerebellum, cerebral cortex, and hippocampus, respectively). This compound has pharmacological actions reminiscent of benzodiazepine receptor partial inverse agonists such as FG 7142 and 3-carboethoxy-beta-carboline. Thus, while not a convulsant, 3-ethoxy-beta-carboline potentiated the convulsant actions of pentylenetetrazole in mice. Furthermore, this compound reduced both the time spent and the total entries in the open arms of an elevated plus maze and also inhibited stress-induced ulcer formation, effects that are also observed with benzodiazepine receptor inverse agonists. These findings suggest that 3-ethoxy-beta-carboline is a partial inverse agonist at benzodiazepine receptors which may prove useful for in vivo studies since it has a higher affinity for benzodiazepine receptors and better solubility than the commonly used partial inverse agonist FG 7142. Furthermore, 3-ethoxy-beta-carboline appears to be less vulnerable to metabolic degradation than ester analogs with a similar pharmacological profile such as 3-carboethoxy-beta-carboline.     

Affinity of beta-carbolines on rat brain benzodiazepine and opiate binding sites

The affinity of beta-carbolines, which may be formed in the body, to benzodiazepine and opiate receptors was studied by measuring their ability to inhibit the binding of [3H]-flunitrazepam and [3H]-dihydromorphine on rat brain synaptosomal membranes. All "aromatized" beta-carbolines studied (norharmane, harmane and 6-methoxyharmane) inhibited the specific binding of [3H]-flunitrazepam in micromolar concentrations, dihydro-beta-carbolines (6-methoxyharmalan, harmalol) were less potent, while all tetrahydro-beta-carbolines showed very low affinity. 6-Hydroxytetrahydroharmane, which is formed by condensation 5HT with acetaldehyde, inhibited [3H]-dihydromorphine binding in micromolar concentration, while norharmane and tetrahydro-beta-carbolines without OH-group showed little affinity. beta-Carbolines are the most potent known natural benzodiazepine receptor ligands. Because they are formed after alcohol drinking, their effects on benzodiazepine and opiate receptors may be connected with alcohol dependence although some beta-carbolines may inhibit 5HT uptake in still lower concentrations.     

Biotinylated 1012-S conjugate as a probe ligand for benzodiazepine receptors: characterization of receptor binding sites and receptor assay for benzodiazepine drugs.

A nonisotopic receptor assay using the biotin-1012-S conjugate was developed and the usefulness of this conjugate as a probe ligand for the benzodiazepine receptor was evaluated. The conjugate was incubated in a receptor suspension, and then the concentration of free conjugate in the supernatant was determined nonisotopically with a solid-phase avidin-biotin binding assay. Studies on the ligand saturation with the conjugate demonstrated that the conjugate has very high affinity and specificity for the receptors and the biotin labeling does not decrease the affinity of 1012-S. This assay method was applied to the characterization of binding sites of benzodiazepine receptors in cow brain. Competition interactions between the conjugate and benzodiazepine drugs gave well-defined dose-response curves. These results confirm the possibility that this conjugate could serve as a probe for the study of receptor-ligand interactions and provide the basis of a new nonisotopic receptor assay for benzodiazepine drugs.     

Characteristics of the benzodiazepine receptors in rats with different predispositions to the development of experimental alcoholism

A study was made of the density and affinity of benzodiazepine receptors in the cortex of the cerebral hemispheres and hippocampus of rats with different predisposition to alcohol consumption. No differences were revealed in the parameters under study in animals with varying duration of ethanol anesthesia and in rats after voluntary consumption of ethanol for 3.5 and 10 months. In a state of abstinence rats with physical dependence manifested a dramatic decrease in the density and affinity of benzodiazepine receptors in the cortex of the cerebral hemispheres. No changes described were detected in the hippocampus. The role of benzodiazepine receptors in the development of abstinence is discussed.     

Acute effects of drug administration and withdrawal on the benzodiazepine receptor

Effects of one week of benzodiazepine drug administration on central benzodiazepine receptor binding characteristics were evaluated in a series of experiments in male Sprague-Dawley rats. Administration of short- and intermediate-acting benzodiazepines was observed to increase the number of available receptor binding sites (B_max) without changing affinity of drug for receptor. Furthermore, these changes did not occur after administration and withdrawal of long-acting benzodiazepines. In addition, there appeared to be a relationship between the affinity of the different benzodiazepines for the receptor and the degree of increase in the number of receptor binding sites. The results may help to explain the relationship between withdrawal of certain benzodiazepine drugs and the occurrence of rebound phenomena in clinical situations.     

Microcalorimetric method to determine competitive binding action of a psychotropic drug (dipotassium chlorazepate) on L-tryptophan · human serum albumin complex

A mathematical treatment and an original microcalorimetric method are developed to verify an eventual competitive binding between any two substances for the same macromolecule. To apply this method, a competitive binding of L-tryptophan and one benzodiazepin (dipotassium chlorazepate) for human serum albumin is perfectly demonstrated.The association constants and the enthalpy variations are equal to 14 000 ± 2000 M^?1 and ?6.6 ± 0.2 kcal/mol for human serum albumin · tryptophan complex and 13 000 ± 1000 M^?1 and ?10.0 ± 0.2 kcal/mol for human serum albumin · chlorazepate complex. In all cases the stoichiometry is equal to one.The binding of tryptophan to human serum albumin is partially stereospecific; the association constant and the enthalpy variation for D-tryptophan complex are equal, respectively, to 1000 ± 200 M^?1 and ?2.6 ± 0.3 kcal/mol.     

Differential modulation by muscimol of [3H] flunitrazepam binding to benzodiazepine binding site subtypes

The effects of the GABA agonist, muscimol on [³H]flunitrazepam binding were examined in cerebellum and hippocampus regions proposed to contain different populations of benzodiazepine binding site subtypes. Quantitative analysis was made of the contribution of different components of [³H]flunitrazepam binding by utilising the selective affinities of propyl β-carboline-3-carboxylate for these sites. The influence of muscimol on each of these components was determined and the results provide clear evidence that GABA receptors interact with only some subtypes of benzodiazepine binding sites; for example, whilst the cerebellar site and the low affinity hippocampal site are influenced, the high affinity site in hippocampus appears to be quite unaffected.     

Uniform regulation of central and peripheral benzodiazepine binding sites by GABA receptor agonists in rats

Central and peripheral benzodiazepine binding sites were studied in vitro after the administration of GABAA and GABAB agonists. Cerebral cortex and kidney homogenates were used in this study. Muscimol (1.5 mg/kg, intraperitoneally) pretreatment significantly increased the affinity of benzodiazepine binding sites not only in the cerebral cortex but also in the kidneys. Similar changes were obtained with (-) and (+) baclofen (5 mg/kg, intraperitoneally), with the only exception that (-) baclofen in addition to changes in the affinity caused a marked decrease in the number of binding sites in both structures studied. The mechanism of action of GABA agonists on peripheral benzodiazepine binding sites is discussed.     

Photoaffinity Labelling of the Benzodiazepine Receptor Compromises the Recognition Site but Not Its Effector Mechanism

When subjected to ultraviolet light flunitrazepam could be irreversibly attached to brain membrane preparations such that the affinity of the benzodiazepine receptor for certain ligands, such as diazepam, becomes markedly reduced. However, the apparent affinity for diazepam is increased by the addition of gamma-aminobutyric acid and sodium chloride, to the same extent in this membrane preparation as in membranes not so pretreated. This observation suggests that photoaffinity labelling of the benzodiazepine receptor with flunitrazepam modifies the recognition characteristics of the receptor but not its effector mechanism.