Diazepam-Sensitive Specific Binding of Phenytoin in Rat Brain

Abstract

[³H]Phenytoin binding to rat cortical membrane was significantly enhanced in the presence of diazepam. This binding is saturable, reversible and displacable by unlabelled phenytoin. Analyses of the binding data either by the Scatchard plot or by the displacement curve revealed a high and a low affinity sites with K_d values of 32 ± 5 nM and 8.5 ± 1.1 μM, respectively. Similar enhancement of [³H]phenytoin binding was observed when diazepam was replaced by Ro 5–4864 (4″-chlorodiazepam) which is selective for the ‘peripheral’ type benzodiazepine binding sites. In contrast, neither the ‘central’ type receptor selective agonist clonazepam nor the antagonist Ro 15–1788 enhanced [³H]phenytoin binding. Therefore, it seems that these phenytoin binding sites in rat cerebral cortex are associated with a benzodiazepine site similar to the ‘peripheral’ type binding site for its selective affinity for Ro 5–4864. However, judging from the micromolar concentrations required for the enhancement of [³H]phenytoin binding, they appear unlikely to be the same ‘peripheral’ type binding sites as measured by [³H]Ro 5–4864 binding (K_d approx. 1 nM). The micromolar affinity benzodiazepine recognition sites are a possibility, if they indeed exist.     

Flunitrazepam Binding to Intact and Homogenized Astrocytes and Neurons in Primary Cultures

[3H]Flunitrazepam binds to intact and homogenized mouse astrocytes and neurons in primary cultures. In intact cells, the binding is to a single, high-affinity, saturable population of benzodiazepine binding sites with a KD of 7 nM and Bmax of 6,033 fmol/mg protein in astrocytic cells and a KD of 5 nM and Bmax of 924 fmol/mg protein in neurons. After homogenization, the Bmax values decrease drastically in both cell types, but most in astrocytes. The temperature and time dependency are different for the two cell types, with a faster association and dissociation in astrocytes than in neurons and a greater temperature sensitivity in the astrocytes. Moreover, flunitrazepam binding sites on neuronal and astrocytic cells have different pharmacological profiles. In intact astrocytic cells, Ro 5-4864 (Ki = 4 nM) is the most potent displacing compound, followed by diazepam (Ki = 6 nM) and clonazepam (Ki = 600 nM). In intact neurons, the relative order of potency of these three compounds is different: diazepam (Ki = 7 nM) is the most potent, followed by clonazepam (Ki = 26 nM) and Ro 5-4864, which has little effect. After homogenization the potency of diazepam decreases. We conclude that both neuronal and astrocytic cells possess high-affinity [3H]flunitrazepam binding sites. The pharmacological profile and kinetic characteristics differ between the two cell types and are further altered by homogenization.     

Characterization of [3H]Ro 5-4864 Binding Sites in Rat Vas Deferens

The presence of benzodiazepine binding sites in rat vas deferens was detected using [3H]Ro 5-4864 as a radioligand. The binding of [3H]Ro 5-4864 to the mitochondrial sites is saturable, reversible, and temperature and time dependent. The association rate constant (k1) was 8.7 +/- 0.7 x 10(7) M-1 min-1, and the dissociation rate constant (k-1) was 0.031 +/- 0.003 min-1. The dissociation constant (KD) determined by saturation binding was 5.22 +/- 0.56 nM. The density of binding was 4,926 +/- 565 fmol/mg of protein. The Hill coefficient of binding was 0.99 +/- 0.01, an indication that [3H]Ro 5-4864 binds to a single site. The [3H]Ro 5-4864 binding was inhibited competitively by Ro 5-4864 and 2-hydroxy-5-nitrobenzyl-6-thioguanosine and noncompetitively by PK 11195, nitrendipine, alpha,beta-methylene-ATP, and carboxyatractyloside and was not affected by clonazepam, dicyclohexylcarbodiimide, or protoporphyrin IX. Our data indicate that [3H]Ro 5-4864 binding sites are not identical to those labeled by PK 11195. These binding sites are modulated by the ADP/ATP mitochondrial carrier, and an interaction of dihydropyridines and [3H]Ro 5-4864 binding sites in rat vas deferens is suggested.     

Labelling of "Peripheral-Type" Benzodiazepine Binding Sites in the Rat Brain By Using [3H]PK 11195, an Isoquinoline Carboxamide Derivative: Kinetic Studies and Autoradiographic Localization

PK 11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide] is a new ligand for the "peripheral-type" benzodiazepine binding sites, chemically unrelated to benzodiazepines. It displaces with a very high potency (IC50 congruent to 10(-9) M) [3H]-RO5-4864 (a benzodiazepine which specifically labels the peripheral-type sites) from its binding sites. [3H]PK 11195 binds to a membrane fraction from rat brain cortex and rat olfactory bulb in a saturable and reversible manner with a very high affinity (KD = 10(-9) M). The number of maximal binding sites was ten times greater in the olfactory bulb than in the brain cortex. The order of potency of several compounds as displacers at 25 degrees C (PK 11195 greater than RO5-4864 greater than diazepam greater than dipyridamole greater than clonazepam) demonstrates that [3H]PK 11195 binds to the peripheral-type benzodiazepine binding sites. The KD value for the [3H]PK 11195 binding is not affected by temperature changes, whereas RO5-4864 and diazepam affinities decrease with increasing temperatures. Autoradiographic images of [3H]PK 11195 binding to rat brain sections show that binding sites are mainly localized in the olfactory bulb, median eminence, choroid plexus, and ependyma. This ligand could be a useful tool to elucidate the physiological and pharmacological relevance of these binding sites.     

Binding of [3H]Ro 5–4864 and [3H]PK 11195 to Cerebral Cortex and Peripheral Tissues of Various Species: Species Differences and Heterogeneity in Peripheral Benzodiazepine Binding Sites

The binding of [3H]PK 11195 and [3H]Ro 5-4864 to membrane preparations from cerebral cortex and peripheral tissues of various species was studied. [3H]PK 11195 (0.05-10 nM) bound with high affinity to rat and calf cerebral cortical and kidney membranes. [3H]Ro 5-4864 (0.05-30 nM) also successfully labeled rat cerebral cortical and kidney membranes, but in calf cerebral cortical and kidney membranes, its binding capacity was only 3 and 4%, respectively, of that of [3H]PK 11195. Displacement studies showed that unlabeled Ro 5-4864, diazepam, and flunitrazepam were much more potent in displacing [3H]PK 11195 from rat cerebral cortex and kidney membranes than from calf tissues. The potency of unlabeled Ro 5-4864 in displacing [3H]PK 11195 from the cerebral cortex of various other species was also tested, and the rank order of potency was rat = guinea pig greater than cat = dog greater than rabbit greater than calf. Analysis of these displacement curves revealed that Ro 5-4864 bound to two populations of binding sites from rat and calf kidney and from rat, guinea pig, rabbit, and calf cerebral cortex but to a single population of binding sites from cat and dog cerebral cortex. Using [3H]PK 11195 as a ligand, the rank order of binding capacity in cerebral cortex of various species was cat greater than calf greater than guinea pig greater than rabbit greater than dog greater than rat, whereas when [3H]Ro 5-4864 was used, the rank order of binding capacity was cat greater than guinea pig greater than rat greater than rabbit greater than calf greater than dog.     

Presence of a peripheral type benzodiazepine binding site on the macrophage; its possible role in immunomodulation

Saturable binding site for 3H-flunitrazepam (KD = 43 +/- 7 nM, Bmax = 391 +/- 58 fmoles/cell, i.e. 250,000 sites/cell) is characterized on Mouse peritoneal inflammatory macrophages. The affinity for different ligands (PK 11195 greater than Ro 5-4864 greater than diazepam greater than flunitrazepam greater than clonazepam greater than Ro 15-1788) shows that this site is of peripheral type. In vivo the humoral response in Mice to Sheep red blood cells was stimulated by administration of 1 mg/kg of PK 11195 (+85%), Ro 5-4864 (+80%) and diazepam (+58%). Clonazepam and Ro 15-1788 are devoid of activity. This suggests that molecules which show affinity for the "peripheral type" benzodiazepine binding site might modulate the immune response.     

Benzodiazepine receptors on human blood platelets

Binding studies conducted on membrane preparation from human platelets using (3H) Ro5-4864 and (3H) diazepam showed specific and saturable binding. Scatchard analysis revealed a single class of binding sites with KD = 10.8 +/- 0.9 nM and Bmax = 775 +/- 105 fmol/mg protein for (3H) Ro5-4864 and KD = 10.5 +/- 1.1 nM and Bmax = 133 +/- 19 fmol/mg for (3H) diazepam. We were unable to detect any GABA binding site on crude membrane preparation, nor did GABA enhance the binding of (3H) Ro5-4864 or (3H) diazepam. This suggests that benzodiazepine receptors are uncoupled to GABA system on human platelets. Ro15-1788, a specific antagonist for "central type" benzodiazepine (BDZ) binding sites was inactive in displacing (3H) Ro5-4864 from membrane receptors, while PK 11195 (a specific ligand for the "peripheral type" receptor) was the most potent of the drugs tested in inhibiting (3H) Ro5-4864 binding. These results indicate that human blood platelets bear "peripheral-type" BDZ receptor. Moreover, we could not detect any (3H) propyl beta carboline specific binding on platelet membranes. Results on benzodiazepine receptors on human circulating lymphocytes are also reported and similarity in pharmacological properties with platelet benzodiazepine receptors is suggested.     

Benzodiazepine antagonist Ro 15-1788: binding characteristics and interaction with drug-induced changes in dopamine turnover and cerebellar cGMP levels

The recently discovered benzodiazepine antagonist Ro 15-1788 was characterized in binding studies, and its potency and selectivity were determined in vivo by interaction with drug-induced changes in dopamine turnover and cerebellar cGMP level. Ro 15-1788 reduced [3H]flunitrazepam binding in the brain in vivo with a potency similar to that of diazepam and effectively inhibited [3H]diazepam binding in vitro (IC50 = 2.3 +/- 0.6 nmol/liter). [3H]Ro 15-1788 bound to tissue fractions of rat cerebral cortex with an apparent dissociation (KD) of 1.0 +/- 0.1 nmol/liter. The in vitro potency of various benzodiazepines in displacing [3H]Ro 15-1788 from its binding site was of the same rank order as found previously in [3H]diazepam binding. Autoradiograms of [3H]Ro 15-1788 binding in sections of rat cerebellum showed the same distribution of radioactivity as with [3H]flunitrazepam. The attenuating effect of diazepam on the chlorpromazine- or stress-induced elevation of homovanillic acid in rat brain was antagonized by Ro 15-1788. Among a series of compounds which either decreased or increased the rat cerebellar cGMP level, only the effect of benzodiazepine receptor ligands (diazepam, zopiclone, CL 218 872) was antagonized by Ro 15-1788. Thus, Ro 15-1788 is a selective benzodiazepine antagonist acting at the level of the benzodiazepine receptor in the central nervous system. Peripheral benzodiazepine binding sites in kidney and schistosomes were not affected by Ro 15-1788.     

γ-Aminobutyric Acid Receptor Complex of Insect CNS: Characterization of a Benzodiazepine Binding Site

The specific binding of [N-methyl-3H]flunitrazepam ([3H]FNZP) to a membrane fraction from the supraoesophageal ganglion of the locust (Schistocerca gregaria) has been measured. The ligand binds reversibly with a KD of 47 nM. The binding is Ca2+-dependent, a property not found for the equivalent binding site in vertebrate brain. The pharmacological characteristics of the locust binding site show similarities to both central and peripheral benzodiazepine receptors in mammals. Thus binding is enhanced by gamma-aminobutyric acid (GABA), a feature of mammalian central receptors, whereas the ligand Ro 5-4864 was more effective in displacing [3H]FNZP than was clonazepam, which is the pattern seen in mammalian peripheral receptors. The locust benzodiazepine binding site was photoaffinity-labelled by [3H]FNZP, and two major proteins of Mr 45K and 59K were specifically labelled. In parallel experiments with rat brain membranes a single major protein of Mr 49K was labelled, a finding in keeping with many reports in the literature. We suggest that the FNZP binding site described here is part of the GABA receptor complex of locust ganglia. The insect receptor appears to have the same general organization as its mammalian counterpart but differs significantly in its detailed properties.     

The bovine peripheral-type benzodiazepine receptor: a receptor with low affinity for benzodiazepines.

The density of bovine peripheral-type benzodiazepine receptors (PBR) in four tissues was highest in adrenal cortex. The adrenal cortex PBR cofractionated with a mitochondrial membrane marker enzyme and could be solubilized with intact ligand binding properties using digitonin. The membrane bound and soluble mitochondrial receptors were pharmacologically characterized and showed the rank order of potency to inhibit [3H]PK 11195 binding was PK 11195 greater than protoporphyrin IX greater than benzodiazepines (clonazepam, diazepam, or Ro5-4864). [3H]PK 11195 binding to bovine adrenal mitochondria was unaffected by diethylpyrocarbonate, a histidine residue modifying reagent that decreased binding to rat liver mitochondria by 70%. [3H]PK 14105 photolabeled the bovine PBR and the Mr was estimated under nondenaturing (200 kDa) and denaturing (17 kDa) conditions. These results demonstrate the bovine peripheral-type benzodiazepine receptor is pharmacologically and biochemically distinct from the rat receptor, but the receptor component photolabeled by an isoquinoline ligand has a similar molecular weight.     

Effect of some potent adenosine uptake inhibitors on benzodiazepine binding in the CNS

A series of nucleoside transport inhibitors has been tested for their ability to displace [³H]diazepam binding to CNS membranes. No correlation between their potency as [³H]adenosine uptake blockers and as inhibitors of [³H]diazepam binding was found, either in rat or guinea-pig brain tissue. Dipyridamole, a potent adenosine transport inhibitor interacted strongly (K_i = 54 nM) with peripheral-type benzodiazepine binding sites (“acceptor sites”) and was 4–5 fold weaker in displacing [³H]methylclonazepam and [³H]Ro15-1788, ligands selective for the specific central benzodiazepine “receptor”. Unlike the benzodiazepines, dipyridamole had no anticonvulsant action against metrazole-induced convulsions in mice. Ro5-4864, a benzodiazepine which selectively interacts with the peripheral-type benzodiazepine binding site, was approximately equipotent with diazepam in inhibiting [³H]adenosine uptake in brain tissue. These results do not support the idea of a very close link between high-affinity central binding sites for clinically-active benzodiazepines and the adenosine uptake site. The possibility of a connection between benzodiazepine “acceptor” sites and the membrane nucleoside transporter is discussed.     

Benzodiazepine Binding to Cultured Human Pituitary Cells

Benzodiazepine receptors were investigated in a cell line of human pituitary cells (18-54,SF) grown in serum-free medium. Preparations of 18-54,SF whole cells and cell membranes were shown to possess saturable [3H]diazepam binding sites. Membrane sites were found to have a KD of 20 nM for diazepam while whole cells possessed a twofold higher value. The KD values determined from Rosenthal, Hill, and kinetic analyses were consistent for each preparation. Whole-cell binding of [3H]diazepam was observed to be more stable than binding to membranes at higher temperatures (37 degrees C) and when longer incubation times (60 min) were employed at 4 degrees C. The rank order potency of various benzodiazepines to inhibit [3H]diazepam binding to whole cells and membranes was Ro 5-4864, flunitrazepam, diazepam, and clonazepam. Representatives of other drug classes did not inhibit this benzodiazepine binding. When 18-54,SF cells were grown for 24 h with 100 nM diazepam and then extensively washed membranes prepared, the KD for diazepam increased to 38 nM whereas the Bmax was unchanged when compared with untreated controls. Overall, these findings indicate that pituitary cells possess a peripheral-type benzodiazepine receptor and that the whole cell receptor differs quantitatively when compared with the membrane receptor.     

Subcellular Localization of "Peripheral-Type" Binding Sites for Benzodiazepines in Rat Brain

The binding of [3H]Ro 5-4864, a specific ligand for "peripheral-type" benzodiazepine binding sites and [3H]Ro 15-1788, a specific ligand for the central benzodiazepine receptors, was determined in subcellular fractions of rat brain. As previously reported, the highest levels of "peripheral-type" benzodiazepine binding sites and benzodiazepine receptors were found in the crude P1 and P2 fractions, respectively. Purification of these crude fractions revealed that high levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding were present in the mitochondrial and synaptosomal fractions. In contrast, the purified nuclei and myelin contained low levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding.     

Differentiation between two ligands for peripheral benzodiazepine binding sites, [3H]RO5-4864 and [3H]PK 11195, by thermodynamic studies

The [3H]PK 11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinolinecarboxamide, binding sites in rat cardiac membranes are saturable, with high affinity, specific GABA-independent and correspond to the peripheral type of benzodiazepine. The order of potency of displacing agents was: PK 11195 greater than RO5-4864 greater than dipyridamole greater than diazepam greater than clonazepam. The Bmax obtained with [3H]PK 11195 was equivalent of the Bmax obtained with [3H]RO5-4864 in the same experimental conditions. However thermodynamic analysis indicates that the [3H]PK 11195 binding was entropy driven whereas the [3H]RO5-4864 binding was enthalpy driven. Consequently PK 11195 might be an antagonist of these binding sites and RO5-4864 an agonist or a partial agonist. The simultaneous use of both drugs might help to elucidate the physiological relevance of peripheral benzodiazepine binding sites.     

Isolation and Characterization of a Rat Brain Triakontatetraneuropeptide, a Posttranslational Product of Diazepam Binding Inhibitor: Specific Action at the Ro 5-4864 Recognition Site

This report describes the purification and characterization from rat brain of triakontatetraneuropeptide (TTN, DBI 17-50), a major biologically active processing product of diazepam binding inhibitor (DBI). Brain TTN was purified by immunoaffinity chromatography with polyclonal octadecaneuropeptide, DBI 33-50) antibodies coupled to CNBr-Sepharose 4B followed by two reverse-phase HPLC steps. The amino acid sequence of the purified peptide is: Thr-Gln-Pro-Thr-Asp-Glu-Glu-Met-Leu-Phe-Ile-Tyr-Ser-His-Phe-Lys-Gln-Ala-Thr-Val - Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys. Synthetic TTN injected intracerebroventricularly into rats induces a proconflict activity (IC50 0.8 nmol/rat) that is prevented by the specific "peripheral" benzodiazepine (BZ) receptor antagonist isoquinoline carboxamide, PK 11195, but not by the "central" BZ receptor antagonist imidazobenzodiazepine, flumazenil. TTN displaces [3H]Ro 5-4864 from synaptic membranes of olfactory bulb with a Ki of approximately 5 microM. TTN also enhances picrotoxinin inhibition of gamma-aminobutyric acid (GABA)-stimulated [3H]flunitrazepam binding. These data suggest that TTN, a natural DBI processing product acting at "Ro 5-4864 preferring" BZ binding site subtypes, might function as a putative neuromodulator of specific GABAA receptor-mediated effects.     

Differential Binding Properties of the Peripheral-Type Benzodiazepine Ligands [3H]PK 11195 and [3H]Ro 5-4864 in Trout and Mouse Brain Membranes

High-affinity binding sites for [3H]PK 11195 have been detected in brain membranes of rainbow trout (Salmo gairdneri) and mouse forebrain, where the densities of receptors were 1,030 and 445 fmol/mg of protein, respectively. Ro 5-4864 (4'-chlorodiazepam) was 2,200-fold less potent as a competitor of [3H]PK 11195 binding in the piscine than the murine membranes. Investigation of the regional distribution of these sites in trout yielded a rank order of density of spinal cord greater than olfactory bulb = optic tectum = rhombencephalon greater than cerebellum greater than telencephalon. This site in trout shared some of the characteristics of the peripheral-type benzodiazepine receptor (PTBR) (also known as the mitochondrial benzodiazepine receptor) in rodents, i.e., high affinity for PK 11195 and the endogenous ligand protoporphyrin IX, but was unique in the low affinity of Ro 5-4864 (41 microM) and diazepam and the relatively high affinity of the calcium channel ligand diltiazem and two central benzodiazepine ligands, CGS 8216 and CGS 9896. The differential affinity for the two prototypic PTBR ligands in trout is similar to that previously observed in calf and human brain membranes. Structural differences for the trout sites are indicated by the relative inability of diethyl pyrocarbonate to modify histidine residues of the binding site in trout as compared with mouse membranes. Heterogeneity of binding of the two prototypic PTBR ligands in mouse brain membranes was indicated by additivity studies, equilibrium competition experiments, and saturation isotherms, which together support the hypothesis that Ro 5-4864 discriminates between two [3H]PK 11195 binding sites having high (nanomolar) and low (micromolar) affinity, respectively.     

High-affinity binding sites for PK 11195, but not for RO5-4864, in porcine aortic smooth muscle

Peripheral benzodiazepine (BZ) binding sites were characterized in porcine aortic smooth muscle membrane preparation. [3H]PK11195 bound with high affinity to the membranes (Kd = 8.6 + 0.9 nM), whereas [3H]Ro5-4864 bound slightly to the membranes. The Ki value of Ro5-4864 obtained from the inhibition of [3H]PK 11195 binding was 1200 + 200 nM, which was 480 times weaker than that obtained in rat kidney. Furthermore, the Ro5-4864 effect was temperature-insensitive. When [3H]PK 11195 binding was examined in porcine, human and rat platelets, Ro5-4864 inhibited the binding in porcine and human platelets one order of magnitude less potently than that in rat platelets. These results suggest that low affinity for Ro5-4864 in porcine aorta smooth muscle originates in porcine tissue, but not in smooth muscle.     

Solubilization of Peripheral-Type Benzodiazepine Binding Sites from Cat Cerebral Cortex

The present study demonstrates for the first time the solubilization of peripheral-type benzodiazepine binding sites (PBS) from cat cerebral cortex. Of all detergents tested [digitonin, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), Tween 20, deoxycholate, and Triton X-100] in the presence of NaCl, the best solubilization (15% of initial activity) was obtained using 0.5% of the zwitterionic detergent CHAPS plus 2 M NaCl. Specific binding of [3H]PK 11195 to membrane-bound and solubilized PBS was saturable, yielding equilibrium dissociation constants (KD) of 1.3 +/- 0.2 and 1.9 +/- 0.3 nM, respectively, and maximal numbers of binding sites of 1,435 +/- 150 and 980 +/- 126 fmol/mg protein, respectively. The KD value of PK 11195 binding to solubilized PBS obtained from experimental kinetic analysis was 0.95 +/- 0.09 nM. The relative potencies of various compounds (PK 11195, Ro 5-4864, diazepam, flunitrazepam, clonazepam, methyl-beta-carboline-3-carboxylate, and Ro 15-1788) in displacing [3H]PK 11195 specific binding from membrane-bound and solubilized PBS were similar. Most of the solubilized binding activity was destroyed by heating at 60 degrees C for 30 min or by treatment with 2 M guanidinium chloride, which indicates the presence of a protein-binding site in the solubilized preparation. Over 85% of the solubilized binding activity was retained after 1 week at 4 degrees C, which will enable future application of purification procedures without major concern for stability of the material.     

Photoaffinity Labeling of Benzodiazepine Receptor Proteins with the Partial Inverse Agonist [3H]Ro 15–4513: A Biochemical and Autoradiographic Study

Photolabeling of the benzodiazepine receptor, which to date has been done with benzodiazepine agonists such as flunitrazepam, can also be achieved with Ro 15-4513, a partial inverse agonist of the benzodiazepine receptor. [3H]Ro 15-4513 specifically and irreversibly labeled a protein with an apparent molecular weight of 51,000 (P51) in cerebellum and at least two proteins with apparent molecular weights of 51,000 (P51) and 55,000 (P55) in hippocampus. Photolabeling was inhibited by 10 microM diazepam but not by 10 microM Ro 5-4864. The BZ1 receptor-selective ligands CL 218872 and beta-carboline-3-carboxylate ethyl ester preferentially inhibited irreversible binding of [3H]Ro 15-4513 to protein P51. Not only these biochemical results but also the distribution and density of [3H]Ro 15-4513 binding sites in rat brain sections were similar to the findings with [3H]flunitrazepam. Thus, the binding sites for agonists and inverse agonists appear to be located on the same proteins. In contrast, whereas [3H]flunitrazepam is known to label only 25% of the benzodiazepine binding sites in brain membranes, all binding sites are photolabeled by [3H]Ro 15-4513. Thus, all benzodiazepine receptor sites are associated with photolabeled proteins with apparent molecular weights of 51,000 and/or 55,000. In cerebellum, an additional protein (MW 57,000) unrelated to the benzodiazepine receptor was labeled by [3H]Ro 15-4513 but not by [3H]flunitrazepam. In brain sections, this component contributed to higher labeling by [3H]Ro 15-4513 in the granular than the molecular layer.