Life Sciences, Vol. 25, pp . 463-470 Printed in the U.S .A .

Pergamon Press

SOLUBILIZATION OF BENZODIAZEPINE BINDING SITE FROM RAT CORTE% M.Ayub Khan Yousufi, John W. Thomas and John F. Tallman Section on Biochemistry and Pharmacology Biological Psychiatry Branch National Institute of Mental Health Bethesda, Maryland 20205 (Received in final form June 19, 1979) Summary The high-affinity binding site for [ 3H]diazepam has been solubilized from rat brain using 0.5X Lubrol-P%. Using a polyethylene glycol (PEG)-y-globulin assay, it has been possible to demonstrate solubilization of about 60X of the binding sites in a single step . The solubilized binding site possesses a RD of 11 nM for [3H]diazepam compared to approximately 4 nM for the membrane-bound form, and binding is to a single class of sites . The order of potency of benzodiazepinea is identical f.or the solubilized receptor and the membrane-bound form . Binding of [ 3H]diazepam is temperature dependent and higher at 4° than 37 °C . Both urea and guanidine-HC1 were capable of totally inhibiting binding, and this inhibition was partly reversible ; neither sulfhydryl groups nor carbohydrate moieties seem to be important for binding. y-Amiaobutyric acid which enhanced [ H]diazepam binding to membrane fractions was without effect on the solubilized binding site . A high-affinity binding site for benzodiazepinea has been identified in brain by several laboratories (1-5) . Thin site appears to be a pharmacologically relevant site of action of the benzodiazepinea because the rank order potency of many drugs of this class in displacing [3H]diazepam (or [3H]fluaitrazepam) binding are identical to their relative action in behavioral teats (2) and their clinical potency (3) . Although no known transmitter ie said to directly compete with [3H]diazepam for the active site, GABA can enhance the binding of [ 3H]diazepam by interacting with a GABA receptor (5-7) which either directly or through an endogenous modulator (8) can alter the affinity of the benzodiazepine binding site for [ 3H]diazepam . In turn, these proteins seem to control an ionophore which affects the firing of the cell (9,10) . To clarify these interactions, solubilization and purification of the [ 3H]diazepam binding component of this complex has been undertaken . Other laboratories have been purifying both the modulator (S) and the GABA receptor (11) ; in the future it may be possible to reconstitute an actively functioning system in vitro . This communication describes the solubilization and properties of the [ 3H]diazepam binding site .

0024-3205/79/050463-07$02 .00/0

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Solubilization of

[3H]diazepam Binding Site

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Materials and Methods Preparation of Soluble Receptor . Male Sprague-Dawley rata (ARS, Madison, Wisc .) (approximately 200-250 g) were decapitated, their brains The cortex was homogenized removed, and the cortex immediately dissected . in 100 volumes (w/v) of 50 mM Tris-HC1 buffer, pH 7 .0 at 4°C using a The homogenate was cenBrinl®ann polytron setting of 5 for 15 seconds . trifuged at 20,000 g for 10 min, the pellet resuspended in another 100 The pellet was resuapended in 10 volumes of buffer, and recentrifuged. volumes based on the original tissue weight of 0.5X Lubrol-PX (Sigma Ch~i.cal Co ., St . Louis, MO) in the same buffer and homogenized for 1 min, starting the homogenizer at low speed and increasing it gradually to the 5 setting for the remaining 15 seconds . This mixture was kept on ice at 4°C for 1/2 hour with occasional shaking and then centrifuged at 105,000 x g for 1 hour at 4°C . The clear supernatant was removed for the binding assay of the soluble receptor . Specific [ 3H]diazepam binding from Lubrol-solubilized material was stable at least for 10 days at 4°C. Incubation Conditions . Typical incubation, unless otherwise specified, contained 100 ul of Lubrol-P% extract (approximately 0.3 mg protein), 2 nM [3H]diazepam (39.9 Ci mmol-1, New England Nuclear) in a total Volume of 500 ul buffered with the Tris-Lubrol. Incubations were carried out for After incubation the samples were assayed 30 min at 4°C in triplicate . according to the method of Cuatracasas (12) by mixing first with 500 yl of O .1X gamma globulin (w/v) and then with 500 U1 of 24% (w/v) polyethylene glycol 6000 (PEG) both in 50 mM Tris-HC1 buffer, pH 7 .0 at 4 °C (no detergent) . The mixture was immediately filtered under low vacuum through GF/B filters (Whatman Co .) ; the filters were rinsed twice with 7 .0 ml of 8X PEG in 50 mM Tris-HC1 buffer at 4°C, pH 7 .0, dried, and 10 ml of scintillation fluid was added . Specific [3H]diazepam binding is defined as total [ 3H]diazepam binding minus binding obtained in the presence of 10-5 M chlordiazepoxide . In a typical assay, specific binding was 2700 ±100 cpm and nonspecific binding was 500 cpm . Binding to m®brave fractions was carried out as previously described (5) . Results The optimal concentration of Lubrol-PR was O .SX for solubilization at a tissue dilution of 1 :10 (w/v) . It should be noted that the tissue proteia and detergent are present is approximately 1 :1 proportions, and this seems to be the critical variable ae less aolubilization is obtained at a gresÈer tissue dilution and the same detergent concentration or at higher detergent concentrations . Under these conditions between 50-65X of the total membrane-bound benzodiazepine binding sites are solubilized, meaning that centrifugation at 100,000 x g for up to 4 hours will not result in a pelleted binding protein. Molecular weight determinations (below) also support the aolubilization of the receptor . Scatchard analysis of binding data obtained at 4°C and 37 °C wen carried out (Fig . 1) . Ia contrast to the membrane-bound binding site which has as apparent RD of 4 n?i (5), the soluble binding site possesses a RD of 11 aM for [3H]diazepam . Additionally, in agree eat with the data obtained with the membrane-bound site (2,6), binding of benzodiazepines ie teaperature dependent, less being bound at higher temperatures than at 4°C. Thus, at 37 °C the S.D is 62 nM and typically about 20% of the binding levels at 4°C are obtained . Unpublished data indicates that the temperature dependence of binding with the soluble site is best fitted with a continuous curve, rather than a series of discontinuous straight lines, suggesting the absence of a single-phase transition .

Vol . 25, No . 5,

1979

Solubilization of

(3H)diazepam Binding Site

465

FIG. 1 Scatchard analysis of [3 H]diazepam binding in the soluble receptor at 4°C (~-~) and 37 °C (~-~) . The assay was carried out according to the described procedure . Bound specifically bound [ 3H)diazepam (fmolea) ; free ~ (3H]diazepam concentration of unbound [ H]diazepam in the incubation medium (nM) . The apparent dissociation constant KD ~ 8 nZf at 4°C and 22 nM at 37°C .

In order to characterize the pharmacological profile of the soluble receptor, a aeries of benzodiazepiaea were studied for their rank-order Fig. 2 shows a potency is inhibiting binding to the soluble receptor .

C7 Z Z G

ô0 Z F a z kj W s D: 0

-bg (BENZODtAZEPiNE) M

FIG. 2 Inhibition of [3H]diazepam specific binding bY various benzodiazeip nea . The aoluble receptor was incubated with 1 nM [3H]diazepam and increasing concentrations of various beazodiazepines . The points are the means of triplicate determinations and the level of specific binding was similar to Fig . 1 .

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Solubillzation of

[3H]diazepam Binding Site

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benzoseries of six drugs of varying relative potencies in inhibiting published are similar to relative potencies diszepine binding; these Of particular interest are clonazevalues and our own unpublished work . have been used to distinguish the "brain-type" and and R05-4864 which pam The soluble receptor from "kidney-type" benzodiazepine binding site (2) . brain retains its pharmacological properties indicating a difference between these sites that probably is intrinsic to their protein structure Additionally, we rather than their localization in the cell membrane . note that using the identical techniques and several detergent concentrations we were unable to solubilize the "kidney receptor," indicating a much tighter intercalation of the kidney site in its membrane . The heat stability of the soluble receptor was examined and compared to the membrane-bound receptor (Fig . 3} . In the membrane brief heating leads to a small initial enhancement of [ 3H]diazepam binding followed by a slow inactivation over time . About 95X of the initial activity remains after 40 min. In contrast, the soluble receptor shows a biphasic inactivation with about 40X of the sites being rapidly inactivated followed by a much slower decline in the rm~aining activity (60X remaining after 40 min) . The molecular weight of the soluble binding site was determined by gel filtration on 5ephadex G-200. The calculated molecular weight of the solubilized receptor was 210,000-230,000 daltons (Fig . 4) . However, it is not known how many molecules of Lubrol-PX or other proteins remain associated with the receptor ; thus, this may represent a somewhat overestimated molecular weight .

120

n x ô ~ô iF d

100

~U G~ ~T

W

V N

6

10

20

~0 TIME fminu0wl

FIG . 3 Heat stability of soluble receptor . Membrane-bound receptor in Tris-HCl and soluble receptor in 0.5X Lubrol-Tris HCl buffer pH 7 .0 were heated at 50 °C for various time intervals and cooled in ark ice bath . Binding to the receptor was performed with 1.5 nM [ H]diazepam at 4°C for 1/2 hour . Precipitation of soluble receptor ligand complex was performed as described in Methods and the membrane-hound receptor was measured ae described (3) . The standard error in the values was less than 10X; the experiment was replicated twice .

Solubilizstion of [SH]diazepam Hinding Site

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467

FIG . 4 Determination of molecular weight of the receptor . Standard proteins of known molecular weight were chromatographed oa Sephadez G-200 (Pharmacia) packed in a column of 80 x 1 .5 cm . The column was eluted with 0 .5Z Lubrol-P% in 50 mM Trie-HC1 pH 7 .0 at 4 °C . The ratio of elution volume to void volume of the various proteins was plotted against the log of the molecular weight (e) . The molecular weight of the receptor was located on the straight line calculated from the regression analysis (o) .

Ae a prelude to purification, several other factors which might influence the stability of the receptor were examined (Table 1) . The receptor at this stage of purification is stable to dialysis for at least 2 days at 4 ° C (cold room) and the preparation itself for greater than 10 days if TABLE 1 Effect of Various Agents on [ 3H]Diazepam Biadin~ to Solubilised Receptor Agent

Z Control Before Dialysis

Specific Hiadiag After Dialysis

Urea 0 .5 M 2 .0 M

54 22

76 32

Guanidine HC1 0 .1 M 1 .0 M

49 2

71 44

GABA (10 5 M) Soluble Receptor Membrane Bound Receptor tNot determined *p