Proc. NatL Acad. Sci. USAVol. 79, pp. 6084-6088, October 1982Neurobiology

Isolation, characterization, and purification to homogeneity ofa rat brain protein (GABA-modulin)

(benzodiazepines/transmitter receptors)

A. GUIDOTTI*, D. R. KONKEL*, B. EBSTEIN*, M. G. CORDA*, B. C. WISE*, H. KRUTZSCHt, J. L. MEEK*,AND E. COSTA*Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032; and tLaboratory ofImmunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20205Communicated by John J. Burns, June 18, 1982

ABSTRACT y-Aminobutyric acid (GABA)-modulin is a brainneuropeptide that appears to modulate specific high-affinity (20nM) GABA recognition sites in brain. When added to crude syn-aptic membranes this peptide inhibits binding of [3H]GABA to thehigh-affinity site and prevents facilitation of [H]diazepam bind-ing elicited by GABA. GABA-modulin has been purified to homo-geneity by ammonium sulfate precipitation, gel chromatography,and reverse-phase HPLC. Homogeneity was confirmed by a va-riety of means, including chromatography under four differentHPLC conditions, two different polyacrylamide gel electropho-reses, and end group analysis. Purified GABA-modulin containsapproximately 126 amino acids and has a molecular weight of16,500. The GABA-modulin molecule contains an abundance ofhydrophilic basic residues, and neither cysteine nor GABA is pres-ent. End group analyses of GABA-modulin showed that histidineis the free COOH terminus and the NH2 terminus is blocked.GABA-modulin specifically blocked both [3H]GABA binding tosynaptic membranes (IC50, 0.5 ,uM) and GABA-stimulated[H]diazepam binding; the binding of [3H]GABA to low-affinitysites was not affected.

The binding of y-aminobutyric acid (GABA) to specific recog-nition sites on postsynaptic neuronal membranes is regulatedby endogenous modulators (1-4). One of these (2, 5) is the pro-tein termed "GABA-modulin" (6), which noncompetitively re-duces the number of high-affinity GABA recognition sites (2,5). This regulation involves an allosteric mechanism that can bemodified by diazepam (5). GABA-modulin also prevents GABA-induced stimulation of [ H]diazepam binding to brain synapticmembranes (7).

Previously GABA-modulin has been extracted from eitherrat brain (2, 7) or NB2A cloned mouse neuroblastoma cells (6).However, purification and characterization of this peptide hasbeen difficult because of the presence of various materials in-terfering with GABA binding, including GABA (8) itself, andbecause of enzymatic degradation of GABA-modulin. This re-port describes the isolation, purification to homogeneity, andamino acid composition of GABA-modulin isolated from ratbrain.

MATERIALS AND METHODSAnimals. Male Sprague-Dawley rats (175-200 g) were ob-

tained from Zivic-Miller (Allison Park, PA).Materials. Sephadex G-100 and G-75 gels were from Phar-

macia. Bio-Gel P-2 and the Bio-Sil ODS-10 (octadecylsilyl) re-verse-phase HPLC column were from Bio-Rad. The Zorbax CNHPLC column was from Du Pont. The Synchropak AX 300 an-ion-exchange HPLC column was from Synchrom (Linden, IN).

Trypsin (treated with tosyllysine chloromethyl ketone) and chy-motrypsin were from Worthington. HPLC chromatography wascarried out with a Spectraphysics system (Santa Clara, CA).

Protein Determination and NH2- and COOH-TerminalAmino Acid Analysis. Proteins were detected colorimetricallyby the Lowry method (9). Automated NH2-terminal analysiswas attempted on a Beckman sequencer, using 0.1 M Quadrolprogram no. 031281 without Polybrene. Released phenylthio-hydantoins were identified by HPLC analysis (10). The COOH-terminal residue was identified by Akabori hydrazinolysis (11).

Polyacrylamide Slab Gel Electrophoresis. Acidic (pH 3.2)urea gels (2.8 mm thick) were prepared according to the pro-cedure described by Panyim and Challdey (12). NaDodSO4 gelswere run according to Laemmli and Favre (13) with minormodifications.GABA-Modulin Assay. This assay measures the ability of tis-

sue extracts to inhibit the binding of [3H]GABA to crude syn-aptic membranes treated with Triton X-100 (0.05%) or AgNO3(0.1 mM) (2, 14). The assay (final volume 1 ml) was carried outin 20 mM potassium phosphate buffer (pH 7.0). Materials wereadded to the reaction tube in the following order: suspensionofsynaptic membranes (0.3-0.4 mg ofprotein), GABA-modulinextract or control buffer (10-100 ,ul), [3H]GABA or [3H]muscimol(various concentrations in 50 1.l of buffer). Nonspecific binding(usually 10% of the total binding) was determined by adding 1mM GABA. After 10 min of incubation at 2-40C the reactionwas terminated by centrifugation (7). The same conditions wereused to study the effect ofGABA-modulin on GABA-stimulated[3H]diazepam binding [for further details see Massotti et aL (7)].The effect of GABA-modulin on [3H]etorphine (15), [3H]-adenosine (16), [3H]imipramine (17), and [3H]quinuclidinylbenzilate (18) binding was studied by published methods, withthe concentration of the ligand in the Kd range.GABA-Modulin lodination. GABA-modulin (250 ,ug of pro-

tein), diluted in 100 ,ul of phosphate-buffered saline, wastreated with approximately 2 mCi (1 Ci = 3.7 X 1010 becque-rels) of Na'"I and 20 ,ul ofchloramine-T (0.2 mg/ml) for 15 sec,followed by 20 j1 of sodium metabisulfite (2 mg/ml). The sam-ple was desalted with a Bio-Gel P-2 column equilibrated with0.1 M acetic acid, and purity of iodinated material was testedby both NaDodSO4/polyacrylamide gel electrophoresis andreverse-phase HPLC.

RESULTSIsolation of GABA-Modulin. The steps employed to extract

GABA-modulin from rat brain are reported in Fig. 1; typically,20 rat brains were used for each preparation. After decapitation,each brain was rapidly (15-30 sec) removed and homogenized

Abbreviation: GABA, y-aminobutyric acid.

The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-nent" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Rat brain

Homogenization in 10 vol of1 M acetic acid (800C)

Centrifugation at 48,000 x g

SPellet Supernatant

pH 7, 30-60% saturated ammoniumsulfate precipitation, centrifugation at48,000 x g

Pellet

Resuspension in 100 vol of 50 mM Triscitrate (pH 7), 60% saturatedammonium sulfate precipitation,centrifugation at 48,000 x g; thisprocedure repeated twice

Supernatant

Supei

Pellet

Resuspensioicentrifugatic

matant Pellet(crude GABA-modulin extract)

n in 0.1 M acetic acid,on at 48,000 x g

FIG. 1. Scheme for preparation of crude GABA-modulin extract.

in hot (80°C) 1 M acetic acid. After the ammonium sulfate pre-cipitation steps (see Fig. 1) an aliquot of the crude GABA-mod-ulin extract was chromatographed in succession on Sephadex

A

100

B

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Proc. Natl. Acad. Sci. USA 79 (1982) 6085

G-100 and Sephadex G-75 columns, both equilibrated with 0.1M acetic acid (Fig. 2), and finally on a Bio-Sil ODS-10 reverse-phase HPLC column equilibrated with 0.1 M NaC1O4/0.1%H3PO4, pH 2.5, eluted with 0-60% (vol/vol) acetonitrile gra-dient (Fig. 2).

Details on the amount of protein, sample volume, recoveryand purification are reported in Table 1. The recovery and de-gree of purification were estimated by adding 0.9 pug of 125I-la-beled GABA-modulin (specific activity 10,000 cpm/ng) to theinitial homogenate. Usually 20 rat brains yielded 0.5 mg of pu-rified GARA-modulin.

Different HPLC or acrylamide gel electrophoresis conditionsand COOH-terminal amino acid analyses were used as criteriato establish the purity of GABA-modulin. As shown in Fig. 3,with 50-500 jig of protein, a single UV absorbing peak wasobserved in four different HPLC conditions.

Furthermore, the purity ofGABA-modulin was assessed withthree different polyacrylamide gel electrophoretic systems: (i)12% NaDodSO4 gel, (ii) 17% NaDodSO4 gel, and (iii) 15% urea/acid pH gel. In all of these conditions, application of 10-200 ,gofprotein produced a single band staining with Coomassie blue(Fig. 4). COOH-terminal amino acid analysis using hydrazin-olysis was adopted as third criteria for purity ofGABA-modulin.This analysis showed that the COOH terminus was free and thathistidine was. the only detectable. amino acid. NH2-terminalamino acid analysis indicated that the NH2-terminus wasblocked. In fact, no sequence could be determined with thenative preparation.

Characteristics of GABA-Modulin. The amino acid compo-sition ofpurified GABA-modulin is shown inTable 2. It containsa relative abundance of hydrophilic basic residues and no cys-teine. No GABA was detected by this analysis or by anothermeans (sensitivity of the other method for GABA was 2.5 pmol)(19). When purified GABA-modulin was incubated for 30 minat 37°C with trypsin or chymotrypsin, the inhibitory activity on[3H]GABA binding was lost.- Injection of the digested materialon reverse-phase HPLC showed that the peak of protein as-

C70

a0:60

.r 50

b 40a

.r 30

~0m 20

= 10m

0

Fraction

F.

F

60

50-

-b40

30 °2Q

20 ¢

-10D0

10 20 30

Volume, ml

FIG. 2. Purification procedure for GABA-modulin. The crude GABA-modulin extract (Fig. 1), derived from 10 rat brains, was carefully resus-pended in 5 ml of 0.1 M acetic acid and, after centrifugation at 48,000 x g for 10 min, the clear supernatant was chromatographed successively on2.5 x 70 cm Sephadex G-100 (A) and Sephadex G-75 (B) columns equilibrated with 0.1 M acetic acid. The columns were developed with 0.1 M aceticacid with a flow rate of 0.2 ml/min, and 5-ml fractions were collected. The upper traces and the left ordinates indicate inhibition of [3H]GABAbinding; the lower trace and the right ordinates indicate protein measured in 200 ,ul with the Lowry method. Shaded areas represent the fractionswith [3H]GABA binding inhibitory activity. Elution markers: D, blue dextran; A, albumin; 0, ovalbumin; C, chymotrypsinogen; R, ribonuclease;ME, 2-mercaptoethanol. Inhibitory activity for [3HIGABA binding was determined by using 100 ,1u of each fraction after it was lyophilized andresuspended in 1 ml of 20 mM potassium phosphate, pH 7. (C) Reverse-phase HPLC of 250 ,ug of partially purified GABA-modulin from SephadexG-100 and G-75. The conditions were Bio-Sil ODS-10 column- (2 x 250 mm), flow rate 1 ml/min, temperature 2000. After sample application, thecolumn was washed for 30 min with the starting buffer (0.1 M NaClO4/0.1% H3PO4). The peptides were eluted with a gradient from 100% startingbuffer to 70% acetonitrile/30% starting buffer. Fractions (1 ml) were collected, neutralized, lyophilized, and analyzed for [3H]GABA binding in-hibitory activity. GABA-modulin activity (approximately 80%) emerges in a 1-ml fraction (hatched bar) corresponding to 53% acetonitrile.

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6086 Neurobiology: Guidotti et aL

Table 1. Purification of GABA-modulin. from 20 rat'brains

Vol, Protein, 125I-Labeled GABAkmodulin, Recovery, Purification,Step ml mg cpm x 10-6 % -fold

1. Homogenate 400 5,200 18 100 -

2. 48,000 x g supernatant 305 1,000 16.6 92 4.63. 48,000 x g supernatant after

30% (NH)2SO4 fractionation 1,000 600 14.8 82 7.14. 30-60% (NH4)2SO4 precipitate

resuspended in buffer 1,000 400 13.2 73 9.55. 60% (NH4)2S04 precipitate

resuspended in buffer 1,000 180 12 66 196. 60% (NH4)2S04 precipitate

resuspended in 0.1 M acetic acid 100 26 11 61 1207. Sephadex G-100 filtrate 40 5 7.2 38 4008. Sephadex G-75 filtrate 30 1.5 2.7 15 5109. BPLC eluate 6 0.5 1.4 8 810

Fold purification was calculated by comparing the specific activity of GABA-modulin at the different stages of purificationwith the specific activity of the initial homogenate.

sociated with GABA-modulin had disappeared and a numberof other peaks were formed. However, none of these peaksmaintained inhibitory activity for [3H]GABA binding. The mo-lecular weight of GABA-modulin was estimated to be approx-imately 16,000 on the basis of amino acid composition, and17,000 on the basis of NaDodSO4 gel electrophoresis (Fig. 4).

Purified GABA-modulin noncompetitively obliterated thebrain membrane sites that bind [3H]GABA or [3H]muscimolwith high affinity (Fig. 5), whereas it failed to change the low-affinity [3H]GABA binding. In addition (Fig. 5 A and B), whenthe 3H label was equally distributed between high- and low-affinity GABA binding sites, the maximal decrease of [3H]GABAbinding elicited by GABA-modulin was approximately 50% ofthe total binding. The concentration ofGABA-modulin that in-hibits 50% (IC50) of the high-affinity binding of [3H]GABA isapproximately 0.5 ,uM. Similar results were obtained whenGABA-modulin was added to solubilized [3H]GABA recogni-tion sites (data not shown). At this, and at 10 times higher con-

A

,/

/.

I

60LU

20~¢ 0

¢)

0

0 10 20 30 40 50 60

Volume, ml

centration, GABA-modulin does not bind [3H]GABA. In fact,when 100 jug ofGABA-modulin was incubated with [3H]GABAand then chromatographed on a small Sephadex G-25 column[see Massotti et al. (7) for details] no [3H]GABA eluted in thevoid volume. We have compared the ability of GABA-modulinto inhibit [3H]GABA binding with that ofother peptides or pro-teins. Albumin (up to 0.5 mg/ml), lysozyme (up to 100 ug/ml),histone (Sigma type II A, up to 100 ug/ml), and small myelinbasic protein from rat (kindly provided by M. Kies, NationalInstitute of Mental Health) (see Fig. 5A) failed to reduce thebinding of [3H]GABA to crude synaptic cortical membranes. Inother experiments, purified GABA-modulin in concentrationsup to 2.5 or 5 ,uM failed to change the binding of [3H]diazepam,[3H]etorphine, [3H]quinuclidinyl benzilate, [3H]imipramine,and [3H]adenosine to crude synaptic membranes. However,GABA-modulin is apotentinhibitor ofGABA-stimulated [3H]di-azepam binding. The IC50 for this effect is approximately 0.5,uM (see Fig. 6A). In the presence of GABA-modulin, the Ka

B FIG. 3. HPLC analyses of purified GABA-modulin. GABA-modulin obtained from re-verse-phase HPLC of Fig. 2 was tested for pu-rity under four different'HPLC conditions: (A)Bio-Sil ODS-10 column equilibrated with 0.1MNaH2PO4/0.2%.H3PO4,pH 2.5. The peptidewas eluted with a 30-minmlinear gradient ofacetonitrile from 0% to 60% (----). GABA-modulin was eluted with 44% acetonitrile.One hundred micrograms of protein was ap-plied to the column. (B) Bio-Sil ODS-10 col-umn developed with 70% (0.1 M NaH2PO4/

\0.2%H3PO4) + 30% acetonitrile under iso-cratic conditions; flow 1 ml/min. GABA-mod-ulin was eluted in 18 min and 45 sec. Fifty

0 10 20 30 micrograms of protein was applied to the col-D umn. (C) Zorbax CN equilibrated with 0.1 MD NaH2PO4/0:2% H3PO4, pH 2.5. Elution was

with a 60-min linear gradient of acetonitrilefrom 0% to 60%. GABA-modulin was elutedwith 40% acetonitrile. One hundred micro-grams of protein-was applied to the column.(D) Synchropak AX-300 oxiranes anion ex-change column equilibrated with 0.02 MNa2HPO4, pH 8.3; flow 1 ml/min. GABA-modulin eluteswith the voidvolume. No other

- proteins were eluted when the column (at thearrow) was eluted with a linear gradient from

;S 0.02 M Na2HPO4 to 0.2 M Na2HP04/1M-ct NaCI4, pH 8.3.------, The gradient. S. Ab

0 20 40 60 0 20 40 60 sorption profile after application of 500 jig ofGABA-modulin; C, absorption profile in con-

Volume, ml trol conditions.

0

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Proc. NatL Acad. Sci. USA 79 (1982) 6087

10 20 50 100

94 _67 _43 _

30~

20.1 _14.4

FIG. 4. Polyacrylamide gel electrophoresis. GABA-modulin ob-tained from reverse-phase HPLC of Fig. 2 was desalted on a 0.5 x 30cm Bio-Gel P-2 column equilibrated with 0.1M acetic acid. The amountof protein in each lane is given in Mug. (A) NaDodSO4/12% polyacryl-amide gel. The molecular weight markers (Mr, labeled x 10-3) usedwere phosphorylase b (94,000), bovine serum albumin (67,000), oval-bumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibi-tor (20,100), and a-lactalbumin (14,400). Proteins move toward theanode. (B) Urea/15% polyacrylamide gel (pH 3.2). Proteins move to-ward the cathode. The concentration of urea was 6.25 M. Coomassiebrilliant blue G was used to stain the protein.

(activation constant) for GABA stimulation of [3H]diazepambinding increases from 0.1 uM to 1.2 AM (Fig. 6B). The mod-ification by GABA-modulin of the GABA-elicited facilitation of[3H]diazepam binding appears to be competitive.

DISCUSSIONAlthough several reports (1-8) have described the presence ofendogenous inhibitors of the specific binding of [3H]GABA toits recognition sites in crude synaptic membranes or in mem-branes from cloned cell cultures (NB2A), identification of themolecular nature of these inhibitors has been at best partial.GABA-modulin is one such inhibitor located in purified syn-aptic membranes (6, 7). The present report describes a methodfor the purification to homogeneity of GABA-modulin from ratbrain. This endogenous inhibitor of[3H]GABA binding is a pep-tide with a molecular weight of approximately 16,500. Duringits preparation, GABA-modulin tended to be sticky, adsorbingto other molecules and eluting from Sephadex columns with

Table 2. Amino acid composition of GABA modulin purifiedfrom rat brain

Relative no.Residue of residuesAsxThrSerGlxProGlyAlaCysValMetfleLeuTyrPheHisLysArg(Trp)

9813101114704236267815

125

The analysis was repeated two times. A Durrum D500 automaticamino acid analyzer was used.* Tryptophan was destroyed during analysis.

them. This coelution may explain the different positions ofGABA-modulin in comparison to that of the correspondingmolecular weight markers in Sephadex G-100 and G-75 col-umns. Completely homogenous preparations ofGABA-modulinwere obtained when the Sephadex eluate was applied to re-verse-phase HPLC. By using the HPLC technique, the ma-terial was purified to homogeneity in a single (30-min) chro-matographic run. HPLC with different columns and bufferconditions, polyacrylamide gel electrophoresis at different pHvalues, analysis ofamino acid composition, and COOH-terminalamino acid analysis concurred to support the view that GABA-modulin was purified to homogeneity. The major peak of pro-tein eluting from the reverse-phase HPLC with 50% acetoni-trile inhibited both binding of [3H]GABA to the high-affinitysite and GABA-induced stimulation of [3H]diazepam bindingwith an IC50 of about 0.5 uM. This concentration was withinthe range of GABA-modulin concentration present in brain. In

ARSBMP

-O-O-- °-- 100

80

Ca 60

GABA-modulin 40

20

0

1 2 3 4 5

GABA-modulin, pM

B

-GABA-

modulin-4-

1 2 3 4 5

Specifically bound [3H]GABA,pmol/mg protein

+GABA-modulin

0 0.5 1.0

Specifically bound[3Hlmuscimol, pmol

FIG. 5. Effect of GABA-modulin on [3H]GABA binding to crude synaptic membrane preparation from rat cerebral cortex. (A) Different con-centrations of GABA-modulin or small basic myelin proteins from rat (RSBMP) (obtained from M. Kies, National Institute of Mental Health) wereincubated (10 min) at 00C with 300 ,gg of Triton X-100-treated membrane proteins; the binding was measured with 20 nM [3H]GABA. Each valueis the mean ± SEM of three experiments run in triplicate. The 100% value is 0.6 pmol of specifically bound [3H]GABA permg of membrane protein.(B) Scatchard plot analysis of the binding of [3H1GABA (10-240 nM) to Triton X-100-treated membranes in presence of 2.5 ,M GABA-modulin(o) or buffer alone (e). B/F, bound (fmol/mg of protein)/free (pmol) [3H]GABA. (C) Scatchard plot analysis of the binding of [3H]muscimol (1-20nM) to AgNO3-treated membranes in presence of 1 ,uM GABA-modulin (e) or buffer (o). Specifically bound [3H]muscimol is expressed in pmol perassay.

AMr 200 100 50 20

B

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._

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100

80.

60

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6088 Neurobiology: Guidotti et al.

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fact, it can be calculated from the recovery studies with '2I-la-beled GABA-modulin (Table 1) that the concentration ofGABA-modulin in rat brain is approximately 6 AM. The action ofGABA-modulin is specific for GABA binding, because at dosesup to 5 ,uM' it failed to influence the binding of several other3H-labeled ligands we tested. In addition, the specificity ofGABA-modulin action is confirmed by the lack ofeffect by otherproteins of similar molecular weight and charge on [3H]GABAbinding.Amino acid composition, anion-exchange chromatography,

and acrylamide gel electrophoresis with urea at acidic pH re-vealed that the protein is basic. We have therefore comparedGABA.modulin structure and activity with those of the mostwell-known basic proteins extracted from brain.' Histones,which have relatively smaller amounts of arginine and lysine,are eluted from a Bio-Sil ODS column with a higher percentof. acetonitrile (higher than 60%), and have no effect on[3H]GABA binding. Two myelin basic proteins are extractedfrom rat brain:- a high molecular weight protein (approximately170 amino acid residues) and a low molecular weight protein(approximately 126 amino acid residues) (20, 21). The high mo-lecular weight form differs from GABA-modulin'in the ratio ofarginine to lysine, has a lower mobility on acidic urea andNaDodSO4/polyacrylamide gels, and has a retention timelonger than that of GABA-modulin when eluted from a Bio-Sil.ODS column with 70% 0.1 M NaH2PO4/30% acetonitrile. Thesmall rat myelin basic protein has some striking similarities toGABA-modulin when considered from the point ofview of theamino acid composition (compare data.of Table 1 with data ofref. 20 and 21). However, the presence of a histidine at theCOOH terminus instead of arginine indicates that GABA-mod-ulin is also different from the small myelin basic protein ex-tracted from rat brain. In addition, the small rat myelin basicprotein (5 ,M) failed to inhibit the binding of [3H]GABA or[3H]muscimol to crude synaptic membranes' (Fig. 5A).

Although the activity of 'GABA-modulin was destroyed bytrypsin or chymotrypsin digestion, we could not exclude thepossibility that the GABA-modulin we had purified was theprecursor of the endogenous modulator; in fact, we have not yetthoroughly studied the activity ofthe GABA-modixlin fragmentsproduced by more limited cleavage.An important question that remains open is whether GAB.A-

modulin facilitates or inhibits the biological activity of GABAreleased by nerve stimulation. Preliminary experiments showthat when GABA-modulin is injected into the cerebral ventri-.cles it exacerbates the convulsionsinduced by isoniazid, sug-gesting that the increase in' free GABA-modulin decreases the

FI.P 6. Effect of GABA-modulin onthe GABA-induced stimulation. of [3HI-diazepambinding. GABAwas addedto the

+GABA-modulin incubation mixture together with 1 nM[3H]diazepam and GABA-modulin. .Thereactionwas stopped after30min. (A) Var-ious amounts of GABA-modulin .were in-cubated with the synaptic membranes inthe presence or absence of 0.1 M GABA.Each value is the mean of three experi-

-GABA-modulin ments run in triplicate. (B) Lineweaver-Burk plot of the net increase in '[3Hdi-

_ ' '0' azepam binding after treatment with0 1 5 10 GABA (0.01-1 MM) in presence or.absence

1/GABA, ,uM of 2.5 $M GABA-modulin.

action of GABA released, by.nerve impulses.Finally, the.question of the mechanism by which GABA-

modulin inhibits the binding of [3H]GABA remains to be ex-plored in detail. Because GABA-modulin inhibits the high-af-finity GABA binding in an apparently noncompetitive fashion,it is proposed that the mechanism is primarily allosteric. In thisregard, the observation that GABA-modulin is.a good substratefor protein kinase may be of interest. It-is therefore possible tostudy whether endogenous CaW, cGMP-, or cAMP-dependentphosphorylation of GABA-modulin is operative in regulatingthe affinity of GABA recognition sites for their endogenouseffector.1. Johnston, G. A. R. & Kennedy, S. M. E. (1978) in Amino Acids

as Chemical Transmitters, ed. Fonnum, F. (Plenum, New York),pp. 507-516.

2. Toffano, G. A., Guidotti, A. & Costa, E. (1978) Proc. Nati Acad.Sci. USA 75,.4024-4028.

3. Greenlee, D. V., Van Ness, P. C. & Olsen, R. W. (1978) Life Sci.22, 1653-1662.

4. Yoneda, Y. & Kuriama, K. (1980) Nature (London).285, 670-672.5. Guidotti, A., Toffano, G. & Costa, E. (1978) Nature, (London)

275, 553-555.6. Baraldi, M., Guidotti, A., Schwartz, J. P. & Costa, E. (1979) Sci-

ence 205, 821-823.7. Massotti, M., Guidotti, A. & Costa, E. (1981) J. Neurosci. 1,

409-418.8. Napias, C., Bergman, P.' C., Van Ness, 'D. V., Greenlee, D. W.

& Olsen, R. W. (1980) Life Sci. 27, 1001-1011.9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.

(1951)J. Bio. Chem. 193, 265-275.10. Gates, F. T., III, Coligan, J. E. & Kindt, T. J. (1979) Biochem-

istry 18, 2267-2272.11. Frankel-Conrat, H. & Tsung, C. M. (1967) Methods Enzymol. 11,

151-155.12. Panyim, S. & Chalkley, R. (1969) Arch. Biochem. Biophys. 130,

337-346.13. Laemmli, U. K. &- Favre, M. (1973) J. Mot Biol 80, 575-599.14. Supavilai, P., Mannonen, A. & Karobath, M. (1982) Neurochem.

Int., in press.15. Simon, E. J., Hiller, J. M. & Edelman, I. (1973) Proc. Natl. Acad.

Sci. USA 70, 1947-1949.16. Snyder, S. H., Katims, J. J., Annau, Z., Bruns, R. F. & Daly, J.

W. (1981) Proc. Nati Acad. Sci. USA 78, 3260-3264.17. Raisman, R., Briley, M. & Langer, S. Z. (1979) Nature (London)

281, 148-150.18. Yamamura, H. I. & Snyder, S. H. (1974) Proc. Natl Acad. Sci.

USA 71, 1725-1729.19. Bertilsson, L. & Costa, E. (1976)J. Chromatogr. 118, 395-402.20. Martenson, R. E., Deibler, G. E. & Kies, M. W. (1970) Biochim.

Biophys. Acta 200, 353-362.21. Dunkley, P. R. & Carnegie, P. R. (1974) Biochemn. J. 141,

243-255.

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