Life Sciences, Vol. 33, Sup. I, 1983, pp. 203-206 Pergamon Press Printed in the U.S.A.

CHROmaTOGRAPHY AND PARTIAL PURIFICATION OF SOLUBILIZED OPIATE RECEPTORS

Andreas Pfeiffer

National Institute of Mental Health, Laboratory of Clinical Science Bethesda, Maryland 20205

(Received in final form June 26, 1983)

SUMMARY

Sheep brain membrane opiate receptors were solubilized using two diffe- rent approaches: (A) Chaps was used to solubilize [3H]buprenorphine la- beled membranes. Macromolecular labeled material had a stokes radius of approximately 90 A. Treatment with phospholipase A2, but not phospholi- pase C, decreased the stokes radius to 50 A indicating the presence and eventual structural role of phospholipids in the solubilized opiate re- ceptor complex.

(B) Digitonin-NaCl was used for solubilization of receptors which were then labeled with [3H]diprenorphine. This complex could be chromatogra- phed on hydroxylapatite, DEAE-sepharose and phenylboronate gels success- ively permitting an approximately 100-fold purification over the solubi- lized starting material.

Current evidence for the existence of multiple types of opiate receptors relies on pharmacological data. The biochemical characterization of the receptor molecules would enable a clearer answer as to the number of distinguishable re- ceptors. Techniques to solubilize active opiate receptors with 3-(3-cholamido- propyl) dimethyl-ammonio-l-propane-sulphonate (Chaps)(1) or with digitonin (2 ,3 ) have been developed. The use of Triton X-100 was also reported (4).Solu- bilized receptors display large stokes radii (I-3), and are likely to represent aggregates of several proteins. The ligand binding component of delta opiate receptors was reported to display a molecular weight of 58000 dalton on SDS polyacrylamide gels after covalent labeling (5). The present work was undertaken to characterize the solubilized receptor com- plex and to delineate techniques suitable for the purification of active opiate receptors.

METHODS and MATERIALS

Fresh sheep brain cortex was obtained locally and homogenized with a glass-tef- lon homogenizer in 0.32 M sucrose followed by sedimentation at 1000 x g for 10 min. The supernatant was centrifuged at 40,000 x g for 40 min and pellets (P2- membranes) were either used immediately or frozen and stored at -70 C until thawed for use.

(A) Chaps: P2-membranes were incubated with InM [3H]buprenorphine (kindly provi- ded by Dr.Herz, Munich) for 60 min at 28C in Tris buffer (50mM,pH 7.5). Unbound ligand was removed by repeated pelleting of the membranes which were then solu- bilized in 10 mM Chaps (i). After centrifugation (105,000 x g) macromolecular bound radioactivity was assayed as described (I) and is referred to as opiate receptor (70-80% specific binding obtained by substracting binding in the pre-

0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.

204 Purification of Opiate Receptors Vol. 33, Sup. I, 1983

sence of 1 uM naloxone). In some experiments the solubilized material was concencentrated by ultrafiltration with Amicon XM-300 membranes. For treatment with phospholipase A 2 or C 5-10 mg of solubilized material was incubated with i ug boiled (I0 min) phospholipase A 2 (Crotalus adamanteus venom) or phosphalipase C (bacillus cereus) in the presence of 1 mM Ca 2+ for 15 min at 37 C. Treatment was terminated by the addition of 5 mM EGTA and i mg/ml albumin. (B) Digitonin: P2-membranes were solubilized using I% digitonin, IM NaCI Tris buffer (3). Unsoluble material was sedimented for 1 h at 105,000 x g. Binding experiments were performed in the supernatant after 10-20fold dilution in Tris buffer at 25 C using [3H]dlprenorphine. Nonspecific binding was measured by the inclusion of I uM unlabeled diprenorphine and accounted for 20-50% of total binding depending on the concentration of receptors and radio- label employed. Materials: [3H]buprenorphine (30 Ci/mmole) was from Reckitt and Coiman, [3H]- diprenorphine (9.3 Ci/mmole) was from Amersham. Hydroxylapatite was from Bio Rad and LKB, DEAE- and Ci-6B-sepharose from Pharmacia and phenylboronate PBA-10 from Amicon. Digitonin was purchased from Sigma or Fisher, (~aps from Calbio- chem. Phospholipases were from Sigma. Naloxone was a gift from Endo. Bremazo- cine was donated by Dr. R.Maurer, Basel.

RESULTS

Opiate receptors were solubilized with Chaps after prelabeling of the mem- brane preparation with [3H]buprenorphine. This ligand was chosen because of its slow dissociation.The t 1/2 was 24h at 25 C and exceeded 1 week at 4C in the solubilized receptor preparation as assessed by addition of i uM unla- beled naloxone and monitoring of the macromolecular bound radioactivity. Binding of buprenorphine to unlabeled solubilized brain membranes was poor suggesting that little reassociation occurred in the prelabeled preparation. The solubilized material migrated close to the void volume of a 1.5 x 75 cm CI-6B column (equilibrated with Tris 50 mM, 0.32M sucrose) in the major pro- tein peak. To test for the possible occurrence of phospholipids in the solu- bilized receptor complex the preparation was treated with phospholipase A 2 or phospholipase C prior to chromatography. The amount of phospholipase employed inhibited 80% of [3H]buprenorphine binding to membranes. Treatment with phos- pholipase A 2 released 30-40% of the bound radioactivity and shifted the peak

o

! I J I

10 FRACTION 80 40

i ,

FIG. I

Effect of phospholipase A 2 or phospholipase C treat- ment on [3H]buprenorphine labeled opiate receptors. The phospholipase C treated preparation coeluted with untreated control prepara- tions.

Vol. 33, Sup. I, 1983 Purification of Opiate Receptors 205

of macromolecular bound radioactivity markedly to the right while treatment with phospholipase C did not alter the position in which macromolecular bound radioactivity eluted (Fig. i). Phospholipase A 2 treatment did not affect the position of the major protein peak thereby allowing for a 2-5fold puri- fication . The phospholipase treatment, moreover, did not affect the slow dissociation of [3H]buprenorphine. The characteristics of opiate receptors were further studied after solubili- zation ~ith digitonin-NaCl. In agreement with earlier reports (2,3) the affini- ty of [~H]diprenorphine and (-)Bremazocine was i nM K d and that of naloxone 20 nM K d. (+)Bremazocine displayed an over 1000fold greater K d than its stereoisomer. Phospholipase A 2 treatment of the solubilized preparation decreased binding by 50-80% at 1 ug/ml. The binding capacity in a typical preparation was 100-150 fmol/mg protein. Binding capacity was retained on hydroxyl apatite columns when the receptor preparation was loaded in 30 mM sodium phosphate buffer, pH 7.25 containing 0.05% digitonin and I00 mM NaCI (NaCI-PD). In a typical experiment i0 pmol of [3H]diprenorphine binding capac- ity were loaded onto a 1.7 x 30cm column and eluted with a gradient from 30-400 mM sodium phosphate. Binding in the eluate fractions was tested after buffer exchange of aliquots into 50 mM Tris digitonin buffer with the aid of G-25 chromatography on PD-10 columns. Two peaks of opiate binding activity were eluted, the first of which migrated with the majority of proteins while the second peak was 2-5fold enriched in opiate binding activity. Fractions from this peak containing 34% of the starting material were concen- trated and desalted by ultrafiltration and then diluted into 18 mM NaCI-PD. This material was applied to a DEAE-sepharose column (1.7 x 30cm) equilibrated in the same buffer, washed with I column volume 35 mM NaCI-PD and eluted with a gradient from 35-300 mM NaCI-PD. Binding was assayed directly in aliquots of the eluate since NaCI was found not to interfere. Binding activity eluted bet- ween 130-260 mM NaCI with the later fractions having a binding capacity of 2.8

S ,o.

u

O.1

1 5 10 ~

FIG.2 ~romatograp~ of d ig i ton in so~bi l iged sheep bra in membranes on D~-sepha- rose ( I .7x30 cm; T r i s -~f fe r pH 7.7, 35~ NaCl, 0.05% d ig i ton in ) . The column was e~ted w i th a grad ient from 35-500~ NaC1 in T r i s -d ig i ton in . ~nd ing was assayed in a I iquots of e luate f rac t ions a f te r desa l t ing on P~IO co l~s us ing [3H]d iprenorphtne . ~e f i r s t two po in ts show unreta ined b ind ing ac t iv i ty A re - tention of approximate~ 80% of the applied material was obtained when DE~- sepharose was used under the conditions described in the text; i.e. Hen the ion exchanger was equilibrated in Na-phosphate ~f fe r ~ 7.25, 35~ NaCI, 0.05% digitonin.

206 Purification of Opiate Receptors Vol. 33, Sup. I, 1983

pmol/mg protein; 1.4 pmol recovery). This material was again concentrated by ultrafiltration and diluted into 50 mM MOPS, NaCI i00 mM, digitonin 0.05% buffer and loaded onto a phenylboronate substituted gel. Opiate binding activi- ty was eluted with a sorbitol gradient from 0-100 mM (8 column volumes) and was found in the later fractions eluting at about 70-90 mM sorbitol. Recovery of the applied material was i pmol or 70%.

Discussion

The properties of opiate receptors solubilized with two different detergents were studied. ~naps solubized receptors behaved as large complexes the stokes radius of which was reduced upon phosholipase A2, but not phospholipase C, treatment.This observation suggests the presence of phospholipids in the deter- gent solubilized complex The reduction in the stokes radius by phospholipase A 2 could either reflect a conformational alteration of the receptor complex or cleavage into smaller subunits. Gel exclusion chromatography under nondena- turing conditions does not permit a distinction between these possibilities. Inhibition of opiate binding to solubililized receptors by phospholipase A 2 has been reported (4) previously with the use of Triton X-100 as detergent. The venom (Russels viper), however, in addition to phospholipase, contains a peptide which inhibits opiate binding with high affinity (6).

Chaps solubilized receptors were retained on DEAE-sepharose, the hydrophobic resign phenylsepharose, and on a chromatofocussing gel. However rather drastic conditions were required to elute radioactivity which was usually dissociated from the receptor.

Digitonin-NaCl solubilized receptors were succesfully chromatographed on hy- droxylapatite and DEAE-sepharose. Purification was achieved by using Na-phos- phate buffers of relatively low pH. Use of K-phosphate buffers resulted in loss while further lowering of the pH resulted in precipitation of binding activity. A phenylboronate gel -which binds glycoproteins- was chosen as a third step because of its virtually complete retention of binding activity. A gradient of sorbitol was employed to achieve some further purifiction. The gel was also excellently suited to exchange detergents since the attached receptors can be washed extensively while bound to the matrix. Purification of opiate receptors solubilized with Triton X-100 has previously been reported (7). Bidlack et al. used an affinity resign and achieved a bind- ding capacity of 40 pmol/mg. The procedure reported here permits a purification to a capacity of approximately i0 pmol/mg protein with the use of conventiona- chromatography.

REFERENCES

(i) W.F.SIMONDS ,G.KOSKI ,R.A.STREATY, L.M.HJELMELAND & W.A.KLEE, Proc. Natl. Acad. Sci., USA, 77, 4623-4627, (1980). (2) U.T.RUEGG, S.CUENOD, J.M.HILLER, T.GIOANNINI, R.D.HOWELLS & E.J.SIMON, Proc. Natl. Acad. Sci., USA, 78, 4635-4638 (1981). (3) T.GIOANNINI, B.FOUCAUD, J.M.HILLER, M.E.HATTEN & E.J.SIMON, Biochem. Bio- phys. Res. Comm., 105, 1128-1134, (1982). (4) J.M.BIDLACK & L.G.ABOOD , Life Sci., 27, 331-340, (1980). (5) W.A.KLEE, W.F.SIMOND$, F.W.SWEAT, T.R.BURKE A.E.JACOBSON & K.C.RICE, Febs Lett., 150, 125-128, (1982). (6) P.BEVAN & P. HIESTAND, J. Biol. (~em., 258, 5319-5326, (1983). (7) J.M.BIDLACK, L.G.ABOOD, P. OSEI-GYMAH, & S.ARCHER, Proc. Natl. Acad. Sci., USA, 78, 636-639, (1981).