Proc. Natl. Acad. Sc. USA Vol. 76, No. 4, pp. 1593-1597, April 1979
Biochemistry
Chemical crosslinking of a solubilized enkephalin macromolecular complex (opioid peptides/protein solubilization/receptor characterization/diimidate crosslinking)
R. SUZANNE ZUKIN AND RICHARD M. KREAM Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461
Communicated by Jerard Hurwitz, January 8, 1979
ABSTRACT Covalently bound [3H1D-Ala2,Met5-enkephalinamide- and '25I-labeled D-Ala2,N-Me-Phe4,Met40)5-ol-enkephalin-macromolecule complexes have been prepared by crosslinking the solubilized noncovalent comrlexes from rat brain. Gel electrophoresis of the partially purified 125I-Iabeled enkephalin-macromolecule complex under nondenaturing conditions results in a single major radioactive peak. The complex has a Stokes radius of a pproximately 48 A as determined by molecular exclusion chromatography this radius corresponds to a molecular weight of 380,000 for a spherical molecule. In preliminary experiments, sodium dodecyl sulfate electrophoresis of the complex shows a major radioactive peak corresponding to a molecutar weight of 35,000. The preparation of these specific covalent enkephalin-macromolecule complexes should be useful in purification of the receptor and in probing the molecular mechanism of opiate action. The actions of opiates upon nervous tissue are mediated by specific receptors that bind opiates with high affinity and discriminate between the biologically active (-)-isomers and the relatively inactive (+)-isomers. This stereospecific binding of opiates has made possible the determination of the distribution of opiate receptors within the nervous system (1, 2), has yielded information on the ionic and other requirements for optimal binding (3-5), and has been widely used as an assay system in
the isolation of endogenous opioid substances (6-8). Binding studies have provided phenomenological evidence for multiple conformational states of the opiate receptor (9, 10) and for heterogeneous subclasses within the receptor population (11). However, there is little information as to the nature of the opiate binding site apart from the fact that it is a membrane-associated protein or proteolipid complex (12-14). One approach that enables preliminary molecular characterization of the receptor and provides a marker during purification is that of labeling membrane-bound receptor prior to extraction with detergent. In 1975 Simon and coworkers introduced this approach for the opiate receptor and used it to solubilize a noncovalent [3H]etorphine macromolecular complex (15). Several lines of evidence indicated that this macromolecule was the opiate receptor. The solubilized, opiate-free receptor did not, however, bind opiates. This report describes the preparation of the putative, covalently bound [3H]enkephalin- and 125I-labeled (125I-)enkephalin-receptor complexes by crosslinking the solubilized noncovalent complexes. The method involves the use of the bifunctional crosslinking reagent, dimethyl suberimidate, an alkyl diimido ester which reacts rapidly and specifically with the unprotonated e-amino group of lysine or NH2-terminal a-amino residue of proteins and peptides (16-18). Suberimidate has been used in a large number of systems to join protein subunits or peptides and proteins within a complex or in close
proximity (18). Use of suberimidate to crosslink the enkephalin-receptor complexes represents a different application in which such a bifunctional reagent is used to attach a protein irreversibly to a radioactive substrate. The covalently bound ligand can then provide a marker to enable preliminary steps in the characterization and purification of the receptor. Preliminary studies of the molecular properties of the suberimidate-crosslinked enkephalin-receptor complex are presented. MATERIALS AND METHODS [3H]D-Ala2,Met5-enkephalin (DALA) (37.7 Ci/mmol, 1 Ci = 3.7 X 10O0 becquerels) and [3H]Met5-enkephalin (42.3 Ci/mmol) were obtained from New England Nuclear. D-Ala2,N-Me-Phe4,Met-(O)5-ol-enkephalin (Sandoz, FK 33824, obtained from Peninsula Laboratories, San Carlos, CA) was iodinated enzymatically to a specific activity of 200 Ci/mmol according to the procedure of Morrison and Bayse (19). The purified 125I-enkephalin cochromatographed with the unlabeled peptide and displayed similar binding characteristics (unpublished results). Solubilization of the noncovalent [3H]enkephalin and 125Ienkephalin complexes was carried out by a modification of the procedure of Simon et al. (15). P2 (mitochondrial/synaptosomal) membranes prepared from whole brains of male Sprague-Dawley rats (150 gm) were incubated at 40C for 45 min with [3H]DALA (8 nM), [3H]Met5-enkephalin (8 nM), or 125I-labeled FK 33-824 (2 nM) in the presence of either levorphanol (10 ,M) or dextrorphan (10 ,M). In the case of [3H]Met5-enkephalin, bacitracin (0.1 mg/ml, Sigma) was included to inhibit proteolysis of the peptide. After incubation, the mixture was centrifuged (30,000 X g, 15 min), and the resulting pellet was resuspended in polyoxyethylene ether detergent (Brij 36T, 1%, Sigma) in phosphate buffer at pH 7.4. The detergent suspension was centrifuged (100,000 X g, 90 min), and the supernatant was then fractionated on a Sephadex G-25 column (Pharmacia) in order to separate [3H]enkephalin from bound ligand. For preparation of covalent [3H]enkephalin-receptor complex, protein fractions from the Sephadex G-25 column containing the highest radioactivity were combined and allowed to react with dimethyl suberimidate (2 mg/ml, Sigma) in 100 mM triethanolamine buffer, pH 8.8, for 30 min at ambient temperature. [3H]etorphine-macromolecule complex was solubilized as described (15). Radioactivity in tritium-containing samples was determined by counting aliquots in Aquasol/toluene (2:1) in a liquid scintillation counter. The sizes of the noncovalent and covalent [3H]DALA- and noncovalent [3H]etorphine-bound complexes were estimated
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviations: DALA, D-Ala2,Met5-enkephalin; FK 33-824, D-Ala2, N-Me-Phe4,Met-(0)5-ol-enkephalin; NaDodSO4, sodium dodecyl sulfate; P2 membranes, mitochondrial/synaptosomal membranes.
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by molecular exclusion chromatography. Column details are given in the figure legends. The covalent 125I-enkephalinmacromolecule complex was purified by a combination of Sephadex G-150 and Sephadex G-200 gel filtration chromatography. The suberimidate-treated sample (2 ml) was fractionated on a Sephadex G-150 column (1.5 X 30cm) eluted with 50 mM Tris-HCI buffer, pH 7.4, at 4VC; 1-ml fractions were collected. The two fractions containing the highest radioactivity (just behind the protein void peak) were combined and applied to a Sephadex G-200 column (1.5 X 30 cm) and eluted with the same buffer at 40C. Protein concentration was monitored by the method of Lowry et al. (20). Polyacrylamide gel electrophoresis was performed under nondenaturing conditions at pH 8.7 in cyclindrical 5% polyacrylamide gels (21) and in the presence of 0.1% sodium dodecyl sulfate (NaDodSO4) in gels of 5% and 10% acrylamide after treatment with 5 mM 2-mercaptoethanol (22). Protein was visualized by staining with Coomassie brilliant blue; radioactivity in slices (2 mm) was monitored directly with a Packard gamma spectrometer. Binding assays were performed by an adaption of the method of Pert and Snyder (23). RESULTS Solubilization of a [3H]Enkephalin-Macromolecule Complex. In Fig. 1 is shown the fractionation by Sephadex G-25 gel filtration of Brij-solubilized P2 membranes preincubated with [33H]DALA (8 nM) in the presence of the inactive opiate 7000
6000
1.0
5000
E DALA > FK 33-824 > levorphanol > naloxone. These relative potencies are essentially the same as those determined for the inhibition of macromolecule-bound soluble counts. In both cases the rank order of potency for these ligands is the same as that determined in the binding assay with P2 membranes under identical conditions using [3HJDALA or [3H]Met5-enkephalin as the radioactive ligands. Incubation of nonradioactive opiates for 1 hr at 4°C with ultracentrifugal supernatants containing [3H]DALAreceptor complex, either untreated or following gel filtration or dialysis to remove excess free ligand, resulted in no displacement of bound radioactivity. Chemical Crosslinking of the Solubiliized [3HjEnkephalin-Macromolecule Complex. Labeled ligand was crosslinked to the receptor by mixing Sephadex column fractions that contained the highest radioactivity with dimethyl suberimidate. In a separate experiment in order to assess covalent labeling, the suberimidate-treated sample was boiled for 20 min, redissolved in 1% NaDodSO4, and once again fractionated on Sephadex G-25 (Fig. 2). Approximately 40% of the applied radioactivity cochromatographed with the protein void peak on this second column; this value was assumed to be the yield of crosslinked receptor. On the other hand, when the highly labeled protein fractions were taken from the first column and boiled and exposed to NaDodSO4 without first allowing them to react with suberimidate, only negligible radioactivity eluted with the protein peak on the second column, and the dextrorphan- and levorphanol-treated preparations were indistinguishable. The treatment with heat and ionic detergent would cause release of any noncovalently bound
E C.)
-Ji I
Fraction no.
FIG. 2. Sephadex G-25 elution profile of Brij extract of P2 membranes following crosslinking and heat-treatment. Fractions 10-12 from the first Sephadex G-25 column (Fig. 1) containing the highest radioactivity and protein concentration were pooled and treated with dimethyl suberimidate (2 mg/ml) in 100 mM triethanolamine buffer, pH 8.8, for 30 min at room temperature. The reaction mixture was frozen for 16 hr, thawed, heated at 100'C for 20 min, and redissolved in 1% NaDodSO4. A 1-ml sample of the resulting suspension was applied to a Sephadex G-25 column (1 X 20 cm) and eluted with 50 mM Tris-HCl, pH 7.4, at 220C; 1-ml fractions were collected. P2 membranes were bound initially with 8 nM [3H]DALA as described for Fig. 1 in the presence of 10 ,M dextrorphan (0) or 10 MAM levorphanol (0). X, Protein concentration.
peptide; therefore these results indicate that stereospecific, covalently bound [3H]enkephalin-macromolecule complex has been obtained in a soluble form. Fractionation by gel filtration of the labeled protein fractions from the second Sephadex G-25 column on three successive Sephadex G-25 columns resulted in no further detectable dissociation of radioactivity. The noncovalent [3H]DALA-macromolecule complex exhibited biphasic dissociation at 250C. Approximately 50% of the originally bound peptide was dissociated within 20 min, but no further dissociation could be detected after a 2-hr total incubation. The half time for dissociation of [3H]Met-enkephalin from rat brain homogenate is 2 hr at 25°C (24). The yield of enkephalin crosslinked to macromolecule in the suberimidate reaction was 40% for [3H]Met-enkephalin and [3H]DALA. The yield was 20% when 125I-FK 33-824 was used. These values were reproducible in 10 independent experiments. It is of interest that, when unsolubilized P2 membrane homogenates incubated with [3H]DALA (8 nM) were treated with suberimidate, the yield of macromolecular crosslinked peptide in the soluble fraction was reduced by more than 1:4 compared with the experiment in which suberimidate was added after solubilization. Preliminary Characterization of the Enkephalin-Macromolecule Complex. The size of the covalently bound [3H]enkephalin-macromolecule complex was estimated by molecular exclusion chromatography on Sepharose 6B (Fig. 3). Radioactivity from the [3H]DALA complex either before or after suberimidate treatment chromatographed at an elution position corresponding to a Stokes radius of 48 A. The corresponding molecular weight for a spherical macromolecule is approximately 380,000. Radioactivity from noncovalent [3H]etorphine-macromolecule complex eluted at the same position. This size estimate agrees with the value for the [3H]etorphine complex determined by Simon et al. (15). The elution position of the noncovalent and covalent [3H]DALA complexes remained unchanged when the protein concentration of the sample was varied 5-fold. Both the [3H]DALA and 125I-FK
Proc. Natl. Acad. Sci. USA 76 (1979)
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DISCUSSION Covalently bound [3H]enkephalin and '25I-enkephalin-macromolecule complexes have been prepared by crosslinking of the solubilized noncovalent complexes by dimethyl suberimidate. Two pieces of evidence suggest that the ligand is bound to a protein in the enkephalin-macromolecule complex. These are the sensitivity of the binding to heat and to trypsin. Moreover, the successful crosslinking of the peptide to the receptor by suberimidate indicates the presence of a reactive amino group as might be expected for the surface lysine residues of protein (12). That the macromolecule is the opiate receptor is suggested by several findings. First, a large excess of levorphanol inhibits the ligand binding much more effectively than does an excess of dextrorphan, and this stereospecific difference is greater in the void volume peak of protein following solubilization and fractionation by gel filtration. Second, the rank order of potency of six opiate ligands in inhibition of total soluble and macromolecule-bound soluble counts is the same as that determined for inhibition by these of [3H]enkephalin binding to brain P2 membranes. Binding affinities for the opiates have been shown to correlate with clinical potency under appropriate conditions (23). Third, no stereospecific bound complex could be detected in the case of P2 membranes from cerebellar tissue, a brain region which has a low density of opiate-binding sites
/ Enkephalin >[HI /
44 a)
complex
0.8
0
Catalase
0.7-
Phosphorylase B
0.61
JjHexokinase
30
50
40
Stokes radius,
60 A
FIG. 3. Size determination of the solubilized noncovalent and covalent [3H]DALA- and noncovalent [3H~etorphine-bound complexes by gel filtration. Samples (100 Ml) were applied to a Sepharose 6B column (0.5 X 10 cm) and eluted with 50 mM Tris-HCl, pH 7.4, at 4°C; 100-$l fractions were collected. Data are expressed as (-log Kavg)112 where Kavg = (Ve - VVt - V); Ve, VO, and Vt referring to the elution volume, void volume, and total volume, respectively (25). VO and Vt values were 600 ,l and 1800 jAl, respectively. Standard proteins were monitored by absorbance at 280 nm, and the radiolabeled complexes were monitored by radioactivity determination.
33-824 covalent complexes eluted in a single peak just behind the void peak on Sephadex G-200; both eluted with the void peak on Sephadex G-150 gel filtration chromatography. Fractionation of the 125I-FK 33-824-bound sample by gel filtration on a Sephadex G-150 column followed by fractionation of the radioactive peak from this column on a Sephadex G-200 column resulted in a 33-fold enrichment in radioactivity of the covalent enkephalin complex (Table 2). Electrophoresis of the covalently bound 125I-enkephalin complex under nondenaturing conditions on 5% polyacrylamide gels showed a single major radioactive peak (Fig. 4 upper). In preliminary experiments, electrophoresis of this complex on NaDodSO4/10% polyacrylamide gels resulted in a single major radioactive peak at a position corresponding to a molecular weight of 35,000 (Fig. 4 lower).
(1).
The size of the solubilized enkephalin-macromolecule complex has been estimated by molecular exclusion chromatography to be 48 A, or 380,00 daltons for the case of a spherical receptor. The finding that the [3H]enkephalin complex and the [3H]etorphine complex comigrate is consistent with binding of the two ligands to the same protein complex. The concentration of Brij present in this complex has not been determined, however, and this could clearly influence the apparent size of the macromolecule. The finding that the noncovalent and covalent enkephalin complexes elute at an identical position suggests that suberimidate has crosslinked the peptide to the same macromolecule to which it was bound in the noncovalent complex. Moreover, the finding of a single peak on native gel electrophoresis of the enkephalin-receptor complex indicates that there is not random covalent attachment of the peptide. The present study represents a new application of diimidate esters in which dimethyl suberimidate was used to join a protein to its peptide substrate. It is of interest that enkephalin has apparently been crosslinked through the a-amino moiety of tyrosine, a residue implicated in binding of the peptide to the opiate receptor (26, 27). In studies of guinea pig brain, methylation of the tyrosine a-amino group enkephalinamide has been shown to enhance its ability to displace [3H]naloxone binding and inhibit contractions of the guinea pig ileum (11).
Specific crosslinking of the opiate receptor to radiolabeled ligand has enabled preliminary characterization of the protein
Table 2. Purification of covalent 125I-FK 33-824-receptor complex
Stereospecific proteinbound
radioactivity, Protein, Fraction
Purification of covalent Specific activity, complex, cpm/mg fmol/mg -fold
cpm
mg
First Sephadex G-25 column (noncovalent complex) Second Sephadex G-25 column
60,000
2.0
30,000
(covalent complex) Sephadex G-150 column Sephadex G-200 column
12,000 6,000 6,000
2.0 0.12 0.03
6,000 48,000 200,000
87.5 30 240 1000
8 33
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Zukin and Kream
Proc. Natl. Acad. Sci. USA 76 (1979)
gands. In addition, after correction for fractional crosslinking, the method provides a quantitative assay for the solubilized receptor. It will be of particular interest to see whether the evidence for somewhat different receptors for opiates and enkephalins (11) can be substantiated by the present techniques. Finally, crosslinking with radiolabeled ligand provides a valuable marker for the receptor protein and provides a first step in the isolation and purification of the enkephalin receptor.
E
E
1597
500
O 400 C,)
C") 300
200
-
Dye front
100
0
2 4 6 8 10
22 26 30 34 38 20 24 28 32 36 40 Slice no.
18
14 12
16
700
600
E
,i500 CN C,)
400-
CY)
300
Dye front
200
100
0
2
4 6 8 10
12
14
16
18
20 Slice
22
24
26
28
30
32
34
36
38 40
no.
FIG. 4. Electrophoretic analyses of the partially purified 1251-FK 33-824-macromolecule complex. (Upper) Electrophoresis of the pooled radioactive fractions from the Sephadex G-150 column on a 5% polyacrylamide cylindrical gel under nondenaturing conditions. Gels were prepared according to the procedure of Davies and Stark (21), and electrophoresis was carried out at 3 mA for 3 hr. (Lower) Electrophoresis of the pooled radioactive fractions from the Sephadex G-150 column on a NaDodSO4/10% polyacrylamide cylindrical gel, prepared according to the procedure of Laemmli (22), at 8 mA for 5 hr. Radioactivity was monitored in 2-mm gel slices by counting directly with a Packard gamma counter. Staining with Coomassie brilliant blue showed a large number of protein bands in both gels. The gel patterns were reproducible in two separate experiments.
and a 33-fold purification of the ligand-receptor complex. The labeled subunit is estimated by NaDodSO4 gel electrophoresis to be 35,000 daltons. Thus, the receptor complex would appear to comprise an aggregate of identical or nonidentical protein constituents. The development of radiolabeled ligands of higher specific activity should increase the sensitivity of the determination of receptor molecular weight and subunit composition by NaDodSO4 gel electrophoresis. Because the crosslinking reaction depends on the accessibility of the bound peptide, evaluation of the efficiency of labeling with different opiate and opioid peptide ligands and with crosslinking agents of varying chain length or reactive groups should provide insight into differences in the bound conformation of the various li-
We thank Dr. E. J. Simon and Hoffman-LaRoche for their respective gifts of etorphine and the opiate enantiomers, dextrorphan and levorphanol, Ms. Gail Federoff for excellent technical assistance, and Drs. M. Kalimi and R. A. North for their helpful criticism of the manuscript. This work was supported by Biomedical Research Support Grant funds and by National Institutes of Health Grant 5T32 GM 07128-04. 1. Kuhar, M. J., Pert, C. B. & Snyder, S. H. (1973) Nature (London) 245,447-450. 2. Hiller, J. M., Pearson, J. & Simon, E. J. (1973) Res. Commun. Chem. Pathol. Pharmacol. 6, 1052-1061. 3. Simon, E. J., Hiller, J. M., Groth, J. & Edelman, I. (1975) J. Pharmacol. Exp. Ther. 192,531-537. 4. Pert, C. B. & Snyder, S. H. (1974) Mol. Pharmacol. 10, 868879. 5. Pasternak, G. W., Snowman, A. M. & Snyder, S. H. (1975) Mol. Pharmacol. 11,735-744. 6. Terenius, L. & Wahlstrom, A. (1975) Acta Pharmacol. Toxicol. 35, Suppl. 1, 55. 7. Simantov, R. & Snyder, S. H. (1976) Proc. Nati. Acad. Sci. USA 73,2515-2519. 8. Cox, B. M., Goldstein, A. & Li, C. H. (1976) Proc. Nati. Acad. Sci. USA 73, 1821-1823. 9. Pasternak, G. W. & Snyder, S. H. (1975) Nature (London) 253, 563-565. 10. Simon, E. J. & Groth, J. (1975) Proc. Natl. Acad. Sci. USA 72, 2404-2408. 11. Lord, J. A., Kosterlitz, H. W., Hughes, J. & Waterfield, A. A. (1977) Nature (London) 267, 495-499. 12. Pert, C. B. & Snyder, S. H. (1973) Science 174, 1011-1014. 13. Simon, E. J., Hiller, J. M. & Edelman, I. (1973) Proc. Nati. Acad. Sci. USA 70, 1947-1949. 14. Pasternak, G. W. & Snyder, S. H. (1974) Mol. Pharmacol. 10, 183-193. 15. Simon, E. J., Hiller, J. M. & Edelman, I. (1975) Science 190, 389-390. 16. Ludwig, M. L. & Hunter, M. J. (1967) Methods Enzymol. 11, 595-604. 17. Davies, G. E. & Stark, G. R. (1970) Proc. Natl. Acad. Sci. USA 66,651-656. 18. Peters, K. & Richards, F. M. (1977) Annu. Rev. Biochem. 46, 523-551. 19. Morrison, M. & Bayse, G. (1970) Biochemistry 9, 2995-3000. 20. Lowry, 0. H., Rosebrough, A. L., Farr, A. L. & Randall, R. J.
(1951) J. Biol. Chem. 193,265-275. 21. Davies, G. E. & Stark, G. R. (1970) Proc. Nati. Acad. Sci. USA 66,651-656. 22. Laemmli, U. K. (1970) Nature (London) 227,680-685. 23. Pert, C. B. & Snyder, S. H. (1974) Science 179, 1011-1013. 24. Simantov, R., Childers, S. R. & Snyder, S. H. (1978) Eur. J. Pharmacol. 47,319-331. 25. Siegel, L. M. & Monty, K. J. (1966) Biochim. Biophys. Acta 112, 346-362. 26. Goldstein, A., Goldstein, J. S. & Cox, B. M. (1975) Life Sci. 17, 1643-1654. 27. Roques, B. P., Garbay-Jaureguiberry, C., Oberlin, R., Anteunis, M. & Lala, A. K. (1976) Nature (London) 262,778-779.
Biochemistry
Chemical crosslinking of a solubilized enkephalin macromolecular complex (opioid peptides/protein solubilization/receptor characterization/diimidate crosslinking)
R. SUZANNE ZUKIN AND RICHARD M. KREAM Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461
Communicated by Jerard Hurwitz, January 8, 1979
ABSTRACT Covalently bound [3H1D-Ala2,Met5-enkephalinamide- and '25I-labeled D-Ala2,N-Me-Phe4,Met40)5-ol-enkephalin-macromolecule complexes have been prepared by crosslinking the solubilized noncovalent comrlexes from rat brain. Gel electrophoresis of the partially purified 125I-Iabeled enkephalin-macromolecule complex under nondenaturing conditions results in a single major radioactive peak. The complex has a Stokes radius of a pproximately 48 A as determined by molecular exclusion chromatography this radius corresponds to a molecular weight of 380,000 for a spherical molecule. In preliminary experiments, sodium dodecyl sulfate electrophoresis of the complex shows a major radioactive peak corresponding to a molecutar weight of 35,000. The preparation of these specific covalent enkephalin-macromolecule complexes should be useful in purification of the receptor and in probing the molecular mechanism of opiate action. The actions of opiates upon nervous tissue are mediated by specific receptors that bind opiates with high affinity and discriminate between the biologically active (-)-isomers and the relatively inactive (+)-isomers. This stereospecific binding of opiates has made possible the determination of the distribution of opiate receptors within the nervous system (1, 2), has yielded information on the ionic and other requirements for optimal binding (3-5), and has been widely used as an assay system in
the isolation of endogenous opioid substances (6-8). Binding studies have provided phenomenological evidence for multiple conformational states of the opiate receptor (9, 10) and for heterogeneous subclasses within the receptor population (11). However, there is little information as to the nature of the opiate binding site apart from the fact that it is a membrane-associated protein or proteolipid complex (12-14). One approach that enables preliminary molecular characterization of the receptor and provides a marker during purification is that of labeling membrane-bound receptor prior to extraction with detergent. In 1975 Simon and coworkers introduced this approach for the opiate receptor and used it to solubilize a noncovalent [3H]etorphine macromolecular complex (15). Several lines of evidence indicated that this macromolecule was the opiate receptor. The solubilized, opiate-free receptor did not, however, bind opiates. This report describes the preparation of the putative, covalently bound [3H]enkephalin- and 125I-labeled (125I-)enkephalin-receptor complexes by crosslinking the solubilized noncovalent complexes. The method involves the use of the bifunctional crosslinking reagent, dimethyl suberimidate, an alkyl diimido ester which reacts rapidly and specifically with the unprotonated e-amino group of lysine or NH2-terminal a-amino residue of proteins and peptides (16-18). Suberimidate has been used in a large number of systems to join protein subunits or peptides and proteins within a complex or in close
proximity (18). Use of suberimidate to crosslink the enkephalin-receptor complexes represents a different application in which such a bifunctional reagent is used to attach a protein irreversibly to a radioactive substrate. The covalently bound ligand can then provide a marker to enable preliminary steps in the characterization and purification of the receptor. Preliminary studies of the molecular properties of the suberimidate-crosslinked enkephalin-receptor complex are presented. MATERIALS AND METHODS [3H]D-Ala2,Met5-enkephalin (DALA) (37.7 Ci/mmol, 1 Ci = 3.7 X 10O0 becquerels) and [3H]Met5-enkephalin (42.3 Ci/mmol) were obtained from New England Nuclear. D-Ala2,N-Me-Phe4,Met-(O)5-ol-enkephalin (Sandoz, FK 33824, obtained from Peninsula Laboratories, San Carlos, CA) was iodinated enzymatically to a specific activity of 200 Ci/mmol according to the procedure of Morrison and Bayse (19). The purified 125I-enkephalin cochromatographed with the unlabeled peptide and displayed similar binding characteristics (unpublished results). Solubilization of the noncovalent [3H]enkephalin and 125Ienkephalin complexes was carried out by a modification of the procedure of Simon et al. (15). P2 (mitochondrial/synaptosomal) membranes prepared from whole brains of male Sprague-Dawley rats (150 gm) were incubated at 40C for 45 min with [3H]DALA (8 nM), [3H]Met5-enkephalin (8 nM), or 125I-labeled FK 33-824 (2 nM) in the presence of either levorphanol (10 ,M) or dextrorphan (10 ,M). In the case of [3H]Met5-enkephalin, bacitracin (0.1 mg/ml, Sigma) was included to inhibit proteolysis of the peptide. After incubation, the mixture was centrifuged (30,000 X g, 15 min), and the resulting pellet was resuspended in polyoxyethylene ether detergent (Brij 36T, 1%, Sigma) in phosphate buffer at pH 7.4. The detergent suspension was centrifuged (100,000 X g, 90 min), and the supernatant was then fractionated on a Sephadex G-25 column (Pharmacia) in order to separate [3H]enkephalin from bound ligand. For preparation of covalent [3H]enkephalin-receptor complex, protein fractions from the Sephadex G-25 column containing the highest radioactivity were combined and allowed to react with dimethyl suberimidate (2 mg/ml, Sigma) in 100 mM triethanolamine buffer, pH 8.8, for 30 min at ambient temperature. [3H]etorphine-macromolecule complex was solubilized as described (15). Radioactivity in tritium-containing samples was determined by counting aliquots in Aquasol/toluene (2:1) in a liquid scintillation counter. The sizes of the noncovalent and covalent [3H]DALA- and noncovalent [3H]etorphine-bound complexes were estimated
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviations: DALA, D-Ala2,Met5-enkephalin; FK 33-824, D-Ala2, N-Me-Phe4,Met-(0)5-ol-enkephalin; NaDodSO4, sodium dodecyl sulfate; P2 membranes, mitochondrial/synaptosomal membranes.
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by molecular exclusion chromatography. Column details are given in the figure legends. The covalent 125I-enkephalinmacromolecule complex was purified by a combination of Sephadex G-150 and Sephadex G-200 gel filtration chromatography. The suberimidate-treated sample (2 ml) was fractionated on a Sephadex G-150 column (1.5 X 30cm) eluted with 50 mM Tris-HCI buffer, pH 7.4, at 4VC; 1-ml fractions were collected. The two fractions containing the highest radioactivity (just behind the protein void peak) were combined and applied to a Sephadex G-200 column (1.5 X 30 cm) and eluted with the same buffer at 40C. Protein concentration was monitored by the method of Lowry et al. (20). Polyacrylamide gel electrophoresis was performed under nondenaturing conditions at pH 8.7 in cyclindrical 5% polyacrylamide gels (21) and in the presence of 0.1% sodium dodecyl sulfate (NaDodSO4) in gels of 5% and 10% acrylamide after treatment with 5 mM 2-mercaptoethanol (22). Protein was visualized by staining with Coomassie brilliant blue; radioactivity in slices (2 mm) was monitored directly with a Packard gamma spectrometer. Binding assays were performed by an adaption of the method of Pert and Snyder (23). RESULTS Solubilization of a [3H]Enkephalin-Macromolecule Complex. In Fig. 1 is shown the fractionation by Sephadex G-25 gel filtration of Brij-solubilized P2 membranes preincubated with [33H]DALA (8 nM) in the presence of the inactive opiate 7000
6000
1.0
5000
E DALA > FK 33-824 > levorphanol > naloxone. These relative potencies are essentially the same as those determined for the inhibition of macromolecule-bound soluble counts. In both cases the rank order of potency for these ligands is the same as that determined in the binding assay with P2 membranes under identical conditions using [3HJDALA or [3H]Met5-enkephalin as the radioactive ligands. Incubation of nonradioactive opiates for 1 hr at 4°C with ultracentrifugal supernatants containing [3H]DALAreceptor complex, either untreated or following gel filtration or dialysis to remove excess free ligand, resulted in no displacement of bound radioactivity. Chemical Crosslinking of the Solubiliized [3HjEnkephalin-Macromolecule Complex. Labeled ligand was crosslinked to the receptor by mixing Sephadex column fractions that contained the highest radioactivity with dimethyl suberimidate. In a separate experiment in order to assess covalent labeling, the suberimidate-treated sample was boiled for 20 min, redissolved in 1% NaDodSO4, and once again fractionated on Sephadex G-25 (Fig. 2). Approximately 40% of the applied radioactivity cochromatographed with the protein void peak on this second column; this value was assumed to be the yield of crosslinked receptor. On the other hand, when the highly labeled protein fractions were taken from the first column and boiled and exposed to NaDodSO4 without first allowing them to react with suberimidate, only negligible radioactivity eluted with the protein peak on the second column, and the dextrorphan- and levorphanol-treated preparations were indistinguishable. The treatment with heat and ionic detergent would cause release of any noncovalently bound
E C.)
-Ji I
Fraction no.
FIG. 2. Sephadex G-25 elution profile of Brij extract of P2 membranes following crosslinking and heat-treatment. Fractions 10-12 from the first Sephadex G-25 column (Fig. 1) containing the highest radioactivity and protein concentration were pooled and treated with dimethyl suberimidate (2 mg/ml) in 100 mM triethanolamine buffer, pH 8.8, for 30 min at room temperature. The reaction mixture was frozen for 16 hr, thawed, heated at 100'C for 20 min, and redissolved in 1% NaDodSO4. A 1-ml sample of the resulting suspension was applied to a Sephadex G-25 column (1 X 20 cm) and eluted with 50 mM Tris-HCl, pH 7.4, at 220C; 1-ml fractions were collected. P2 membranes were bound initially with 8 nM [3H]DALA as described for Fig. 1 in the presence of 10 ,M dextrorphan (0) or 10 MAM levorphanol (0). X, Protein concentration.
peptide; therefore these results indicate that stereospecific, covalently bound [3H]enkephalin-macromolecule complex has been obtained in a soluble form. Fractionation by gel filtration of the labeled protein fractions from the second Sephadex G-25 column on three successive Sephadex G-25 columns resulted in no further detectable dissociation of radioactivity. The noncovalent [3H]DALA-macromolecule complex exhibited biphasic dissociation at 250C. Approximately 50% of the originally bound peptide was dissociated within 20 min, but no further dissociation could be detected after a 2-hr total incubation. The half time for dissociation of [3H]Met-enkephalin from rat brain homogenate is 2 hr at 25°C (24). The yield of enkephalin crosslinked to macromolecule in the suberimidate reaction was 40% for [3H]Met-enkephalin and [3H]DALA. The yield was 20% when 125I-FK 33-824 was used. These values were reproducible in 10 independent experiments. It is of interest that, when unsolubilized P2 membrane homogenates incubated with [3H]DALA (8 nM) were treated with suberimidate, the yield of macromolecular crosslinked peptide in the soluble fraction was reduced by more than 1:4 compared with the experiment in which suberimidate was added after solubilization. Preliminary Characterization of the Enkephalin-Macromolecule Complex. The size of the covalently bound [3H]enkephalin-macromolecule complex was estimated by molecular exclusion chromatography on Sepharose 6B (Fig. 3). Radioactivity from the [3H]DALA complex either before or after suberimidate treatment chromatographed at an elution position corresponding to a Stokes radius of 48 A. The corresponding molecular weight for a spherical macromolecule is approximately 380,000. Radioactivity from noncovalent [3H]etorphine-macromolecule complex eluted at the same position. This size estimate agrees with the value for the [3H]etorphine complex determined by Simon et al. (15). The elution position of the noncovalent and covalent [3H]DALA complexes remained unchanged when the protein concentration of the sample was varied 5-fold. Both the [3H]DALA and 125I-FK
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Zukin and Kream
1596
DISCUSSION Covalently bound [3H]enkephalin and '25I-enkephalin-macromolecule complexes have been prepared by crosslinking of the solubilized noncovalent complexes by dimethyl suberimidate. Two pieces of evidence suggest that the ligand is bound to a protein in the enkephalin-macromolecule complex. These are the sensitivity of the binding to heat and to trypsin. Moreover, the successful crosslinking of the peptide to the receptor by suberimidate indicates the presence of a reactive amino group as might be expected for the surface lysine residues of protein (12). That the macromolecule is the opiate receptor is suggested by several findings. First, a large excess of levorphanol inhibits the ligand binding much more effectively than does an excess of dextrorphan, and this stereospecific difference is greater in the void volume peak of protein following solubilization and fractionation by gel filtration. Second, the rank order of potency of six opiate ligands in inhibition of total soluble and macromolecule-bound soluble counts is the same as that determined for inhibition by these of [3H]enkephalin binding to brain P2 membranes. Binding affinities for the opiates have been shown to correlate with clinical potency under appropriate conditions (23). Third, no stereospecific bound complex could be detected in the case of P2 membranes from cerebellar tissue, a brain region which has a low density of opiate-binding sites
/ Enkephalin >[HI /
44 a)
complex
0.8
0
Catalase
0.7-
Phosphorylase B
0.61
JjHexokinase
30
50
40
Stokes radius,
60 A
FIG. 3. Size determination of the solubilized noncovalent and covalent [3H]DALA- and noncovalent [3H~etorphine-bound complexes by gel filtration. Samples (100 Ml) were applied to a Sepharose 6B column (0.5 X 10 cm) and eluted with 50 mM Tris-HCl, pH 7.4, at 4°C; 100-$l fractions were collected. Data are expressed as (-log Kavg)112 where Kavg = (Ve - VVt - V); Ve, VO, and Vt referring to the elution volume, void volume, and total volume, respectively (25). VO and Vt values were 600 ,l and 1800 jAl, respectively. Standard proteins were monitored by absorbance at 280 nm, and the radiolabeled complexes were monitored by radioactivity determination.
33-824 covalent complexes eluted in a single peak just behind the void peak on Sephadex G-200; both eluted with the void peak on Sephadex G-150 gel filtration chromatography. Fractionation of the 125I-FK 33-824-bound sample by gel filtration on a Sephadex G-150 column followed by fractionation of the radioactive peak from this column on a Sephadex G-200 column resulted in a 33-fold enrichment in radioactivity of the covalent enkephalin complex (Table 2). Electrophoresis of the covalently bound 125I-enkephalin complex under nondenaturing conditions on 5% polyacrylamide gels showed a single major radioactive peak (Fig. 4 upper). In preliminary experiments, electrophoresis of this complex on NaDodSO4/10% polyacrylamide gels resulted in a single major radioactive peak at a position corresponding to a molecular weight of 35,000 (Fig. 4 lower).
(1).
The size of the solubilized enkephalin-macromolecule complex has been estimated by molecular exclusion chromatography to be 48 A, or 380,00 daltons for the case of a spherical receptor. The finding that the [3H]enkephalin complex and the [3H]etorphine complex comigrate is consistent with binding of the two ligands to the same protein complex. The concentration of Brij present in this complex has not been determined, however, and this could clearly influence the apparent size of the macromolecule. The finding that the noncovalent and covalent enkephalin complexes elute at an identical position suggests that suberimidate has crosslinked the peptide to the same macromolecule to which it was bound in the noncovalent complex. Moreover, the finding of a single peak on native gel electrophoresis of the enkephalin-receptor complex indicates that there is not random covalent attachment of the peptide. The present study represents a new application of diimidate esters in which dimethyl suberimidate was used to join a protein to its peptide substrate. It is of interest that enkephalin has apparently been crosslinked through the a-amino moiety of tyrosine, a residue implicated in binding of the peptide to the opiate receptor (26, 27). In studies of guinea pig brain, methylation of the tyrosine a-amino group enkephalinamide has been shown to enhance its ability to displace [3H]naloxone binding and inhibit contractions of the guinea pig ileum (11).
Specific crosslinking of the opiate receptor to radiolabeled ligand has enabled preliminary characterization of the protein
Table 2. Purification of covalent 125I-FK 33-824-receptor complex
Stereospecific proteinbound
radioactivity, Protein, Fraction
Purification of covalent Specific activity, complex, cpm/mg fmol/mg -fold
cpm
mg
First Sephadex G-25 column (noncovalent complex) Second Sephadex G-25 column
60,000
2.0
30,000
(covalent complex) Sephadex G-150 column Sephadex G-200 column
12,000 6,000 6,000
2.0 0.12 0.03
6,000 48,000 200,000
87.5 30 240 1000
8 33
Biochemistry:
Zukin and Kream
Proc. Natl. Acad. Sci. USA 76 (1979)
gands. In addition, after correction for fractional crosslinking, the method provides a quantitative assay for the solubilized receptor. It will be of particular interest to see whether the evidence for somewhat different receptors for opiates and enkephalins (11) can be substantiated by the present techniques. Finally, crosslinking with radiolabeled ligand provides a valuable marker for the receptor protein and provides a first step in the isolation and purification of the enkephalin receptor.
E
E
1597
500
O 400 C,)
C") 300
200
-
Dye front
100
0
2 4 6 8 10
22 26 30 34 38 20 24 28 32 36 40 Slice no.
18
14 12
16
700
600
E
,i500 CN C,)
400-
CY)
300
Dye front
200
100
0
2
4 6 8 10
12
14
16
18
20 Slice
22
24
26
28
30
32
34
36
38 40
no.
FIG. 4. Electrophoretic analyses of the partially purified 1251-FK 33-824-macromolecule complex. (Upper) Electrophoresis of the pooled radioactive fractions from the Sephadex G-150 column on a 5% polyacrylamide cylindrical gel under nondenaturing conditions. Gels were prepared according to the procedure of Davies and Stark (21), and electrophoresis was carried out at 3 mA for 3 hr. (Lower) Electrophoresis of the pooled radioactive fractions from the Sephadex G-150 column on a NaDodSO4/10% polyacrylamide cylindrical gel, prepared according to the procedure of Laemmli (22), at 8 mA for 5 hr. Radioactivity was monitored in 2-mm gel slices by counting directly with a Packard gamma counter. Staining with Coomassie brilliant blue showed a large number of protein bands in both gels. The gel patterns were reproducible in two separate experiments.
and a 33-fold purification of the ligand-receptor complex. The labeled subunit is estimated by NaDodSO4 gel electrophoresis to be 35,000 daltons. Thus, the receptor complex would appear to comprise an aggregate of identical or nonidentical protein constituents. The development of radiolabeled ligands of higher specific activity should increase the sensitivity of the determination of receptor molecular weight and subunit composition by NaDodSO4 gel electrophoresis. Because the crosslinking reaction depends on the accessibility of the bound peptide, evaluation of the efficiency of labeling with different opiate and opioid peptide ligands and with crosslinking agents of varying chain length or reactive groups should provide insight into differences in the bound conformation of the various li-
We thank Dr. E. J. Simon and Hoffman-LaRoche for their respective gifts of etorphine and the opiate enantiomers, dextrorphan and levorphanol, Ms. Gail Federoff for excellent technical assistance, and Drs. M. Kalimi and R. A. North for their helpful criticism of the manuscript. This work was supported by Biomedical Research Support Grant funds and by National Institutes of Health Grant 5T32 GM 07128-04. 1. Kuhar, M. J., Pert, C. B. & Snyder, S. H. (1973) Nature (London) 245,447-450. 2. Hiller, J. M., Pearson, J. & Simon, E. J. (1973) Res. Commun. Chem. Pathol. Pharmacol. 6, 1052-1061. 3. Simon, E. J., Hiller, J. M., Groth, J. & Edelman, I. (1975) J. Pharmacol. Exp. Ther. 192,531-537. 4. Pert, C. B. & Snyder, S. H. (1974) Mol. Pharmacol. 10, 868879. 5. Pasternak, G. W., Snowman, A. M. & Snyder, S. H. (1975) Mol. Pharmacol. 11,735-744. 6. Terenius, L. & Wahlstrom, A. (1975) Acta Pharmacol. Toxicol. 35, Suppl. 1, 55. 7. Simantov, R. & Snyder, S. H. (1976) Proc. Nati. Acad. Sci. USA 73,2515-2519. 8. Cox, B. M., Goldstein, A. & Li, C. H. (1976) Proc. Nati. Acad. Sci. USA 73, 1821-1823. 9. Pasternak, G. W. & Snyder, S. H. (1975) Nature (London) 253, 563-565. 10. Simon, E. J. & Groth, J. (1975) Proc. Natl. Acad. Sci. USA 72, 2404-2408. 11. Lord, J. A., Kosterlitz, H. W., Hughes, J. & Waterfield, A. A. (1977) Nature (London) 267, 495-499. 12. Pert, C. B. & Snyder, S. H. (1973) Science 174, 1011-1014. 13. Simon, E. J., Hiller, J. M. & Edelman, I. (1973) Proc. Nati. Acad. Sci. USA 70, 1947-1949. 14. Pasternak, G. W. & Snyder, S. H. (1974) Mol. Pharmacol. 10, 183-193. 15. Simon, E. J., Hiller, J. M. & Edelman, I. (1975) Science 190, 389-390. 16. Ludwig, M. L. & Hunter, M. J. (1967) Methods Enzymol. 11, 595-604. 17. Davies, G. E. & Stark, G. R. (1970) Proc. Natl. Acad. Sci. USA 66,651-656. 18. Peters, K. & Richards, F. M. (1977) Annu. Rev. Biochem. 46, 523-551. 19. Morrison, M. & Bayse, G. (1970) Biochemistry 9, 2995-3000. 20. Lowry, 0. H., Rosebrough, A. L., Farr, A. L. & Randall, R. J.
(1951) J. Biol. Chem. 193,265-275. 21. Davies, G. E. & Stark, G. R. (1970) Proc. Nati. Acad. Sci. USA 66,651-656. 22. Laemmli, U. K. (1970) Nature (London) 227,680-685. 23. Pert, C. B. & Snyder, S. H. (1974) Science 179, 1011-1013. 24. Simantov, R., Childers, S. R. & Snyder, S. H. (1978) Eur. J. Pharmacol. 47,319-331. 25. Siegel, L. M. & Monty, K. J. (1966) Biochim. Biophys. Acta 112, 346-362. 26. Goldstein, A., Goldstein, J. S. & Cox, B. M. (1975) Life Sci. 17, 1643-1654. 27. Roques, B. P., Garbay-Jaureguiberry, C., Oberlin, R., Anteunis, M. & Lala, A. K. (1976) Nature (London) 262,778-779.
