Vol. 178, No. 3, 1991 August 15, 1991
AFFINITY
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1147-1152
LABELING OF MELATONIN
BINDING SITES IN THE HAMSTER BRAIN
Yossi Anis and Nava Zisapel” Department of Biochemistry,The George S. Wise Faculty of Life sciences,Tel Aviv University, Tel Aviv 69978,1srael Received
June
27,
1991
SUMMARY: N-Bromoacetyl-2-iodo-5methoxytryptamine (BIM), a novel derivative of the biologically active melatonin analog, 2-iodomelatonin,was used to identify melatonin binding proteins in synaptosomesfrom Syrian hamsterbrain. Incubation of the synaptosomes with BIM resultedin a concentration dependent,irreversibleinhibition of 2-1251-iodomelatonin binding. The radioactive form of BIM, N-Bromoacetyl-Z ‘251-iodo-5-methoxytryptamine(‘251-BIM), became covalently attached to three proteins in the synaptosomes, in a concentration dependentmanner. These proteins had apparent molecular weight values of 92, 55 and 45 kilodaltons. The incorporation of ‘251-BIM into all three proteins was inhibited by BIM> 2-iodomelatonin> melatonin whereasthe melatonin antagonistN-(1,4 dinitrophenyl)-5methoxytryptamine (MG23) selectively inhibited the labeling of the 45 kDa protein. These resultsindicate that the 92, 55 and 45 KDa polypeptides are melatonin binding proteins. o 1991 press,I~=. A~~SIU
In mammalsthat usechangesin the photoperiod to time their reproduction and thermoregulation, the phase and duration of the nocturnal production of melatonin by the pineal gland are of critical importance (reviewed in 1,2). A number of studies implicated the brain, especiallythe medial preoptic and supraretrochiasmaticareasas the main sites of melatonin’sneuroendocrine activities (3). Melatonin binding sites have been characterized and quantified in membraneand synaptosomalpreparationsand slicesfrom the Syrian, Djungarian and Syberian hamsterbrain by useof 2-‘251-iodomelatonin ( ‘251-melatonin),a potent melatoninanalog(4-8). Thesesitesexhibited low (Kd ca 80 nM), intermediate (Kd ca 3 nM) and high affinity (Kd 20-40 PM) for 1251melatonin. The apparent molecular massesof melatonin receptors in the hamsterhypothalamus and chick retina have been determined by target size analysisto be 30 and 44 kilodaltons, respectively (9), but the molecular structure of these sites has not been elucidated. We have developed (to be describedelsewhere) a novel, chemically reactive melatonin derivative, NBromoacetyl-2-[‘251]-iodo-5-methoxytryptamine (‘251-BIM). This particular ligand hasbeen chosen becauseof its structural similarity to 1251-melatonin, and of the very high speciBcradioactivity which could be obtained with ‘251-labeledligands. We report here on the identification of melatonin binding proteins in the hamsterbrain using this compoundas an affinity label.
* To whom correspondenceshould be addressed. 0006-291X/91
1147
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Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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2-[‘251]-iodomelatonin ( ‘251-melatonin) and unlabeled 2-iodomelatonin were prepared by iodination of melatonin (Sigma, St Louis, MO) as described (10.11). 2-[‘251]-iodomelatonin was diluted radioisotopically with 100 fold excess of unlabeled 2-iodomelatonin to yield a specific radioactivity of 20 Ci/mmol. ML-23 was prepared as described (12). The preparation of BIM and its radioactive form, N-Bromoacetyl-Z t1251]-iodo-5-methoxytryptamine (12’I-BIM) will be described elsewhere. Male Syrian hamsters (Mesocricetus auratus; animal facilities, Tel Aviv University) were maintained on a daily 14 h light:10 h darkness schedule (lights-on 05 h; cool white fluorescent illumination) at 24+2OC. The animals (2 months old), were decapitated between 18-19 h and their brains were rapidly removed and suspended in 10 ml/g ice cold 0.32 M sucrose. Crude synaptosomal pellets were prepared as described (6) and suspended in 2 volumes of 50 mM TrisHCl buffer, pH 7.4 containing 5 mM CaC$. The following experiments were performed: A) Aliquots of the synaptosomal preparations (0.4 mg protein/ml; 13) were incubated at 37’~ with 10-14-10-3 M BIM, for 30 min, in the presence of 50 nM 1251-melatonin in the presence or absence of 50 PM melatonin. Membranes were then collected by vacuum filtration on GFK filters as described (6) and the radioactivity determined. Specific binding was defined as that displaced by 50 /.LM melatonin. B) Aliquots of the synaptosomal preparations (0.4 mg protein/ml; 13) were incubated with 10-‘4-10-3 M BIM, for 30 min at 37’~. Membranes were then collected by centrifugation (lOOOOg, 10 min), and washed extensively by 4 repeated cycles of resuspension in the Tris buffer followed by centrifugation. The membranes were then resuspended in the Tris buffer and the reversible equilibrium binding of ‘251-melatonin was assessed: aliquots of the membranes were incubated in triplicates with 50 nM ‘251-melatonin at 37OC for 30 min in the absence or presence of unlabeled melatonin (50 PM). Membranes were then collected by vacuum filtration on GF/C filters as described [2] and the radioactivity determined. Specific binding was defined as that displaced b 50 PM melatonin. C) Aliquots of the synaptosomal preparations were incubated with 4 nM ’ Y51-BIM (2100 Ci/mmol) in the absence or presence of various indoles (0-10m3 M) for O-30 min at 37OC. The reaction was terminated by the addition of sodium-dodecyl sulfate (SDS) sample buffer (36 mM Tris-HCL, pH 6.8, 3% wt/vol SDS, 5% v/v 2mercaptoethanol and 10% v/v glycerol) and subjected to electrophoresis on 7.5% polyacrylamide gels (14). The gels were subsequently dried and subjected to autoradiography. RESULTS AND DISCUSSION The concentration dependency of the BIM-mediated decrease in ‘251-melatonin binding is shown in Fig 1. Incubation of the synaptosomes with various BIM concentrations during the equilibration
Fipure 1. Concentration dependency of the inhibition by BIM of speeitic 1251-melatonin binding. A) Synaptosomes were treated with BIM in the presence of ‘251-melatonin (50 nM) and in the absence or presence of melatonin (50 PM). B) Synaptosomes were treated with BIM, washed extensively and then incubated with ‘251-melatonin (50 nM) in the absence or presence of melatonin (50 PM). Specific ‘251-melatonin bound was defined as that displaced by 50 PM melatonin. 1148
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Fipure 2. Concentration dependency of the incorporation of “51-BIM into synaptosomal proteins. Synaptosomes were treated with 0.5 (a), 1 (b), 2 (c) and 4 (d) nM ‘251-BIM for 30 min. Right panel: Autoradiograms of the els. The 90, 55 and 45 kDa labeled proteins are depicted. Left panel: The incorporation of ’ f ‘I-BIM into the 90 (o), 45 (0) and 55 (a) kDa proteins was quantified by densitometry.
with the reversible ligand ‘251-melatonin led to a dose dependent decrease in the amount of 1251melatonin bound: about 20% of ‘251-melatonin binding was inhibited by l-10 nM BIM, and further elevation of BIM concentration to 10pM completely inhibited specific ‘251-melatonin binding. These data indicated competition between BIM and ‘251-melatonin on synaptosomal melatonin binding sites. Treatment of the synaptosomes with various BIM concentrations for 30 min followed by extensive washing of the membranes prior to the equilibration with
1251-
melatonin, also resulted in a concentration dependent decrease in ‘251-melatonin binding, indicating irreversible
inhibition of the binding sites. The percentage of irreversible inhibition of lz51-
melatonin binding increased with increasing BIM concentration, approaching a plateau of ca 10% inhibition between 5 and 100 nM. Further elevation of BIM concentration between 0.1-10 PM resulted in a further concentration-dependent concentration
loss of ‘251-melatonin binding capacity. The biphasic
dependency indicates that BIM forms two types of reversible complexes with
melatonin binding sites, prior to the covalent modification: a high and a low affinity type, one accounting for lo-20% and the other for SO-90% of the synaptosomal ‘2511-melatonin binding sites, respectively. We have concentrated on analysis of the high affinity type. Following incubation of the hamster brain synaptosomes with 0.1-4 nM ‘251-BIM for 30 min, the synaptosomal polypeptides
were separated by SDS polyacrylamide gel electrophoresis
and the
radiolabeled proteins were detected by autoradiography (Fig 2). Only three polypeptides repeatedly incorporated the label, with apparent molecular masses of 92, 55 and 4.5 kilodaltons &Da). An additional labeled polypeptide with apparent molecular weight of 37 kDa was not always present. Preliminary peptide-mapping studies indicated that the three proteins were distinct rather than degradation products of the 92 kDa protein. The incorporation of the label into the three proteins increased greatly at low and slightly at higher “‘I-BIM
concentrations approaching a
plateau above 4 nM (Fig 2). These data suggest a positive correlation between the incorporation 1149
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Time, min
Fieure 3. Kinetics of the incorporation of ‘251-BIM (4 nM) into synaptosomal proteins. Synaptosomes were treated with S nM ‘251-B1M for 1 (a), 2 (b), 5 (c), 10 (d) and 15 (e) min. Right panel: Autoradiograms of the gels. The 90, 55 and 45 kDa labeled proteins are depicted. Left panel: The amount of 1251-BIMincorporated into the 90 (0) and 45 (0) kDa proteins was quantified by slicing the gels and counting. Figure 4. Incorporation of ‘251-BIM (4 nM) into synaptosomal proteins. Left panel: in the absence (a) and presence of 10 PM of ML-23 (b), melatonin (c), 2-iodomelatonin (d), and BIM (e). Reight panel: in the absence (n) or presence of various concentrations of ML-23 or 2iodomelatonin (2-IM). The 90, 55 and 45 kDa labeled proteins are depicted. of ‘251-BIM into the 92, 55 and 45 kDa synaptosomal proteins, and the inactivation of a particular class of ‘251-melatonin binding sites which forms a high affinity complex with BIM. The time course of incorporation
of 12’I-BIM
(4 nM) was followed by slicing the gels and
counting the amount of radioactivity incorporated into the specific synaptosomal protein bands (Fig 3). The rate of incorporation was found to be similar for all three proteins and to follow an apparent first order kinetics (k=0.17 min“). The ability of various indole compounds to protect against the incorporation of 12’I-BIM (4 nM) into the 92, 55 and 45 kDa proteins during 20 min incubation periods was investigated (Fig 4). Unlabeled BIM, 2-iodomelatonin and melatonin (10 nM-10 PM) inhibited 12’I-BIM labeling of all three proteins in a dose-dependent manner. 2-iodomelatonin was more potent than melatonin, which suits well the order of potency of these ligands to inhibit reversible ‘251-melatonin binding in hamster brain synaptosomes [7]. Interestingly, ML-23, previously found to negate melatoninmediated responses in the rat both in vivo and in vitro (12) and to differentially inhibit some melatonin responses in the female and male hamsters (15, 16) selectively and potently prevented the incorporation of 12’I-BIM into the 45 kDa protein. The molecular mass of the 45 kDa melatonin binding protein is compatible with the value of ca 44 kDa reported for melatonin receptors in the chick retina, obtained from irradiation inactivation studies (9). These sites have been classified as ‘high affinity
12’I-melatonin binding sites (17).
In hamster synaptosomes, the ‘low affinity’ sites are much more abundant than the ‘high affinity’ ‘*‘I-melatonin
sites (4-8). As shown here, the sites which become inactivated by nanomolar
concentrations of BIM are a minor fraction of the ‘251-melatonin binding sites in the hamster synaptosomes. Taken together, these observations suggest that the 45 kDa protein is related to the ‘high affinity’ type of ‘251-melatonin binding sites. This could not be directly assessed since 1150
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in the hamster synaptosomes the ‘high affinity ’ ‘251-melatonin binding could not be measured separately from the ‘low afftnity’ sites. Yet, ML-23 which was very potent in protecting the 45 kDa protein against alkylation by 12’I-BIM, has been found to be more potent than melatonin in displacing ‘251-melatonin from the ‘low affinity’ sites in rat brain synaptosomes (14) but less potent than melatonin in displacing ‘251-melatonin from the ‘high affinity sites in the sheep pars tuberalis (18). It has been shown that the extent of obstruction of a protein by a protective ligand against the attacking reagent is not always related to its equilibrium association constant with the protein (19) and ML-23 might thus protect the ‘high affinity ’ ‘251-melatonin binding sites more efficiently than melatonin despite having a lower affinity toward the melatonin binding site. The value reported for the hamster hypothalamus using the irradiation inactivation technique, i.e. 30 kDa (9) did not correspond to any of the polypeptides which became consistently labeled by nanomolar concentrations of ‘251-BIM. A simple explanation is, that a 30 kDa protein is related to the inactivation of a type of sites which accounts for about 8090% of the “51-melatonin binding in the hamster brain synaptosomes and could be inactivated by high (over 1 PM) BIM concentrations
(Fig 1). The nature of the proteins associated with this type of sites was not
investigated in the present study. It remains to be determined if the 92, 55 and 45 kDa polypeptides represent subunits of a melatonin receptor or distinct receptor subtypes. The results demonstrate the potency of BIM as a selective affinity label for melatonin binding proteins according to the following criteria: 1) formation of reversible complexes with the sites; 2) specific
protection against labeling by BIM, melatonin,
2-iodomelatonin and ML-23.
3) exclusive labeling of proteins which also recognize melatonin receptor ligands.
Acknowledgment: This work was supported in part by the Israel Academy of Sciences and Humanities, Basic Research Foundation.
REFERENCES 1. Reiter, R.j. (1981) Am. J. Anat. 13, 287-313. 2. Heldmaier, G., Boeckler, H, Buchberger, A, Klaus, S., Puchalski, W., Steinlechner, S. and Weisinger, H. (1986) in: living in the coldPhysiological and biochemical adaptations (H.C. Heller et al. eds.) elsevier Publishing Co. Inc. pp 361-372. 3. Glass J.D. (1988) Pin. Res. Rev. 6, 219-259. 4. Duncan, M.J., Takahashi, J.S. and Dubocovich, M.L. (1988) Endocrinology 122, X25-1830. 5. Weaver, D.R., Namboodiri, M.A.A. and Reppert, SM., (1988) FEBS Lett. 228, 123-127. 6 Anis, Y., Nir, I. and Zisapel, N. (1989) Molec. Cell. Endocrinol. 67, 121-129. 7. Anis Y. and Zisapel, N. (1991) Molec Cell. Endocrinol. 76, 23-34. 8. Weaver D.R. Weaver, Provencio, I., Carbon, L.L. and Reppert S.M. (1991) Endocrinology 128, 1086-1092. 9. Pickering, D.S., Niles, L.P. and Jung, C.Y. (1990) Neurosci. Res. Commun. 6, 11-18. 10. Vakkuri, O., Lamsa, E., Rahkamaa, E., Routsalainen, H. and Leppaluoto, J. (1984) Analyt. Biochem. 142, 284-289. (Copenhagen) 11. Vakkuri, O., Leppaluoto, J. and Vuolteenaho, 0. (1984) Acta Endocrinol. 106, 152-157. 12. Laudon, M., Yaron, Z. and Zisapel, N. (1988) J. Endocr. 116, 43-53. 1151
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13. Markwell, M.A.K., Haas, SM., Bieber, L.C., Talbert, N.E. (1978) Analyt. Biochem. 87, 206-210. 14. Laemmli,U.K. (1970) Nature (London) 227, 680685. 15. Nordio, M., Vaughan, M.K, Zisapel, N., Migliaccio, S., van Jaarsveld,A. and Reiter, R.J. (1989) Proc. Sot. Fixp. Biol. Med. 37, 321-325. 16. Buzzell, G-R., Menendez-Pelaez,Troiani, M.E., McNeill, M.E. and Reiter, R.J., (1990) J. Pineal Res. 8, 229-235. 17. Dubocovich, M., Shankar, G. and Mickel, M. (1987) Eur. J. Pharmacol.162, 289-299. 18. Morgan, P.J., Williams, L.M., Davidson, G., Lawson, W., and Howell, H.E. (1989) J. Neuroendocrinol. 1, l-4. 19. Singer, S.J. (1967) Adv. Protein Chem. 22, l-20.
1152
AFFINITY
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1147-1152
LABELING OF MELATONIN
BINDING SITES IN THE HAMSTER BRAIN
Yossi Anis and Nava Zisapel” Department of Biochemistry,The George S. Wise Faculty of Life sciences,Tel Aviv University, Tel Aviv 69978,1srael Received
June
27,
1991
SUMMARY: N-Bromoacetyl-2-iodo-5methoxytryptamine (BIM), a novel derivative of the biologically active melatonin analog, 2-iodomelatonin,was used to identify melatonin binding proteins in synaptosomesfrom Syrian hamsterbrain. Incubation of the synaptosomes with BIM resultedin a concentration dependent,irreversibleinhibition of 2-1251-iodomelatonin binding. The radioactive form of BIM, N-Bromoacetyl-Z ‘251-iodo-5-methoxytryptamine(‘251-BIM), became covalently attached to three proteins in the synaptosomes, in a concentration dependentmanner. These proteins had apparent molecular weight values of 92, 55 and 45 kilodaltons. The incorporation of ‘251-BIM into all three proteins was inhibited by BIM> 2-iodomelatonin> melatonin whereasthe melatonin antagonistN-(1,4 dinitrophenyl)-5methoxytryptamine (MG23) selectively inhibited the labeling of the 45 kDa protein. These resultsindicate that the 92, 55 and 45 KDa polypeptides are melatonin binding proteins. o 1991 press,I~=. A~~SIU
In mammalsthat usechangesin the photoperiod to time their reproduction and thermoregulation, the phase and duration of the nocturnal production of melatonin by the pineal gland are of critical importance (reviewed in 1,2). A number of studies implicated the brain, especiallythe medial preoptic and supraretrochiasmaticareasas the main sites of melatonin’sneuroendocrine activities (3). Melatonin binding sites have been characterized and quantified in membraneand synaptosomalpreparationsand slicesfrom the Syrian, Djungarian and Syberian hamsterbrain by useof 2-‘251-iodomelatonin ( ‘251-melatonin),a potent melatoninanalog(4-8). Thesesitesexhibited low (Kd ca 80 nM), intermediate (Kd ca 3 nM) and high affinity (Kd 20-40 PM) for 1251melatonin. The apparent molecular massesof melatonin receptors in the hamsterhypothalamus and chick retina have been determined by target size analysisto be 30 and 44 kilodaltons, respectively (9), but the molecular structure of these sites has not been elucidated. We have developed (to be describedelsewhere) a novel, chemically reactive melatonin derivative, NBromoacetyl-2-[‘251]-iodo-5-methoxytryptamine (‘251-BIM). This particular ligand hasbeen chosen becauseof its structural similarity to 1251-melatonin, and of the very high speciBcradioactivity which could be obtained with ‘251-labeledligands. We report here on the identification of melatonin binding proteins in the hamsterbrain using this compoundas an affinity label.
* To whom correspondenceshould be addressed. 0006-291X/91
1147
$1.50
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
178,
No.
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3, 1991
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AND METHODS
2-[‘251]-iodomelatonin ( ‘251-melatonin) and unlabeled 2-iodomelatonin were prepared by iodination of melatonin (Sigma, St Louis, MO) as described (10.11). 2-[‘251]-iodomelatonin was diluted radioisotopically with 100 fold excess of unlabeled 2-iodomelatonin to yield a specific radioactivity of 20 Ci/mmol. ML-23 was prepared as described (12). The preparation of BIM and its radioactive form, N-Bromoacetyl-Z t1251]-iodo-5-methoxytryptamine (12’I-BIM) will be described elsewhere. Male Syrian hamsters (Mesocricetus auratus; animal facilities, Tel Aviv University) were maintained on a daily 14 h light:10 h darkness schedule (lights-on 05 h; cool white fluorescent illumination) at 24+2OC. The animals (2 months old), were decapitated between 18-19 h and their brains were rapidly removed and suspended in 10 ml/g ice cold 0.32 M sucrose. Crude synaptosomal pellets were prepared as described (6) and suspended in 2 volumes of 50 mM TrisHCl buffer, pH 7.4 containing 5 mM CaC$. The following experiments were performed: A) Aliquots of the synaptosomal preparations (0.4 mg protein/ml; 13) were incubated at 37’~ with 10-14-10-3 M BIM, for 30 min, in the presence of 50 nM 1251-melatonin in the presence or absence of 50 PM melatonin. Membranes were then collected by vacuum filtration on GFK filters as described (6) and the radioactivity determined. Specific binding was defined as that displaced by 50 /.LM melatonin. B) Aliquots of the synaptosomal preparations (0.4 mg protein/ml; 13) were incubated with 10-‘4-10-3 M BIM, for 30 min at 37’~. Membranes were then collected by centrifugation (lOOOOg, 10 min), and washed extensively by 4 repeated cycles of resuspension in the Tris buffer followed by centrifugation. The membranes were then resuspended in the Tris buffer and the reversible equilibrium binding of ‘251-melatonin was assessed: aliquots of the membranes were incubated in triplicates with 50 nM ‘251-melatonin at 37OC for 30 min in the absence or presence of unlabeled melatonin (50 PM). Membranes were then collected by vacuum filtration on GF/C filters as described [2] and the radioactivity determined. Specific binding was defined as that displaced b 50 PM melatonin. C) Aliquots of the synaptosomal preparations were incubated with 4 nM ’ Y51-BIM (2100 Ci/mmol) in the absence or presence of various indoles (0-10m3 M) for O-30 min at 37OC. The reaction was terminated by the addition of sodium-dodecyl sulfate (SDS) sample buffer (36 mM Tris-HCL, pH 6.8, 3% wt/vol SDS, 5% v/v 2mercaptoethanol and 10% v/v glycerol) and subjected to electrophoresis on 7.5% polyacrylamide gels (14). The gels were subsequently dried and subjected to autoradiography. RESULTS AND DISCUSSION The concentration dependency of the BIM-mediated decrease in ‘251-melatonin binding is shown in Fig 1. Incubation of the synaptosomes with various BIM concentrations during the equilibration
Fipure 1. Concentration dependency of the inhibition by BIM of speeitic 1251-melatonin binding. A) Synaptosomes were treated with BIM in the presence of ‘251-melatonin (50 nM) and in the absence or presence of melatonin (50 PM). B) Synaptosomes were treated with BIM, washed extensively and then incubated with ‘251-melatonin (50 nM) in the absence or presence of melatonin (50 PM). Specific ‘251-melatonin bound was defined as that displaced by 50 PM melatonin. 1148
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Fipure 2. Concentration dependency of the incorporation of “51-BIM into synaptosomal proteins. Synaptosomes were treated with 0.5 (a), 1 (b), 2 (c) and 4 (d) nM ‘251-BIM for 30 min. Right panel: Autoradiograms of the els. The 90, 55 and 45 kDa labeled proteins are depicted. Left panel: The incorporation of ’ f ‘I-BIM into the 90 (o), 45 (0) and 55 (a) kDa proteins was quantified by densitometry.
with the reversible ligand ‘251-melatonin led to a dose dependent decrease in the amount of 1251melatonin bound: about 20% of ‘251-melatonin binding was inhibited by l-10 nM BIM, and further elevation of BIM concentration to 10pM completely inhibited specific ‘251-melatonin binding. These data indicated competition between BIM and ‘251-melatonin on synaptosomal melatonin binding sites. Treatment of the synaptosomes with various BIM concentrations for 30 min followed by extensive washing of the membranes prior to the equilibration with
1251-
melatonin, also resulted in a concentration dependent decrease in ‘251-melatonin binding, indicating irreversible
inhibition of the binding sites. The percentage of irreversible inhibition of lz51-
melatonin binding increased with increasing BIM concentration, approaching a plateau of ca 10% inhibition between 5 and 100 nM. Further elevation of BIM concentration between 0.1-10 PM resulted in a further concentration-dependent concentration
loss of ‘251-melatonin binding capacity. The biphasic
dependency indicates that BIM forms two types of reversible complexes with
melatonin binding sites, prior to the covalent modification: a high and a low affinity type, one accounting for lo-20% and the other for SO-90% of the synaptosomal ‘2511-melatonin binding sites, respectively. We have concentrated on analysis of the high affinity type. Following incubation of the hamster brain synaptosomes with 0.1-4 nM ‘251-BIM for 30 min, the synaptosomal polypeptides
were separated by SDS polyacrylamide gel electrophoresis
and the
radiolabeled proteins were detected by autoradiography (Fig 2). Only three polypeptides repeatedly incorporated the label, with apparent molecular masses of 92, 55 and 4.5 kilodaltons &Da). An additional labeled polypeptide with apparent molecular weight of 37 kDa was not always present. Preliminary peptide-mapping studies indicated that the three proteins were distinct rather than degradation products of the 92 kDa protein. The incorporation of the label into the three proteins increased greatly at low and slightly at higher “‘I-BIM
concentrations approaching a
plateau above 4 nM (Fig 2). These data suggest a positive correlation between the incorporation 1149
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Time, min
Fieure 3. Kinetics of the incorporation of ‘251-BIM (4 nM) into synaptosomal proteins. Synaptosomes were treated with S nM ‘251-B1M for 1 (a), 2 (b), 5 (c), 10 (d) and 15 (e) min. Right panel: Autoradiograms of the gels. The 90, 55 and 45 kDa labeled proteins are depicted. Left panel: The amount of 1251-BIMincorporated into the 90 (0) and 45 (0) kDa proteins was quantified by slicing the gels and counting. Figure 4. Incorporation of ‘251-BIM (4 nM) into synaptosomal proteins. Left panel: in the absence (a) and presence of 10 PM of ML-23 (b), melatonin (c), 2-iodomelatonin (d), and BIM (e). Reight panel: in the absence (n) or presence of various concentrations of ML-23 or 2iodomelatonin (2-IM). The 90, 55 and 45 kDa labeled proteins are depicted. of ‘251-BIM into the 92, 55 and 45 kDa synaptosomal proteins, and the inactivation of a particular class of ‘251-melatonin binding sites which forms a high affinity complex with BIM. The time course of incorporation
of 12’I-BIM
(4 nM) was followed by slicing the gels and
counting the amount of radioactivity incorporated into the specific synaptosomal protein bands (Fig 3). The rate of incorporation was found to be similar for all three proteins and to follow an apparent first order kinetics (k=0.17 min“). The ability of various indole compounds to protect against the incorporation of 12’I-BIM (4 nM) into the 92, 55 and 45 kDa proteins during 20 min incubation periods was investigated (Fig 4). Unlabeled BIM, 2-iodomelatonin and melatonin (10 nM-10 PM) inhibited 12’I-BIM labeling of all three proteins in a dose-dependent manner. 2-iodomelatonin was more potent than melatonin, which suits well the order of potency of these ligands to inhibit reversible ‘251-melatonin binding in hamster brain synaptosomes [7]. Interestingly, ML-23, previously found to negate melatoninmediated responses in the rat both in vivo and in vitro (12) and to differentially inhibit some melatonin responses in the female and male hamsters (15, 16) selectively and potently prevented the incorporation of 12’I-BIM into the 45 kDa protein. The molecular mass of the 45 kDa melatonin binding protein is compatible with the value of ca 44 kDa reported for melatonin receptors in the chick retina, obtained from irradiation inactivation studies (9). These sites have been classified as ‘high affinity
12’I-melatonin binding sites (17).
In hamster synaptosomes, the ‘low affinity’ sites are much more abundant than the ‘high affinity’ ‘*‘I-melatonin
sites (4-8). As shown here, the sites which become inactivated by nanomolar
concentrations of BIM are a minor fraction of the ‘251-melatonin binding sites in the hamster synaptosomes. Taken together, these observations suggest that the 45 kDa protein is related to the ‘high affinity’ type of ‘251-melatonin binding sites. This could not be directly assessed since 1150
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1991
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in the hamster synaptosomes the ‘high affinity ’ ‘251-melatonin binding could not be measured separately from the ‘low afftnity’ sites. Yet, ML-23 which was very potent in protecting the 45 kDa protein against alkylation by 12’I-BIM, has been found to be more potent than melatonin in displacing ‘251-melatonin from the ‘low affinity’ sites in rat brain synaptosomes (14) but less potent than melatonin in displacing ‘251-melatonin from the ‘high affinity sites in the sheep pars tuberalis (18). It has been shown that the extent of obstruction of a protein by a protective ligand against the attacking reagent is not always related to its equilibrium association constant with the protein (19) and ML-23 might thus protect the ‘high affinity ’ ‘251-melatonin binding sites more efficiently than melatonin despite having a lower affinity toward the melatonin binding site. The value reported for the hamster hypothalamus using the irradiation inactivation technique, i.e. 30 kDa (9) did not correspond to any of the polypeptides which became consistently labeled by nanomolar concentrations of ‘251-BIM. A simple explanation is, that a 30 kDa protein is related to the inactivation of a type of sites which accounts for about 8090% of the “51-melatonin binding in the hamster brain synaptosomes and could be inactivated by high (over 1 PM) BIM concentrations
(Fig 1). The nature of the proteins associated with this type of sites was not
investigated in the present study. It remains to be determined if the 92, 55 and 45 kDa polypeptides represent subunits of a melatonin receptor or distinct receptor subtypes. The results demonstrate the potency of BIM as a selective affinity label for melatonin binding proteins according to the following criteria: 1) formation of reversible complexes with the sites; 2) specific
protection against labeling by BIM, melatonin,
2-iodomelatonin and ML-23.
3) exclusive labeling of proteins which also recognize melatonin receptor ligands.
Acknowledgment: This work was supported in part by the Israel Academy of Sciences and Humanities, Basic Research Foundation.
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