Vol. 182, No. 2, 1992 January 31, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 722-726
Monoclonal Antibody to GABA Binding Protein, a Possible GABAB Receptor Hiroshi Nakayasu’, Hiroshi Mizutanit, Kazumitu Hanai*, Hiroshi Kimura* and Kinya Kuriyama’* ‘Department of Pharmacology, Kyoto Prefectural University of Medicine, Kyoto 602, Japan *Institute of Molecular Neurobiology, Shiga University of Medical Science, Otsu 52@21, Japan
Received
December
11,
1991
SUMMARY:
A monoclonal antibody has been raised against a partially puritied preparation for the GABAB receptor. The antibody recognized a protein of about 80 kDa in bovine brain synaptic membrane. bnmunoabsorbent agarose beads conjugated with the antibody were able to remove, without visible changes in electrophoresed profiles of total proteins, over 90% of the baclofen suppressive GABA binding activity (designated herein, GABAB receptor binding activity) in the solubilized synaptic membrane fraction. Moreover, the addition of GB-1 antibody directly inhibited the GABA binding activity in the crude synaptic membrane fraction. These results indicate that the monoclonal antibody obtained here recognizes the GABA binding protein, or more specifically a GABAa receptor, 0 1992 Academic Press, Inc.
GABA is known to be one of inhibitory neurotransmitters in a wide variety of vertebrate and invertebrate nervous systems (1,2). Possible changes of GABAergic function have been repeatedly documented in such brain disorders as Huntington’s disease (3,4). GABA receptors are generally classified into two subtypes; GABAA (ionotropic) (5.6) and GABAn (metabotropic) (7,8). Although molecular bases of the GABAA receptor are accumulating rapidly, GABAB receptor(s) is so far neither purified nor cloned. GABAB receptor(s) is believed to play important roles in controlling adenylate cyclase activity (9), phosphatidylinositol turnover (lo), and calcium ion influx (11) via corresponding G protein. Previously, we reported partial purification of the GABAB receptor using
*To whom correspondence should be addressed. Abbreviations; GABA, y-aminobutyric acid; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-lpropanesulfonate; PMSF, Phenymethysulfonyl fluoride; CNBr, Cyanogen bromide. 0006-291X/92 Copyright All rights
$1.50
0 199.2 by Academic Press, of reproduction in any form
Inc. reserved.
722
Vol.
182, No. 2, 1992
a GABAB
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
agonist baclofen as an affinity ligand (12). Subsequently, we have been trying to produce
antibody against the receptor protein aiming for. the application in both cDNA cloning and immunocytochemistry. We now report the preparation and characterization of a monoclonal antibody (GB-1) which recognized a 80 kDa protein, as a possible candidate for GABAB
MATERIALS
receptors.
AND METHODS
Preparation of Monoclonal Antibody. A partially purified GABAB receptor preparation was extracted from about 50 bovine brains according to our previous paper (12). Briefly, a fraction from a baclofen-affinity column (0.5 M KC1 eluate) was concentrated and injected into five Balblc mice at day 1 (20 rig/mouse, with complete adjuvant), at day 29 (10 ng with incomplete adjuvant) and finally at day 43 (20 ng without adjuvant). Titers of antisera were determined by Western blots. Two mice showing high titers were used for cell fusion with P3Ul myeloma cells at day 46. The hybridomas were selected in a HAT medium for 2 weeks. Positive clones were detected by one dimensional Western blot analysesusing the above mentioned receptor preparation. The clones were transferred into 96 well plates for selection by a limited dilution method. Strongly positive clones were then cloned again, and the recloned hybridomas were injected into six other mice (107 cells/ mouse) in order to produce ascitic antibody. Two weeks after the injection, the ascitic fluid was collected and then kept at -80 ‘C until use. The resulting antibody (GB-1) was an IgM type as identified by a mouse-type subisotyping kit (BioRad, Co.) and. by sodium dodecyl sulfate / polyacrylamide gel electrophoresis (SDS-PAGE). Preparation of Antibody Conjugated Beads. The ascitic fluid which contained about 10 mg IgM was concentrated by ammonium sulfate precipitation, and dialyzed against 10 mM borate buffer containing 0.15 M NaCl, pH 8.5. It was then incubated for 8 h with 2.5 ml of CNBr-activated Sepharose (Pharmacia, Co.) in the same buffer at 4 ‘C, washed once with the buffer, then the active groups on the beads were blocked by incubating with 1 M Tris-HCl buffer, pH 8.5. Almost all proteins were adsorbed on the beads asjudged by the elimination of the absorbance at 280 nm in the supematant. The beads were washed with 2 M NaCI, then with 0.1 M glycine-HCl buffer (pH 2.3), and with 10 mM Tris-HCl buffer containing 0.15 M NaCl, pH 7.4. The antibody bound agarose beads were suspendedwith an equal volume of 100% glycerol and kept at -20 ‘C. SDS-PAGE and Immunoblots SDS-PAGE and Western blotting were carried out by the method of (13). Nitrocellulose membranes were blocked by Tween 20. Proteins on the blots were stained by colloidal gold particles of 15nm (14). For immunoblots, the membranes were incubated with the ascitic fluid (1: 10,000 dilution) in 1OmM Tris-HCl, pH 7.4, containing 0.15M NaCl and 0.05% Tween 20, washed 4 times with the same buffer, then incubated for 2h with alkaline phosphatase conjugated secondary antibody. The localization of the immunoreactive protein(s) were visualized using bromochloroindolyl phosphate and nitroblue tetrazolium (15).
RESULTS AND DISCUSSION Specificity of Monoclonal Antibody. First, the specificity of the monoclonal antibody was examined. Gn Western blot analysesusing the crude preparation of bovine synaptic membrane (Fig. l), the antibody recognized only one protein band among over one hundred protein moieties. The molecular weight of the immuno-positive protein was about 80 kDa. A similar protein in both size 723
Vol.
182,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
-94 -67 -43 -30 -21
Fig. L Specificityof Monoclonal Antibody (GB-1). Bovine synapticmembranewas prepared accordingto (20). Lane 1, immunoblot (12% gel) of synapticmembranewithout primary antibody; 2, the sameimmunoblot with GB-I monoclonal antibody; 3, protein stainingof the sameblot by colloidalgold; 4, molecularweight standards.
and immunoreactivity has been detected in our recent study for much extensive purification (to be reported elsewhere). The 61 kDa protein reported earlier (16) might be a partially degraded form of the 80 kDa antigen. Absorption of GABA binding activity by Monocional Antibody GB-I Conjugated Beads.
The supernatant solubilized from freshly prepared extract of bovine synaptic membrane contained a high level of GABA binding activity. Since this binding is specifically antagonized by baclofen, it seemed to occur via GABAB receptor-mediated mechanisms (17). As shown in Fig. 2, the GB-1 conjugated immunoabsorbent cleared the binding activity in the supematant. Moreover, no immunore.active protein was observed in the absorbed supematant, while the protein protile on SDSPAGE did not seem to alter (Fig. 2). It was strongly suggested therefore that the activity removed by the GB-1 bearing beads was a small amount of protein with high GABA binding capacity having the same specificity as the GABAB receptors.. Inhibition of GABAe binding activity by monoclonal antibody GB-1. When GB-1 was added
to the crude synaptic membrane fraction, specific GABA binding activity was decreased dosedependently (Fig. 3). This inhibition appears to be caused by a direct interaction of GB-1 with the GABA binding protein, becausepurified GB-1 IgM was used and because control IgM did not affect the binding.
Because such an inhibition by the GB-I IgM occurred without the addition of a
detergent, it is unlikely that the recognition site of this monoclonal antibody resides on the intracellular domain of GABAB receptor where it interacts with GTP binding protein (18). Although the mechanism of the inhibition is not clear at present, competitive inhibition seemsunlikely. The site of immunorecognition with GB-1 may be close to the GABA binding site near enough to distort by physical interference. 724
Vol.
182, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
‘? 0
0 20
80
26 E aa
280
Antibody ConjugatedBeads Added (~1) 1
2345678
Es?4 3 .e
9
946743-
02
302%
-e+
03 "0
" 0
5
GB-1 IgM Control IgM I 20
I 40
Monoclonal Antibody Added (~1)
Fig. 2. Depletion of GABAB binding activity from a solubilized preparation of bovine synaptic membrane. The synaptic membrane fraction was solubilized in 50 mM Tris-HCI, pH 7.4, containing 2 % CHAPS, 1 mM PMSF, 0.02 mM leupeptin and 0.02 mlvl antipain. GB-1 monoclonal antibody conjugated beads were washed with the same buffer. The beads (lanes 2 and 6,0 pl; 3 and 7,20 pl; 4 and 8, 80 pl; 5 and 9, 280 pl) were added to solubilized synaptic membrane, and incubated overnight at 4 ‘C. After centrifugation, the GABAB receptor binding activity was measured using each supematant(upper panel). Protein staining (9% gel, lanes 2-5) and GB-1 antibody staining (lanes 6-9) using each supematant (20 pl for protein staining and 180 pl for immunoblot) were performed as described in text. Fin. 3. Inhibition of GABAB receptor binding activity by GB-1 monoclonal antibody. Synaptic membrane fraction was incubated overnight at 4 ‘C with the purified GB-1 IgM (closed circle) or with the equal amount of control IgM from non-immunized mice (open circle). GABAB receptor binding activity was measured as described in our previous paper (12).
It is noteworthy that the GB-1 antibody removed nearly 90% of GABA binding activity of the same specificity as GABAB receptors. The result suggests that the baclofen-suppressible GABA binding activity is exclusively due to this immunoreactive protein. This suggests the existence of only one subtype of GABAB receptors. Although possible subtypes cannot be denied, the wellknown high and low affinities in GABAB receptor (19), for example, may also be explained by such a monopolistic GABAB receptor as a result of interaction with various GTP binding proteins or other modifying proteins. 125
Vol.
182,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
In conclusion, the GB-1 monoclonal antibody specifically recognize a GABA binding protein of about 80 kDa of the same binding specificity as GABAB receptors. Evidently many studies are urgently required using GB-1 antibody including immunohistochemical localization, cDNA cloning and sequencing of the immunopositive protein. Further confirmation by receptor expression on eukaryotic cell membrane should allow a final identification.
Acknowledgment. This work was supported, in part, by Grants-in-aid from the Ministry of Education, Science and Culture, Japan.
REFERENCES 1. Kuriyama, K., Haber, B., Sisken, B., and Roberts, E. (1966) Proc. Natl. Acad. Sci. USA., 55, 846-852. 2. Bormann, J. (1988) Trends Neurosci., 11, 112-l 16. 3. Kanazawa, I., Kimura, M., Murata, M., Tanaka, Y., and Cho, F. (1990) Brain, 113,509535. 4. Yoshida, M., Rabin, A., and Anderson, M. (1972) Exp. Brain Res., 15.333-347. 5. Olsen, R.W. (1981), J. Neurochem., 37, 1-13. 6. Hirouchi, M., Taguchi, J.-I., Ueha, T., and Kuriyama, K. (1987) Biochem. Biophys. Res. Commun., 146, 1471-1477, 7. Hill, D.R. and Bowery, N.G. (1981) Nature, 290, 149-152. 8. Ohmori, Y, Hirouchi, M., Taguchi, J.-I., and Kuriyama, K.(1990) J. Neurochem., 54, 80-85. 9. Wojcik, W.J., and Neff, N.H. (1984) Mol. Pharmacol., 25, 24-28. 10. Crawford, M.L.A., and Young, J.M. (1988) J. Neurochem., 51, 1441-1447. 11. Holz, G.G., Rane. S.G., and Dunlap, K. (1986) Nature, 319, 670-672. 12. Ohmori, Y., and Kuriyama, K. (1990) Biochem. Biophys. Res. Commun., 172,22-27. 13. Nakayasu, H. and Berezney, R. (1991) Proc. Nat]. Acad. Sci. USA., 88, 10312-10316. 14. Mammal of Enprotech (MA., USA) for ISS Protein Gold. 15. Blake, M.S., Johnston, K.H.. Russel-Johnes, G.J., and Gotschlich, E.C. (1984) Anal. Biochem., 136, 175-179. 16. Kuriyama, K., Mizutani, H., Ohmori, Y., and Nakayasu, H. (1991) Alfred Benzon Sympsium, 32, 14-15. 17. Bowery, N.G., Hill, D.R., and Hudson, A.L. (1983) Br. J. Pharmacol., 78, 191-206. 18. Asano, T., Ui, M., and Ogasawara, N. (1985) J. Biol. Chem., 260, 12653-12658. 19. Dutar, P., and Nicoll, R.A. (1988) Neuron, 1,585-591. 20. Zukin, S.R., Young, A.B., and Synder, S.H. (1974) Proc. Natl. Acad. Sci., USA. 71,48024807.
726
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 722-726
Monoclonal Antibody to GABA Binding Protein, a Possible GABAB Receptor Hiroshi Nakayasu’, Hiroshi Mizutanit, Kazumitu Hanai*, Hiroshi Kimura* and Kinya Kuriyama’* ‘Department of Pharmacology, Kyoto Prefectural University of Medicine, Kyoto 602, Japan *Institute of Molecular Neurobiology, Shiga University of Medical Science, Otsu 52@21, Japan
Received
December
11,
1991
SUMMARY:
A monoclonal antibody has been raised against a partially puritied preparation for the GABAB receptor. The antibody recognized a protein of about 80 kDa in bovine brain synaptic membrane. bnmunoabsorbent agarose beads conjugated with the antibody were able to remove, without visible changes in electrophoresed profiles of total proteins, over 90% of the baclofen suppressive GABA binding activity (designated herein, GABAB receptor binding activity) in the solubilized synaptic membrane fraction. Moreover, the addition of GB-1 antibody directly inhibited the GABA binding activity in the crude synaptic membrane fraction. These results indicate that the monoclonal antibody obtained here recognizes the GABA binding protein, or more specifically a GABAa receptor, 0 1992 Academic Press, Inc.
GABA is known to be one of inhibitory neurotransmitters in a wide variety of vertebrate and invertebrate nervous systems (1,2). Possible changes of GABAergic function have been repeatedly documented in such brain disorders as Huntington’s disease (3,4). GABA receptors are generally classified into two subtypes; GABAA (ionotropic) (5.6) and GABAn (metabotropic) (7,8). Although molecular bases of the GABAA receptor are accumulating rapidly, GABAB receptor(s) is so far neither purified nor cloned. GABAB receptor(s) is believed to play important roles in controlling adenylate cyclase activity (9), phosphatidylinositol turnover (lo), and calcium ion influx (11) via corresponding G protein. Previously, we reported partial purification of the GABAB receptor using
*To whom correspondence should be addressed. Abbreviations; GABA, y-aminobutyric acid; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-lpropanesulfonate; PMSF, Phenymethysulfonyl fluoride; CNBr, Cyanogen bromide. 0006-291X/92 Copyright All rights
$1.50
0 199.2 by Academic Press, of reproduction in any form
Inc. reserved.
722
Vol.
182, No. 2, 1992
a GABAB
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
agonist baclofen as an affinity ligand (12). Subsequently, we have been trying to produce
antibody against the receptor protein aiming for. the application in both cDNA cloning and immunocytochemistry. We now report the preparation and characterization of a monoclonal antibody (GB-1) which recognized a 80 kDa protein, as a possible candidate for GABAB
MATERIALS
receptors.
AND METHODS
Preparation of Monoclonal Antibody. A partially purified GABAB receptor preparation was extracted from about 50 bovine brains according to our previous paper (12). Briefly, a fraction from a baclofen-affinity column (0.5 M KC1 eluate) was concentrated and injected into five Balblc mice at day 1 (20 rig/mouse, with complete adjuvant), at day 29 (10 ng with incomplete adjuvant) and finally at day 43 (20 ng without adjuvant). Titers of antisera were determined by Western blots. Two mice showing high titers were used for cell fusion with P3Ul myeloma cells at day 46. The hybridomas were selected in a HAT medium for 2 weeks. Positive clones were detected by one dimensional Western blot analysesusing the above mentioned receptor preparation. The clones were transferred into 96 well plates for selection by a limited dilution method. Strongly positive clones were then cloned again, and the recloned hybridomas were injected into six other mice (107 cells/ mouse) in order to produce ascitic antibody. Two weeks after the injection, the ascitic fluid was collected and then kept at -80 ‘C until use. The resulting antibody (GB-1) was an IgM type as identified by a mouse-type subisotyping kit (BioRad, Co.) and. by sodium dodecyl sulfate / polyacrylamide gel electrophoresis (SDS-PAGE). Preparation of Antibody Conjugated Beads. The ascitic fluid which contained about 10 mg IgM was concentrated by ammonium sulfate precipitation, and dialyzed against 10 mM borate buffer containing 0.15 M NaCl, pH 8.5. It was then incubated for 8 h with 2.5 ml of CNBr-activated Sepharose (Pharmacia, Co.) in the same buffer at 4 ‘C, washed once with the buffer, then the active groups on the beads were blocked by incubating with 1 M Tris-HCl buffer, pH 8.5. Almost all proteins were adsorbed on the beads asjudged by the elimination of the absorbance at 280 nm in the supematant. The beads were washed with 2 M NaCI, then with 0.1 M glycine-HCl buffer (pH 2.3), and with 10 mM Tris-HCl buffer containing 0.15 M NaCl, pH 7.4. The antibody bound agarose beads were suspendedwith an equal volume of 100% glycerol and kept at -20 ‘C. SDS-PAGE and Immunoblots SDS-PAGE and Western blotting were carried out by the method of (13). Nitrocellulose membranes were blocked by Tween 20. Proteins on the blots were stained by colloidal gold particles of 15nm (14). For immunoblots, the membranes were incubated with the ascitic fluid (1: 10,000 dilution) in 1OmM Tris-HCl, pH 7.4, containing 0.15M NaCl and 0.05% Tween 20, washed 4 times with the same buffer, then incubated for 2h with alkaline phosphatase conjugated secondary antibody. The localization of the immunoreactive protein(s) were visualized using bromochloroindolyl phosphate and nitroblue tetrazolium (15).
RESULTS AND DISCUSSION Specificity of Monoclonal Antibody. First, the specificity of the monoclonal antibody was examined. Gn Western blot analysesusing the crude preparation of bovine synaptic membrane (Fig. l), the antibody recognized only one protein band among over one hundred protein moieties. The molecular weight of the immuno-positive protein was about 80 kDa. A similar protein in both size 723
Vol.
182,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
-94 -67 -43 -30 -21
Fig. L Specificityof Monoclonal Antibody (GB-1). Bovine synapticmembranewas prepared accordingto (20). Lane 1, immunoblot (12% gel) of synapticmembranewithout primary antibody; 2, the sameimmunoblot with GB-I monoclonal antibody; 3, protein stainingof the sameblot by colloidalgold; 4, molecularweight standards.
and immunoreactivity has been detected in our recent study for much extensive purification (to be reported elsewhere). The 61 kDa protein reported earlier (16) might be a partially degraded form of the 80 kDa antigen. Absorption of GABA binding activity by Monocional Antibody GB-I Conjugated Beads.
The supernatant solubilized from freshly prepared extract of bovine synaptic membrane contained a high level of GABA binding activity. Since this binding is specifically antagonized by baclofen, it seemed to occur via GABAB receptor-mediated mechanisms (17). As shown in Fig. 2, the GB-1 conjugated immunoabsorbent cleared the binding activity in the supematant. Moreover, no immunore.active protein was observed in the absorbed supematant, while the protein protile on SDSPAGE did not seem to alter (Fig. 2). It was strongly suggested therefore that the activity removed by the GB-1 bearing beads was a small amount of protein with high GABA binding capacity having the same specificity as the GABAB receptors.. Inhibition of GABAe binding activity by monoclonal antibody GB-1. When GB-1 was added
to the crude synaptic membrane fraction, specific GABA binding activity was decreased dosedependently (Fig. 3). This inhibition appears to be caused by a direct interaction of GB-1 with the GABA binding protein, becausepurified GB-1 IgM was used and because control IgM did not affect the binding.
Because such an inhibition by the GB-I IgM occurred without the addition of a
detergent, it is unlikely that the recognition site of this monoclonal antibody resides on the intracellular domain of GABAB receptor where it interacts with GTP binding protein (18). Although the mechanism of the inhibition is not clear at present, competitive inhibition seemsunlikely. The site of immunorecognition with GB-1 may be close to the GABA binding site near enough to distort by physical interference. 724
Vol.
182, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
‘? 0
0 20
80
26 E aa
280
Antibody ConjugatedBeads Added (~1) 1
2345678
Es?4 3 .e
9
946743-
02
302%
-e+
03 "0
" 0
5
GB-1 IgM Control IgM I 20
I 40
Monoclonal Antibody Added (~1)
Fig. 2. Depletion of GABAB binding activity from a solubilized preparation of bovine synaptic membrane. The synaptic membrane fraction was solubilized in 50 mM Tris-HCI, pH 7.4, containing 2 % CHAPS, 1 mM PMSF, 0.02 mM leupeptin and 0.02 mlvl antipain. GB-1 monoclonal antibody conjugated beads were washed with the same buffer. The beads (lanes 2 and 6,0 pl; 3 and 7,20 pl; 4 and 8, 80 pl; 5 and 9, 280 pl) were added to solubilized synaptic membrane, and incubated overnight at 4 ‘C. After centrifugation, the GABAB receptor binding activity was measured using each supematant(upper panel). Protein staining (9% gel, lanes 2-5) and GB-1 antibody staining (lanes 6-9) using each supematant (20 pl for protein staining and 180 pl for immunoblot) were performed as described in text. Fin. 3. Inhibition of GABAB receptor binding activity by GB-1 monoclonal antibody. Synaptic membrane fraction was incubated overnight at 4 ‘C with the purified GB-1 IgM (closed circle) or with the equal amount of control IgM from non-immunized mice (open circle). GABAB receptor binding activity was measured as described in our previous paper (12).
It is noteworthy that the GB-1 antibody removed nearly 90% of GABA binding activity of the same specificity as GABAB receptors. The result suggests that the baclofen-suppressible GABA binding activity is exclusively due to this immunoreactive protein. This suggests the existence of only one subtype of GABAB receptors. Although possible subtypes cannot be denied, the wellknown high and low affinities in GABAB receptor (19), for example, may also be explained by such a monopolistic GABAB receptor as a result of interaction with various GTP binding proteins or other modifying proteins. 125
Vol.
182,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
In conclusion, the GB-1 monoclonal antibody specifically recognize a GABA binding protein of about 80 kDa of the same binding specificity as GABAB receptors. Evidently many studies are urgently required using GB-1 antibody including immunohistochemical localization, cDNA cloning and sequencing of the immunopositive protein. Further confirmation by receptor expression on eukaryotic cell membrane should allow a final identification.
Acknowledgment. This work was supported, in part, by Grants-in-aid from the Ministry of Education, Science and Culture, Japan.
REFERENCES 1. Kuriyama, K., Haber, B., Sisken, B., and Roberts, E. (1966) Proc. Natl. Acad. Sci. USA., 55, 846-852. 2. Bormann, J. (1988) Trends Neurosci., 11, 112-l 16. 3. Kanazawa, I., Kimura, M., Murata, M., Tanaka, Y., and Cho, F. (1990) Brain, 113,509535. 4. Yoshida, M., Rabin, A., and Anderson, M. (1972) Exp. Brain Res., 15.333-347. 5. Olsen, R.W. (1981), J. Neurochem., 37, 1-13. 6. Hirouchi, M., Taguchi, J.-I., Ueha, T., and Kuriyama, K. (1987) Biochem. Biophys. Res. Commun., 146, 1471-1477, 7. Hill, D.R. and Bowery, N.G. (1981) Nature, 290, 149-152. 8. Ohmori, Y, Hirouchi, M., Taguchi, J.-I., and Kuriyama, K.(1990) J. Neurochem., 54, 80-85. 9. Wojcik, W.J., and Neff, N.H. (1984) Mol. Pharmacol., 25, 24-28. 10. Crawford, M.L.A., and Young, J.M. (1988) J. Neurochem., 51, 1441-1447. 11. Holz, G.G., Rane. S.G., and Dunlap, K. (1986) Nature, 319, 670-672. 12. Ohmori, Y., and Kuriyama, K. (1990) Biochem. Biophys. Res. Commun., 172,22-27. 13. Nakayasu, H. and Berezney, R. (1991) Proc. Nat]. Acad. Sci. USA., 88, 10312-10316. 14. Mammal of Enprotech (MA., USA) for ISS Protein Gold. 15. Blake, M.S., Johnston, K.H.. Russel-Johnes, G.J., and Gotschlich, E.C. (1984) Anal. Biochem., 136, 175-179. 16. Kuriyama, K., Mizutani, H., Ohmori, Y., and Nakayasu, H. (1991) Alfred Benzon Sympsium, 32, 14-15. 17. Bowery, N.G., Hill, D.R., and Hudson, A.L. (1983) Br. J. Pharmacol., 78, 191-206. 18. Asano, T., Ui, M., and Ogasawara, N. (1985) J. Biol. Chem., 260, 12653-12658. 19. Dutar, P., and Nicoll, R.A. (1988) Neuron, 1,585-591. 20. Zukin, S.R., Young, A.B., and Synder, S.H. (1974) Proc. Natl. Acad. Sci., USA. 71,48024807.
726
