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Characterization of an antiserum against the bovine glycine receptor
$158 CHARACTERIZATION
OF
AN
ANTISERUM
AGAINST
THE
BOVINE
GLYCINE
RECEPTOR
TAKESHI ~URAKAMI I*, TOSH~UKI ARAKI I*, SATOSH~
H A ~ I H I R A I*, A K I O W A N A ~ A I*, S A D A O SHIOSAKA-, SABURO AIMOTO ~ and MASAYA TOHYAMA-. -Dept. of Anatomy, -Dept of ~euroanatomy, Ins. of Higher Nervous Activity, Osaka University Medical School, Peptide Center, Ins. for Protein Research, Osaka University. Specific polyclonal antibodies to the bovine glycine receptor ( g l y R) w e r e produced and characterized. The glycine receptor was solubilized with Triton X I00 a n d p u r i f i e d using aminostryohnine agarose. Polyclonal antibodies against gly R were raised in r a b b i t s b y i n j e c t i n g a total of 60-80 ug of proteins. The specificity of the serum was established from a variety of tests including immunoprecipitation, immunoaffinity chromatography, dot immunoassay, ELISA tests and Western immunoblottings. Although precipitating reactions were not detected, complexes that were formed between the IgG and the receptor could be detected by protein A-Sepharose, and quantitative immunoprecipitation was observed. The antibodies cross-reacted with the glycine receptors from rat and rabbit brains, but not with the GABA-A receptor. It w a s p o s s i b l e to purify gly R by immunoaffinity chromatography using agarose coupled with IgG from the immunized animal, as w ~ l l as u s i n g a m i n o s t r i c h n i n e agarose. The antiserum blocked the binding o f (~H) s t r y c h n i n e to gly R without changing the dissociation constance (Kd). S p e c i f i c i t y of the antiserum was further indicated by a dot immunoassay a n d E L I S A t e s t s in w h i c h t h e i n t e n s i t y of the reaction product was proportional to the amount of the gly R protein and applied I g G . In t h e W e s t e r n blotting experiments, intense staining was observed on 58K polypeptide among three subunits (48K, 5 8 K a n d 93K) o f g l y R.
APPARENT BINDING ACTIVITY OF [3H]GLUTATHIONE
IN SYNAPTIC MEMBRANES OF THE RAT BRAIN.
KIYOKAZU OGITA and YUKIO YONEDA, Department of Pharmacology, Setsunan University, Hirakta, Osaka 573-01, Japan
Faculty of Pharmaceutical
Sciences,
Reduced (GSH) as well as oxidized (GSSG) forms of glutathione significantly displaced the specific bindlng of [3H]L-glutamlc acid (G]u) to synaptic membranous preparations of the rat brain. This inhlbltion occurred independently of the incubatlon temperature in a concentration-dependent manner at the concentratlon range from 1 to 1,000 ~M. An apparent binding activity of [3H]GSH was detected in the synaptlc membranous preparatlons. Thls apparent binding was displaced in a concentration-dependent fashion not on]y by GSH, but also by GSSG. The apparent binding found in the presence of an excess of GSH was considered to be nonspecifica!ly bound [~H]GSH. The apparent specific blnding occupied more than 70% of the total bidding. The apparent specific binding was about 2 times as high at 30°C as that at 2°C. The apparent blnding increased linearly wlth increasing concentrations of synaptic membranous proteins up to 500 Dg. More than 90% (95.7 ± 3.7%) of the radloactivlty in the incubation medium after incubation at 30°C for 60 mln comlgrated wlth the authentic GSH when analyzed by thin layer chromatography on cellulose-coated plates with peno1:H20 (75:25 at 22 ± 2°C) as a solvent system. No significant radioactivity was detected in the spot corresponding to the authentic GSSG. The apparent binding was saturable wlth increasing concentrations of [3H]GSH over the concentration range from i00 to i0,000 nM. Scatchard analysis of these data revealed that the apparent binding sites conslsted of two independent components at 30°C, such as high affinity sites and low affinity sites, while being comprised of a single hlgh afflnlty constituent at 2°C. The apparent binding was profoundly inhlbited in a concentrationdependent manner by the addition of GSH derivatives without SH-moieties, includlng S-methy]-glutathione and S-hexyl-glutathione. The binding activity was slmllarly inhlblted by y-L-GIu-L-GIu and ~-L-G]u-LGlu, whereas ¥-L-GIu-GIy displaced the apparent binding more potently than y-D-Glu-Gly. No slgnificant a]teratlon of the apparent binding was induced by the pretreatment of synaptlc membranous preparations wlth Trlton X-100 at 0.08-0.4%. These results suggest that the apparent bindlng of [3H]GSH may not be attributable to the disulfide bonding between membranous SH-residues and SH-moieties in the ligand, and may indeed represent the specific binding sites of [3H]GSH in the brain. The functlona] slgnlflcance of g]utathlone may need to be re-evaluated. (Supported by a grant 62771981 from the Mlnlstry of Educatlon, Science and Cu]ture, Japan.)
OF
AN
ANTISERUM
AGAINST
THE
BOVINE
GLYCINE
RECEPTOR
TAKESHI ~URAKAMI I*, TOSH~UKI ARAKI I*, SATOSH~
H A ~ I H I R A I*, A K I O W A N A ~ A I*, S A D A O SHIOSAKA-, SABURO AIMOTO ~ and MASAYA TOHYAMA-. -Dept. of Anatomy, -Dept of ~euroanatomy, Ins. of Higher Nervous Activity, Osaka University Medical School, Peptide Center, Ins. for Protein Research, Osaka University. Specific polyclonal antibodies to the bovine glycine receptor ( g l y R) w e r e produced and characterized. The glycine receptor was solubilized with Triton X I00 a n d p u r i f i e d using aminostryohnine agarose. Polyclonal antibodies against gly R were raised in r a b b i t s b y i n j e c t i n g a total of 60-80 ug of proteins. The specificity of the serum was established from a variety of tests including immunoprecipitation, immunoaffinity chromatography, dot immunoassay, ELISA tests and Western immunoblottings. Although precipitating reactions were not detected, complexes that were formed between the IgG and the receptor could be detected by protein A-Sepharose, and quantitative immunoprecipitation was observed. The antibodies cross-reacted with the glycine receptors from rat and rabbit brains, but not with the GABA-A receptor. It w a s p o s s i b l e to purify gly R by immunoaffinity chromatography using agarose coupled with IgG from the immunized animal, as w ~ l l as u s i n g a m i n o s t r i c h n i n e agarose. The antiserum blocked the binding o f (~H) s t r y c h n i n e to gly R without changing the dissociation constance (Kd). S p e c i f i c i t y of the antiserum was further indicated by a dot immunoassay a n d E L I S A t e s t s in w h i c h t h e i n t e n s i t y of the reaction product was proportional to the amount of the gly R protein and applied I g G . In t h e W e s t e r n blotting experiments, intense staining was observed on 58K polypeptide among three subunits (48K, 5 8 K a n d 93K) o f g l y R.
APPARENT BINDING ACTIVITY OF [3H]GLUTATHIONE
IN SYNAPTIC MEMBRANES OF THE RAT BRAIN.
KIYOKAZU OGITA and YUKIO YONEDA, Department of Pharmacology, Setsunan University, Hirakta, Osaka 573-01, Japan
Faculty of Pharmaceutical
Sciences,
Reduced (GSH) as well as oxidized (GSSG) forms of glutathione significantly displaced the specific bindlng of [3H]L-glutamlc acid (G]u) to synaptic membranous preparations of the rat brain. This inhlbltion occurred independently of the incubatlon temperature in a concentration-dependent manner at the concentratlon range from 1 to 1,000 ~M. An apparent binding activity of [3H]GSH was detected in the synaptlc membranous preparatlons. Thls apparent binding was displaced in a concentration-dependent fashion not on]y by GSH, but also by GSSG. The apparent binding found in the presence of an excess of GSH was considered to be nonspecifica!ly bound [~H]GSH. The apparent specific blnding occupied more than 70% of the total bidding. The apparent specific binding was about 2 times as high at 30°C as that at 2°C. The apparent blnding increased linearly wlth increasing concentrations of synaptic membranous proteins up to 500 Dg. More than 90% (95.7 ± 3.7%) of the radloactivlty in the incubation medium after incubation at 30°C for 60 mln comlgrated wlth the authentic GSH when analyzed by thin layer chromatography on cellulose-coated plates with peno1:H20 (75:25 at 22 ± 2°C) as a solvent system. No significant radioactivity was detected in the spot corresponding to the authentic GSSG. The apparent binding was saturable wlth increasing concentrations of [3H]GSH over the concentration range from i00 to i0,000 nM. Scatchard analysis of these data revealed that the apparent binding sites conslsted of two independent components at 30°C, such as high affinity sites and low affinity sites, while being comprised of a single hlgh afflnlty constituent at 2°C. The apparent binding was profoundly inhlbited in a concentrationdependent manner by the addition of GSH derivatives without SH-moieties, includlng S-methy]-glutathione and S-hexyl-glutathione. The binding activity was slmllarly inhlblted by y-L-GIu-L-GIu and ~-L-G]u-LGlu, whereas ¥-L-GIu-GIy displaced the apparent binding more potently than y-D-Glu-Gly. No slgnificant a]teratlon of the apparent binding was induced by the pretreatment of synaptlc membranous preparations wlth Trlton X-100 at 0.08-0.4%. These results suggest that the apparent bindlng of [3H]GSH may not be attributable to the disulfide bonding between membranous SH-residues and SH-moieties in the ligand, and may indeed represent the specific binding sites of [3H]GSH in the brain. The functlona] slgnlflcance of g]utathlone may need to be re-evaluated. (Supported by a grant 62771981 from the Mlnlstry of Educatlon, Science and Cu]ture, Japan.)