| Project/Area Number |
63571039
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biological pharmacy
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
YATSUNAMI Kimio Kyoto University, Pharmaceutical Sciences, Assistant, 薬学部, 助手 (30191141)
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| Co-Investigator(Kenkyū-buntansha) |
ICHIKAWA Atsushi Kyoto University, Pharmaceutical Sciences, Professor, 薬学部, 教授 (10025695)
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| Project Period (FY) |
1988 – 1989
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| Project Status |
Completed (Fiscal Year 1989)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1989: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1988: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | Prostaglandin E / Receptor / Kidney medulla / GTP binding protein / GTP |
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| Research Abstract |
Prostaglandin (PG)E_1 receptor from rat kidney medulla, partially purified through a sucrose gradient centrifugation, was shown to increase its binding activity to agonist in the presence of guanine nucleotides. The stimulation of PGE_1 binding was not due to the increase in B_<max> but due to the decrease in Kd value. Pertussis toxin but not cholera toxin abolished the stimulatory effect of PGE_1 binding by guanine nucleotides.
The PGE_1 receptor, solubilized with 6% digitonin, was purified through three successive steps of column chromatographies (Sepharose CL-6B, Wheat germ agglutinin (WGA)-Agarose and DEAE-Toyopearl), and fractions were examined for ^<35>S-GTPrS binding. The radioactivities of ^3H-PGE_1-bound receptor and ^<35>S-GTPrS binding activity were co-eluted on each column chromatography. The purified PGE_1 receptor contained a substrate for ADP-ribosylation by pertussis toxin. The PGE_1 receptor associated with a guanine nucleotide binding protein was subjected to a native gradient polyacrylamide gel electrophoresis. The activities of ^3H-PGE_1, binding and of ^<35>S-GTPrS binding migrated with a molecular weight of about 400 KD. The protein eluted from the gel was analyzed by a SDS-polyacrylamide gel electrophoresis to detect three peptides with molecular weights of 140, 80 and 50 KDs. These results suggest that PGE_1 receptor of rat kidney medulla exists as a associated form with a pertussis toxin-sensitive guanine nucleotide binding protein and that PGE_1 receptor associated with the pertussis toxin sensitive substrate is responsible for a guanine nucleotide-induced stimulation of PGE_1 binding.
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