Grant-in-Aid for Developmental Scientific Research (B)
|Allocation Type||Single-year Grants |
|Research Institution||Tohoku University |
YONEKURA Hideto Tohoku University, School of Medicine, Department of Biochemistry, Associate Professor, 医学部, 助教授 (80240373)
NATA Koji Tohoku University, School of Medicine, Department of Biochemistry, Research Asso, 医学部, 助手 (90202233)
TAKASAWA Shin Tohoku University, School of Medicine, Department of Biochemistry, Lecturer, 医学部, 講師 (50187944)
OKAMOTO Hiroshi Tohoku University, School of Medicine, Department of Biochemistry, Professor, 医学部, 教授 (60025632)
|Project Period (FY)
1993 – 1995
Completed (Fiscal Year 1995)
|Budget Amount *help
¥16,900,000 (Direct Cost: ¥16,900,000)
Fiscal Year 1995: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1994: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 1993: ¥9,900,000 (Direct Cost: ¥9,900,000)
|Keywords||Cyclic ADP-ribose / Second messenger / Ca^<2+> mobilization / Pancreatic islet beta-cells / Insulin secretion / CD38 / Quantitative method / microsome / cyclicADP-ribose / second messenger / Cs^<2+>mobilization / insulin secretion / 抗cyclicADP-ribose抗体 / cyclic ADP-ribose / Ca2+ mobilization / 測定法 / サクシニルcyclic ADP-ribose / 抗cyclic ADP-ribose抗体|
Recently, cyclic ADP-ribose, a metabolite from NAD^+, has been shown to be a second messenger for intracellular Ca^<2+> mobilization and to play an essential role in insulin secretion from pancreatic beta-cells. Thus, a quantitative method for determining cyclic ADP-ribose concentration has been required for studying of physiological functions of cyclic ADP-ribose in mammalian cells. The aim of this project was to establish a sensitive method for determining cyclic ADP-ribose concentration.
In this project,
(1)We have purified ADP-ribosyl cyclase, which converts NAD^+ to cyclic ADP-ribose, from Aplysia kurodai.
(2)We synthesized cyclic ADP-ribose from NAD^+ using the purified Aplysia ADP-ribosyl cyclase and purified cyclic ADP-ribose by HPLC.
(3)Using cyclic ADP-ribose synthesized by Aplysia ADP-ribosyl cyclase as a standard, we have developed a quantitative method for determining cyclic ADP-ribose concentration by monitoring the release of Ca^<2+> from mammalian cell (such as rat cerebell
um or islet cells) microsomes in a cell free system.
(4)We found that the addition of calmodulin to microsomes sensitized and activated the cyclic ADP-ribose-mediated Ca^<2+> release.
(5)By addition of calmodulin to the cell free cyclic ADP-ribose assay system, we have established a sensitive method for determining cyclic ADP-ribose concentration.
(6)We have also developed a method for preparing the cell extract, in which the cyclic ADP-ribose concentration is determined.
Using above cyclic ADP-ribose assay system,
(7)We found that cyclic ADP-ribose is generated in pancreatic islets by glucose stimulation.
(8)We showed that CD38, which has been reported to be a human leukocyte antigen, has both ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities.
(9)Moreover, we found that ATP,generated in the process of glucose metabolism, inhibits the cyclic ADP-ribose hydrolase activity, resulting in the increased formation of cyclic ADP-ribose.
(10)We produced transgenic mice overexpressing CD38 in islet beta-cells and obtained the first evidance that CD38 has a regulatory role in insulin secretion by glucose in pancreatic beta-cells. Less