1995 Fiscal Year Final Research Report Summary

Studies of substrate recognition mechanism on brain glutamate decarboxylase

Research Project

Project/Area Number	06670155
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	General medical chemistry
Research Institution	KYOTO UNIVERSITY
Principal Investigator	UENO Hiroshi Kyoto University, Fac.of Agriculture Associate Professor, 農学部, 助教授 (30241160)
Project Period (FY)	1994 – 1995
Keywords	Neurotransmitter / GABA / GAD / HDC
Research Abstract	Rat brain glutamate decarboxylase (GAD) isozyme having molecular weight of 65,000 was expressed in Saccharomyces cerevisiae having a specific activity of 50 nmol/min/mg. The expressed enzyme reacted with anti-GAD65 monoclonal antibody and showed a 65 kDa band on SDS-PAGE and Western blot analysis. Detailed culture conditions were examined. Human histidine decarboxylase (HDC) is a close relative of GAD.In order to compare the substrate specificity of two decarboxylases, we have expressed HDC in Saccharomyces cerevisiae. The expression system exhibited 200 pmol/min/mg histamine producing activity which is far superior than any tissue samples. SDS-PAGE analysis gave 74 kDa band corresponding to precursor form of HDC. The expression system we have developed will provide strong hand on further progress in characterizing decarboxylases.