CHANGES IN CONFORMATION OF CHLOROPLAST ATP SYNTHASE CF_0CF_1

Research Project

Project/Area Number	63580158
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	代謝生物化学
Research Institution	TEIKYO UNIVERSITY
Principal Investigator	TAKAKI Mizuho TEIKYO UNIV. PHARM. RESEARCH ASSOCIATE, 薬学部, 助手 (00112764)
Project Period (FY)	1988 – 1989
Project Status	Completed(Fiscal Year 1989)
Budget Amount *help	¥2,100,000 (Direct Cost : ¥2,100,000) Fiscal Year 1989 : ¥700,000 (Direct Cost : ¥700,000) Fiscal Year 1988 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Keywords	CHLOROPLAST / ATP SYNTHASE / CF_0CF_1 / epsilon SUBUNIT / LYSINE RESIDUE / PYRIDOXAL 5'PHOSPHATE / CONFORMATIONAL CHANGE / ACTIVE STATES / ξサブユニット / γサブユニット / ピリドキサールリン酸
Research Abstract	ATP synthase (CF^0 CF^1) of chloroplast catalyzes synthesis of ATP coupled to an electrochemical gradient of protons. The catalytic portion, CF^1, consists of five different subunits alpha, beta, gamma, delta, and epsilon. Energization of chloroplast thylakoids activates CF^0 CF^1, to catalyze ATP synthesis and ATP hydrolysis. In the activation processes, CF^0 CF^1, seems to be in several distinct states. This suggests distinct conformations of CF^0 CF^1, in the activation processes. To know the distinctions among the conformations of CF^0 CF^1, it is useful to map the amino acid residues which are under the influence of the changes in conformation of CF^0 CF^1. If the surroundings of one amino acid residue change greatly accompanying the changes in conformation of CF^0 CF^1, the reactivity of the amino acid residue is expected to alter. In this report, the changes in reactivity of lysine residues in the epsilon subunit of CF^1 were studied by using pyridoxal 5'-phosphate. Incubation of spinach chloroplast thylakoids with pyridoxal 5'-phosphate modified the epsilon subunit of ATP synthase CF^0 CF^1. Illumination of thylakoids stimulated the modification of one specific amino acid residue of the epsilon subunit by a factor of three. Endoproteinase Glu-C treatment of the isolated epsilon subunit and fractionation of the peptides by high performance liquid chromatography revealed a major fluorescent peptide with a sequence GKRQKIE. Further treatment of this peptide with Endoproteinase Arg-C gave a strongly fluorescent tripeptide GXR. From the primary structure of the epsilon subunit, the specifically modified residue was deduced to be Lys-109. This suggests the energy-dependent conformational changes in the epsilon subunit which change the surroundings of Lys-109 and alter the reactivity of this residue.