The research will focus on the terminal reactions of mitochondrial oxidative phosphorylation dealing with the utilization of the electrochemical proton gradient for the phosphorylation of ADP. The process is carried out by a vesicular H+-ATPase consisting of a hydrophobic membrane-embedded segment (Fo) and a hydrophilic, extrinsic multiprotein complex, F1-ATPase. Among the proteins of Fo is a 15,000 dalton, dithiol protein (coupling factor B or FB) which is essential for vectorial H+ translocation in Fo-F1. In its absence or when it is inhibited, the H+ release appears to be scalar, and ATP synthesis and the reverse H+ pumping reaction is blocked. Thus, detailed knowledge of the properties of FB and its mechanism of action are of crucial importance to a full understanding of one of the last unresolved major problems in metabolism.
The aims i nclude determination of the primary structure of FB by the cDNA cloning method, identification of proteins homologous with FB in the H+-ATPase preparations of other species, identification of the nearest neighbor proteins of FB, identification of another thiol protein involved in H+ conduction in Fo, and study of the mechanism of H+ conduction in Fo.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM013641-23
Application #
3268532
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1977-07-01
Project End
1992-06-30
Budget Start
1990-07-01
Budget End
1992-06-30
Support Year
23
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Boston Biomedical Research Institute
Department
Type
DUNS #
058893371
City
Watertown
State
MA
Country
United States
Zip Code
02472
Javed, A A; Ogata, K; Sanadi, D R (1991) Human mitochondrial ATP synthase: cloning cDNA for the nuclear-encoded precursor of coupling factor 6. Gene 97:307-10
Ying, W L; Emerson, J; Clarke, M J et al. (1991) Inhibition of mitochondrial calcium ion transport by an oxo-bridged dinuclear ruthenium ammine complex. Biochemistry 30:4949-52
Javed, A A; Joshi, S (1990) Targeted DNA sequencing: rapid identification of DNA clones by sequencing DNA using mixed oligodeoxynucleotide probes as primers. Biotechniques 9:28-32
Kantham, L; Raychowdhury, R; Ogata, K K et al. (1990) Amino-terminal amino acid sequence of beef heart mitochondrial coupling factor B. FEBS Lett 277:105-8
Stephenson, G; Sanadi, D R (1989) Evidence that coupling factor B is bound to the matrix side of the inner mitochondrial membrane. Biochem Int 19:1087-94
Huang, Y; Kantham, L; Sanadi, D R (1987) Coupling factor B is a component of the Fo proton channel of mitochondrial H+-ATPase. J Biol Chem 262:3007-10
Joshi, S; Hughes, J B; Torok, K et al. (1985) Resolution and reconstitution of H+ -ATPase complex from beef heart mitochondria. Membr Biochem 5:309-25
Joshi, S; Kantham, L; Kaplay, S et al. (1985) Monoclonal antibodies to mitochondrial coupling factor B. FEBS Lett 179:143-7
Huang, Y; Pringle, M J; Sanadi, D R (1985) Diamide blocks H+ conductance in mitochondrial H+-ATPase by oxidizing FB dithiol. FEBS Lett 192:83-7