The objective is to study the structure and mechanism of action of two membrane-bound enzymes of mitochondria: the energy-linked nicotinamide nucleotide transhydrogenase (TH), and the phospholipid-requiring D(-)Beta-hydroxybutyrate dehydrogenase (BDH). TH is structurally the simplest of the mitochondrial energy-transducing enzymes. It is a single polypeptide which occurs in the membrane and isolated state apparently as a homodimer. Our goal is to understand the mechanism of energy transduction by TH. Proposed studies include: (i) isolation and sequence analysis of active site peptides labeled with (a) [3H]p-fluorosulfonylbenzoyl-5'-adenosine (FSBA) at the NAD(H) site, (b) FSBA at the NADP(H) site, and (c) [14C]DCCD at the NAD(H) site; (ii) effects of substrates (particularly NADP and NADPH) and certain inhibitors on TH conformation; (iii) test of alternating site catalysis as a possible explanation of the apparent half-site reactivity of TH; (iv) production of antibodies to various TH fragments and of monoclonals to different TH epitopes for study of enzyme topography in the mitochondrial inner membrane and possible identification of segments concerned with scalar catalysis and proton translocation; (v) deduce the amino acid sequence of TH from the corresponding cDNA sequence. BDH requires lecithin as cofactor; hence it is a model enzyme for study of protein-lipid interaction. Proposed studies include: (i) isolationa nd sequence analysis of active site peptides labeled with (a) arylazido-[3H]Beta-alanyl NAD (N3-NAD), (b) [14C]N-ethylmaleimide, and (c) [14C]DCCD; (ii) labeling of BDH with arylazidophospholipids, isolation and sequence analysis of labeled peptides; (iii) study of role of essential residues (arginyl, thiol, carboxyl) in catalysis. BDH activity is low to negligible in various hepatoma mitochondria. We also plan to investigate whether this is due to low enzyme level (by ELISA immunoassays) or altered phospholipids of hepatoma mitochondria, and to study how these mitochondria cope with virtual lack of BDH function (do they not produce acetoacetate, do they unlike normal liver mitochondria contain succinyl-CoA:acetoacetate CoA-transferase, or are able to metabolize acetoacetate by an alternate route?).

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM024887-11
Application #
3272612
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1981-08-01
Project End
1991-07-31
Budget Start
1988-08-01
Budget End
1989-07-31
Support Year
11
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
San Diego
State
CA
Country
United States
Zip Code
92037
Studley, W K; Yamaguchi, M; Hatefi, Y et al. (1999) Phylogenetic analyses of proton-translocating transhydrogenases. Microb Comp Genomics 4:173-86
Yamaguchi, M; Hatefi, Y (1997) High cyclic transhydrogenase activity catalyzed by expressed and reconstituted nucleotide-binding domains of Rhodospirillum rubrum transhydrogenase. Biochim Biophys Acta 1318:225-34
Hatefi, Y; Yamaguchi, M (1996) Nicotinamide nucleotide transhydrogenase: a model for utilization of substrate binding energy for proton translocation. FASEB J 10:444-52
Yamaguchi, M; Hatefi, Y (1995) Proton-translocating nicotinamide nucleotide transhydrogenase of Escherichia coli. Involvement of aspartate 213 in the membrane-intercalating domain of the beta subunit in energy transduction. J Biol Chem 270:16653-9
Yamaguchi, M; Hatefi, Y (1995) Proton-translocating nicotinamide nucleotide transhydrogenase. Reconstitution of the extramembranous nucleotide-binding domains. J Biol Chem 270:28165-8
Milgrom, Y M; Hatefi, Y (1994) Studies on reconstitution of the Rhodospirillum rubrum nicotinamide nucleotide transhydrogenase. Biochem Mol Biol Int 34:1099-108
Yamaguchi, M; Hatefi, Y (1994) Energy-transducing nicotinamide nucleotide transhydrogenase: nucleotide sequences of the genes and predicted amino acid sequences of the subunits of the enzyme from Rhodospirillum rubrum. J Bioenerg Biomembr 26:435-45
Yamaguchi, M; Hatefi, Y (1993) Energy-transducing nicotinamide nucleotide transhydrogenase. Nucleotide binding properties of the purified enzyme and proteolytic fragments. J Biol Chem 268:17871-7
Yamaguchi, M; Hatefi, Y (1991) Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase. Membrane topography of the bovine enzyme. J Biol Chem 266:5728-35
Yamaguchi, M; Hatefi, Y (1991) Mitochondrial energy-transducing nicotinamide nucleotide transhydrogenase. Purification and properties of the proteinase K-bisected enzyme. J Biol Chem 266:17020-5

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