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?).
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