Ronald R. Fisher

Purification and partial characterization of bovine heart mitochondrial pyridine dinucleotide transhydrogenase

Abstract Bovine heart mitochondrial pyridine dinucleotide transhydrogenase has been purified to near-homogeneity by a six-step procedure. The final preparation is characterized by a single major band with minor contaminants on sodium dodecyl sulfate polyacrylamide gels. The minimal molecular weight is estimated to be 120,000. The protein of the major band is identified as the transhydrogenase by its (a) protection against trypsinolysis with NADH and enhanced degradation in the presence of NADPH, (b) inhibition by low concentrations of palmitoyl-CoA and by Mg 2+ , and (c) pH-rate profile. The specific activity of purified transhydrogenase is increased over twofold after sonication with mitochondrial phospholipids. The enzyme contains no flavin and is not contaminated with cytochromes, NADH dehydrogenase, or NADPH dehydrogenase.

Uptake of oxidized folates by rat liver mitochondria

Folate, dihydrofolate, and methotrexate are rapidly taken up by rat liver mitochondria. The apparent maximal matrix folate concentration is about 2.5-fold that of the suspending medium, whereas dihydrofolate and methotrexate equilibrate across the inner membrane. Fully reduced folates, including tetrahydrofolate, 5-methyltetrahydrofolate, and 5,10-methylenetetrahydrofolate penetrate only the intermembrane space. Addition of dihydrofolate or methotrexate effects a rapid release of pre-loaded folate, and external methotrexate promotes the release of pre-loaded dihydrofolate. The extent of dihydrofolate uptake is enhanced by addition of folate. These results suggest that oxidized folates are transported to the matrix by a carrier-mediated mechanism.

The subunit structure of bovine heart mitochondrial transhydrogenase

Reaction of purified bovine heart transhydrogenase with bifunctional cross-linking reagents dimethyl adipimidate, dimethyl pimelimidate, dimethyl suberimidate, and dithiobis(succinimidyl propionate) results in the appearance of a dimer band on sodium dodecyl sulfate polyacrylamide gels with no higher oligomers formed. Treatment of the enzyme with 6 M urea led to inactivation and prevented cross-linking by dimethyl suberimidate. Transhydrogenase reconstituted into phosphatidylcholine proteoliposomes also yielded a dimer band on cross-linking. These data indicate that soluble and functionally reconstituted transhydrogenase possesses a dimeric structure.

Evidence that reconstituted bovine heart mitochondrial transhydrogenase functions as a proton pump

Inner mitochondrial membrane-bound pyridine dinucleotide transhydrogenase (EC 1.6.1 .l) comprises an energy-coupling site of the respiratory chain [ 1 ] . Mitchell and Moyle [2] demonstrated an uptake of protons into bovine-heart submitochondrial particles coupled to the reduction of NAD’ by NADPH, while Skulachev et al. [3] found the reaction to be coupled to the generation of a membrane potential having the same polarity (positive inside the vesicles) as that generated by respiration or ATP hydrolysis. At high [NADPH] [NAD’] / [NADP’] [NADH] ratios, transhydrogenation is coupled to ATP synthesis from ADP and Pi [4]. It has been proposed that transhydrogenase functions directly as a reversible proton pump catalyzing the following reaction in submitochondrial particles [ l ,S-71 :

Modification of bovine heart mitochondrial transhydrogenase with tetranitromethane

Modification of pyridine dinucleotide transhydrogenase with tetranitromethane resulted in inhibition of its activity. Development of a membrane potential in submitochondrial particles during the reduction of 3-acetylpyridine adenine dinucleotide (AcPyAD+) by NADPH decreased to nearly the same extent as the transhydrogenase rate on tetranitromethane treatment of the membrane. Kinetics of the inactivation of homogeneous transhydrogenase and the enzyme reconstituted into phosphatidylcholine liposomes indicate that a single essential residue was modified per active monomer. NADP+, NADPH and NADH gave substantial protection against tetranitromethane inactivation of both the nonenergy-linked and energy-linked transhydrogenase reactions of submitochondrial particles and the NADPH leads to AcPyAD+ reaction of reconstituted enzyme. NAD+ had no effect on inactivation. Tetranitromethane modification of reconstituted transhydrogenase resulted in a decrease in the rate of coupled H+ translocation that was comparable to the decrease in the rate of NADPH leads to AcPyAD+ transhydrogenation. It is concluded that tetranitromethane modification controls the H+ translocation process solely through its effect on catalytic activity, rather than through alteration of a separate H+-binding domain. Nitrotyrosine was not found in tetranitromethane-treated transhydrogenase. Both 5,5-dithiobis(2-nitrobenzoate)-accessible and buried sulfhydryl groups were modified with tetranitromethane. NADH and NADPH prevented sulfhydryl reactivity toward tetranitromethane. These data indicate that the inhibition seen with tetranitromethane results from the modification of a cysteine residue.

Nuclear magnetic resonance studies on pyridine dinucleotides: The pH dependence of the carbon-13 nuclear magnetic resonance of NAD+ analogs☆

Abstract The pH dependence of the 13 C chemical shifts for nicotinamide adenine dinucleotide (NAD + ), thionicotinamide adenine dinucleotide (TNAD + ), pyridine adenine dinucleotide (PyrAD + ), N -methyl-nicotinamide adenine dinucleotide (N-Me-NAD + ), acetylpyridine adenine dinucleotide (AcPyAD + ), nicotinamide hypoxanthine dinucleotide (NHD + ), and nicotinamide adenine dinucleotide phosphate (NADP + ) are reported. In these analogs the 13 C chemical shifts of the pyridinium moiety reflect the p K a of the opposing purine base, while the 13 C chemical shift dependence on pD for the pyridinium carbons of nicotinamide mononucleotide (NMN + ) and adenosine monophosphate (AMP), 1,4-dihydronicotinamide adenine dinucleotide (NADH), 1,4-dihydronicotinamide adenine dinucleotide phosphate (NADPH), and nicotinic acid adenine dinucleotide (N(a)AD + ) are not influenced by the adenine ring in the pD range tested. Through the use of 13 C-labeled NAD + , the source of the pH dependence of the 13 C chemical shifts was shown to be intramolecular in origin. However, serious doubt is cast on the utility of employing the pD dependence of chemical shift data to determine the nature of solution conformers or their relative populations.

The effect of metal ions on mitochondrial pyridine dinucleotide transhydrogenase.

Bovine heart submitochondrial particle transhydrogenase is inhibited by cations in a concentration and pH-dependent manner, and non-energy-linked transhydrogenation is inhibited to a greater extent by metals than the energy-linked reaction. The inhibition of the enzyme by Mg2+ is competitive with the NADP substrate and non-competitive with the NAD substrate. Mg2+ stimulates inactivation of the enzyme by 5,5-dithiobis(2-nitrobenzoic acid), and protects against thermal and proteolytic inactivation. This suggests that Mg2+ binding in the NADP site alters transhydrogenase to a more thermostable conformation, which is less susceptible to attack by trypsin and more reactive with 5,5-dithiobis(2-nitrobenzoic acid). Other cation inhibitors mimic Mg2+ in these properties. The order of effectiveness of the inhibitors tested is La3+ greater than Mn2+ greater than Ca2+ congruent to Mg2+ greater than Sr2+ greater than Na+ congruent to K+. This order is described by the Irving-Williams order for the stability of metal-ligand complexes, suggesting that carboxylates or amines may comprise the inhibitory cation binding site.

Evidence for substrate-induced conformational changes in mitochondrial transhydrogenase.

The rate of this nonenergy-linked reaction is enhanced by several fold in the forward direction and the apparent equilibrium constant increases from 1 to values near 500 when the membrane oxidative phosphorylation system is energized by electron transport or ATP hydrolysis [ 21. Three principal mechanisms have been proposed to explain the mode of energy input to the transhydrogenase reaction. (a) The chemical mechanism suggests that one of the transhydrogenase substrates reacts with a nonphosphorylated high-energy intermediate of oxidative phosphorylation to form a high-energy pyridine dinucleotide intermediate. Concomitant with hydride ion transfer, the energized substrate is cleaved exothem-&ly to the corresponding product [3]. (b) The conformational mechanism postulates that energization of the oxidative phosphorylation system results in a conversion of an inactive form of the transhydrogenase (TH) to an active form (TH*) [4,5]. Kinetic data indicate that the relative level of active to inactive conformers may also be determined by the prevailing [NADH] [NADP’]/ [NAD’] [NADPH] ratio, according to eq. 2

Nuclear magnetic resonance studies on pyridine dinucleotides. Carbon-13 assignments and structure determination of NADHX and the primary acid product of NADH.

Abstract The 13 C spectra of β-NADH, NADHX, and the primary acid product of NADH were obtained and assigned. The conversion of the NADHX isomers to the two isomers of NADH acid product is demonstrated through the use of 13 C-enriched compounds. The structure of NADHX is assigned as β-6-hydroxy-1,4,5,6-tetrahydronicotinamide adenine dinucleotide and the structures of the primary acid products of NADH are assigned as α- O 2′ -6B-cyclotetrahydronicotinamide adenine dinucleotide and α- O 2′ -6A-cyclotetrahydronicotinamide adenine dinucleotide. The structures of NADHX and the major isomer of the primary acid product, derived from studies of model compounds, are consistent with those proposed by Oppenheimer and Kaplan [ Biochemistry (1974) 13 , 4675, 4685] . However, the spectra of 13 C-enriched primary acid product also demonstrated the existence of the A isomer which was not observed in the latter 1 H study. The A and B isomers were found to exist in the same ratio even when the primary acid product was formed directly from NADHX. This observation is discussed in terms of the previously proposed mechanism for the acid decomposition of NADH.

Rhodamine 123 inhibits import of rat liver mitochondrial transhydrogenase

Rhodamine 123, a laser dye, has been demonstrated to inhibit import of the precursor to pyridine dinucleotide transhydrogenase into mitochondria in rat liver cells. When rat hepatocytes were labeled with 35[S] methionine in the presence of 0.4 mM rhodamine 123, the precursor to transhydrogenase was found to have a half-life in the cytoplasm of 15 minutes as opposed to a half-life of 1-2 minutes when cells were radiolabeled in the absence of the dye. To clarify the mechanism of import inhibition, studies were initiated to assess the effect of rhodamine 123 on mitochondrial respiration. Upon addition of the dye to a mitochondrial suspension, respiration was initially enhanced, then inhibited. The inability of FCCP, a classical uncoupler, to enhance respiration during the inhibitory phase suggests that rhodamine 123 is primarily inhibiting respiration through the electron transport system rather than through the ATPase. These results suggest that rhodamine 123 may inhibit import of the transhydrogenase precursor into mitochondria by disrupting components in the mitochondrial membrane necessary for efficient import.