Selective solubilization of nicotinamide nucleotide transhydrogenase from the mitochondrial inner membrane

Nicotinamide nucleotide transhydrogenase was solubilized from beef heart submitochondrial particles employing Triton X-100 or lysolecithin. Lysolecithin was considerably more efficient and selective and released over 80 % of the transhydrogenase acdtivity from the membrane together with succinate dehydrogenase. Solubilization of NADH dehydrogenase and cytochrome oxidase was more efficiently accomplished with Triton than with lysolecithin. Both detergents released ATPase to various extents. Transhydrogenase remaining bound to particles after treatment with lysolecithin still catalyzed energy-linked transhydrogenation.     

Resolution and reconstitution of mitochondrial nicotinamide nucleotide transhydrogenase.

Nicotinamide nucleotide transhydrogenase from bovine heart mitochondria was solubilized with cholate and partially purified by ammoniumsulphate fractionation and density gradient centrifugation. Compared to submitochondrial particles this preparation contained less than 10% of oligomycin-sensitive ATPase and cytochromes. When reconstituted with purified mitochondrial phosphatidylcholine, the enzyme catalyzed a reduction of NAD⁺ by NADPH that was stimulated by uncouplers and which showed a concomitent uncoupler-sensitive uptake of the lipophilic anion tetraphenylboron, indicating the generation of a membrane potential. It is concluded that transhydrogenase can energize the vesicles directly without the intervention of ATPase or cytochromes.     

Energy-linked nicotinamide-nucleotide transhydrogenase. Characterization of reconstituted ATP-driven transhydrogenase from beef heart mitochondria

The interaction between pure transhydrogenase and ATPase (Complex V) from beef heart mitochondria was investigated with transhydrogenase-ATPase vesicles in which the two proteins were co-reconstituted by dialysis or dilution procedures. In addition to phosphatidylcholine and phosphatidylethanolamine, reconstitution required phosphatidylserine and lysophosphatidylcholine. Transhydrogenase-ATPase vesicles catalyzed a 20-30-fold stimulation of the reduction of NADP+ or thio-NADP+ by NADH and a 70-fold shift of the apparent equilibrium expressed as the nicotinamide nucleotide ratio [NADPH][NAD+]/[NADP+][NADH]. In both of these respects, the transhydrogenase-ATPase vesicles were severalfold more efficient than beef heart submitochondrial particles. By measuring the ATP-driven transhydrogenase and the oligomycin-sensitive ATPase activities simultaneously and under the same conditions at low ATP concentrations, i.e. below 15 microM, the ATP-driven transhydrogenase/oligomycin-sensitive ATPase activity ratio was found to be about 3. This value is consistent with the stoichiometries of three protons translocated per ATP hydrolyzed and one proton translocated per NADPH formed and with a mechanism where the two enzymes interact through a delocalized proton-motive force.     

Pyridine nucleotide transhydrogenations in yeast

Though previously described as very low or absent in yeast, we find significant pyridine nucleotide transhydrogenation (NADPH + acetyl pyridine-NAD+----NADP+ + acetyl pyridine-NADH) activity in yeast extracts when assayed at pH 8-9, and describe here the subcellular distribution and separation of the various molecular forms contributing to the total activity in two yeast species. Gentle subcellular fractionation reveals transhydrogenase activity only in the cytosolic fraction of both Saccharomyces cerevisiae and Candida utilis while intact mitochondria and microsomes are without activity. On sucrose gradient centrifugation, this soluble cytosolic activity proves to be primarily in a high-molecular-weight (greater than 10(6)) band which has salmon-colored fluorescence on uv illumination. Sonication of the particulate subcellular fractions solubilizes substantial transhydrogenase activity from mitochondria of C. utilis (but not from S. cerevisiae) which on sucrose gradients consists of both high (greater than 10(6))- and low-molecular-weight active fractions, each with yellow-green fluorescence. Ammonium sulfate fractionation and sucrose gradient centrifugation of protein solubilized from whole yeast of both species by vigorous homogenization with glass beads confirms the presence and fluorescence of these various molecular weight forms. The relationship of these activities to other enzymatic activities (especially the mitochondrial external NADH dehydrogenase) is discussed.     

Solubilization by lysolecithin and purification of the plasma membrane ATPase of the yeast Schizosaccharomyces pombe.

Purified plasma membranes of Schizosaccharomyces pombe were obtained by precipitation at pH 5.2 of a crude particulate fraction, followed by differential centrifugations and isopycnic centrifugation in a discontinuous sucrose gradient. The specific activity of the Mg2+-requiring plasma membrane ATPase activity (EC 3.6.1.3) was enriched from 0.3 mumol min-1 x mg-1 of protein in the homogenate to 26 in the purified membranes. The optimal conditions for solubilization of the ATPase activity by lysolecithin were found to be: 2 mg/ml of lysolecithin, a lysolecithin to protein ratio of 8 at pH 7.5, and 15 degrees C in the presence of 1 mM ATP and 1 mM ethylenediaminetetraacetic acid. A 6- to 7-fold purification of the solubilized ATPase activity was obtained by centrifugation of the lysolecithin extract in sucrose gradient. Part of the ATPase activity which was inactivated during the centrifugation in the sucrose gradient could be restored by addition of a micellar solution of 50 microgram of lysolecithin/ml during the assay. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the purified enzyme showed only one band of Mr = 105,000 stained with Coomassie blue. Another ATPase component of apparent molecular weight lower than 10,000 was stained by periodic Schiff reagent but not colored by Coomassie blue. The purified enzyme was 85% inhibited by 50 micrometer N,N'-dicyclohexylcarbodiimide and 94% inhibited by 53 microgram of Dio-9/ml.     

Purification and partial characterization of bovine heart mitochondrial pyridine dinucleotide transhydrogenase.

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 Mg²⁺, 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.     

Particle-bound enzymes in the bloodstream form of Trypanosoma brucei.

We have screened the bloodstream form of Trypanosoma brucei for the presence of enzymes that could serve as markers for the microbodies and the highly repressed mitochondrion of this organism. None of seven known microbody enzymes were detected at all, but glycerol-3-phosphate oxidase, ATPase, isocitrate dehydrogenase, acid phosphatase and part of the hyperoxide dismutase and malate dehydrogenase activities were found to be particle-bound after fractionation of homogenates by differential centrifugation. Part of the ATPase activity was sensitive to oligomycin, an inhibitor of oxidative phosphorylation. This oligomycin-sensitive activity can serve as a specific marker for the mitochondria. More than 80% of the NAD+-linked glycerol-3-phosphate dehydrogenase in T. brucei was found to be particulate and latent. The enzyme could be activated by Triton X-100, by the combined action of sonication and salt, but not by salt alone, and partially by freezing and thawing. We conclude that the NAD+-linked glycerol-3-phosphate dehydrogenase is located inside an organelle.     

Purification and preliminary characterization of mitochondrial complex I (NADH: ubiquinone reductase) from broad bean (Vicia faba L.).

NADH:ubiquinone reductase (EC 1.6.19.3), or complex I, was isolated from broad bean (Vicia faba L.) mitochondria. Osmotic shock and sequential treatment with 0.2% (v/v) Triton X-100 and 0.5% (w/v) [3-cholamidopropyl)dimethylammonio]-1-propanesulfate (CHAPS) removed all other NADH dehydrogenase activities. Complex I was solubilized in the presence of 4% Triton X-100 and then purified by sucrose-gradient centrifugation in the presence of the same detergent. The second purification step was hydroxylapatite chromatography. Substitution of CHAPS for Triton X-100 helped remove contaminants such as ATPase. The high molecular mass complex is composed of at least 26 subunits with molecular masses ranging from 6000 to 75,000 kD. The purified complex I reduced ferricyanide and ubiquinone analogs but not cytochrome c. NADPH could not substitute for NADH as an electron donor. The KM for NADH was 20 microM at the optimum pH of 8.0. The NH2-terminal sequence of several subunits was determined, revealing the ambiguous nature of the 42-kD subunit.     

Purification and properties of the proton-translocating adenosine triphosphatase complex of bovine heart mitochondria.

1. The proton-translocating adenosine triphosphatase (ATPase) of bovine heart mitochondria was highly purified by extraction of submitochondrial particles with cholate, fractionation with ammonium sulfate, and sucrose gradient centrifugation in the presence of methanol, deoxycholate, and lysolecithin. 2. The preparation had a very low content of phospholipids, respiratory components, and adenine nucleotide transporter. The ATPase activity (14 o 16 micromoles/min/mg at 30 degrees) was dependent on addition of phospholipids. The purified enzyme was reconstituted with phospholipids, coupling factor 1 (F1), and the oligomycin sensitivity-conferring protein (OSCP) yielding vesicles with highly active 32Pi-ATP exchange (up to 260 nanomoles/min/mg at 30 degrees), and a proton pump driven by ATP. Site III oxidative phosphorylation was reconstituted when purified cytochrome oxidase was included. 3.The 32Pi-ATP exchange of the reconstituted vesicles was sensitive to both rutamycin and dichylohexylcarbodiimide but the ATPase activity was sensitive to rutamycin and not to dicyclohexylcarbodiimide. 4. In sodium dodecyl sulfate-acrylamide gel scans of the complex, the subunits of F1, OSCP, and three other major bands with apparent molecular weights of 32,000, 23,000, and about 11,000 were noted. Three other minor bands with estimated molecular weights of 80,000, 70,000, and 52,000 were also detected. These bands apparently represent residual trace amounts of respiratory components. Quantitative assays of individual respiratory components revealed between 0 and 3% contamination. 5. We conclude that the rutamycin-sensitive ATPase complex functions as a reversible ATP-driven proton pump.     

Fragmentation of mitochondrial H+-ATPase hydrophobic complex]

The subunits with molecular weights of 30 000, 10 000 and 20 000 + 19 000 have been obtained by fractionation of the hydrophobic part of the oligomycin-sensitive ATPase complex on Sephadex G-200 and Sephadex G-150 in the presence of sodium dodecyl sulfate.     

Identification of the NADH-NAD+ transhydrogenase peptide of the mitochondrial NADH-CoQ reductase (Complex I). A photodependent labeling study utilizing arylazido-beta-alanyl NAD+

Incubation of Complex I (NADH-CoQ reductase) of ox heart mitochondria at 4 degrees C in the presence of 0.5 M NaClO4 followed by ammonium sulfate fractionation of the solubilized proteins results in the isolation of a resolved preparation still capable of catalyzing NADH-NAD+ transhydrogenation but having only low levels of NADH dehydrogenase activity. A number of NAD(H) analogues, including the photoaffinity probes, arylazido-beta-alanyl NAD+ (A3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]propionyl]NAD+ and arylazido-beta-alanyl AcPyAD+ (A3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]propionyl]AcPyAD+ can be utilized as substrates for transhydrogenation in this preparation. A further incubation (10 min) of the resolved NADH-NAD+ transhydrogenase in the presence of 0.5 M NaClO4, but now at 30 degrees C, results in the complete loss of this transhydrogenase activity. Photoaffinity labeling experiments utilizing arylazido-[3-3H]beta-alanyl NAD+ and arylazido-[3-3H]beta-alanyl AcPyAD+ with the resolved NADH-NAD+ transhydrogenase preparation prior to and following NaClO4 (30 degrees C) treatment indicates that the 42,000 molecular weight component of Complex I is the pyridine nucleotide binding site responsible for the major NADH-NAD+ (DD) transhydrogenase activity of Complex I.     

Reconstitution of cytochrome oxidase vesicles and conferral of sensitivity to energy transfer inhibitors

Summary 1. Phospholipids and cytochrome oxidase solubilized with chotate were reconstituted either by dialysis or by dilution of the detergent. The reconstituted cytochrome oxidase vesicles oxidized ascorbate-cytochromec at a rate which was low, insensitive to energy transfer inhibitors and markedly stimulated by uncouplers of oxidative phosphorylation. The rate of reconstitution was dependent on pH, on the concentration of cholate and on the presence of high concentrations of monovalent ions or low concentrations of divalent ions. The integrity of the cytochrome oxidase vesicles was retained after freeze-drying, provided sucrose was present during the process. 2. Reconstitution with pure phospholipids revealed that cardiolipin was required for the marked stimulation of respiration by uncouplers. 3. Cytochrome oxidase vesicles were reconstituted in the presence of hydrophobic mitochondrial proteins which contained oligomycin-sensitive ATPase. The resulting vesicles oxidized ascorbate-cytochromec at a rapid rate which was not enhanced by uncouplers. Addition of an energy transfer inhibitor such as rutamycin resulted in a partial inhibition of respiration which was released by uncouplers. 4. Cytochrome oxidase vesicles reconstituted in the presence of phenol red were rather impermeable to protons and became very permeable on addition of uncouplers. When the reconstitution was performed in the presence of the hydrophobic proteins from mitochondria, proton translocation became partially sensitive to rutamycin. 5. These observations are consistent with some of the formulations of the chemiosmotic hypothesis.     

Studies on rat liver mitochondria: 4. Enzyme activities in mitochondria preserved at 0-4 degrees C

Rat liver mitochondria, stored with the energy-linked functions preserved or in aging conditions, were used to assay the activity of various enzymes during five days. The preservation of energy-linked functions was monitored by the respiratory control coefficient. ATPase, cytochrome oxidase and NADH dehydrogenase showed increased activity when the energy-linked functions were preserved. In aging conditions, cytochrome oxidase, NADH dehydrogenase and ATPase showed decreased activity. The ATPase activity increased only when mitochondria were stored in the presence of inhibitors of the electron transport chain. The activity of NADH oxidase did not change, and succinate oxidase and succinate dehydrogenase showed a small decrease in their activity. The enzymes of the matrix, alpha-ketoglutarate dehydrogenase, malate dehydrogenase and aspartate aminotransferase showed little decrease in activity under either of the conditions of storage. The total protein content decreased slightly under both conditions of storage. These results show that the activity of the enzymes analysed was maintained at reasonable levels, when the energy-linked functions of isolated mitochondria were preserved.     

Energy-linked transhydrogenase. Effects of valinomycin and nigericin on the ATP-driven transhydrogenase reaction catalyzed by reconstituted transhydrogenase-ATPase vesicles

Reconstituted transhydrogenase-ATPase vesicles obtained with purified beef heart transhydrogenase and oligomycin-sensitive ATPase were investigated with respect to the mode of interaction between the two proton pumps, with special reference to the relative contributions of the membrane potential and proton gradient using valinomycin and nigericin in the presence of potassium. In the absence of ionophores and at low ATP concentrations, below 20 microM, the ATPase generated a proton motive force which was predominantly due to a membrane potential, whereas at saturating concentrations of ATP the proton gradient was the predominant component. The ATP-dependence of the rate of the ATP-driven transhydrogenase reaction showed apparent Km values in the low and high ATP concentration range of about 3 and 56 microM, respectively, with a corresponding difference in Vmax of about 3-fold. It is concluded that the reconstituted transhydrogenase can utilize both a membrane potential and a proton gradient, separately or combined, where the relative contributions of these components depend on the activity of the ATPase. In the reconstituted vesicles, the maximally active transhydrogenase is apparently driven by an electrochemical proton gradient where the membrane potential and the proton gradient contribute one-third and two-thirds, respectively. The rate-dependent relative generation of a membrane potential and pH gradient presumably reflects the proton pump characteristics of the ATPase and/or buffering/permeability characteristics of the vesicles rather than the properties of the transhydrogenase per se. These results are discussed in relation to current models for transhydrogenase-linked proton translocation.     

The role of lipid in the energy-dependent transhydrogenase systems ofEscherichia coli

The energy-dependent and independent transhydrogenase activities and the NADH oxidase of membrane particles ofEscherichia coli WS1 were inactivated by phospholipase A fromCrotalus terrificus. Ca²⁺-activated ATPase was stimulated by this treatment. Although these results suggest that phospholipid is involved in the transhydrogenase systems, trypsin treatment produced similar results. Proteolytic activity was not detected in the phospholipase preparation but its presence could not be ruled out. Membranes containing different unsaturated fatty-acid components were obtained by growing the fatty-acid auxotroph,E. coli K1060, on linoleic, oleic, or elaidic acids. Discontinuities in the Arrhenius plots of the activities of NADH oxidase, Ca²⁺-activated ATPase, energy-dependent and independent transhydrogenases, were observed at definite temperatures (transition temperatures). With the exception of NADH oxidase, the transition temperatures could not be correlated with those expected for phase changes in the phospholipids of the membranes. Transition temperatures were also found when a lipid-free, purified ATPase was used. It is concluded that phase changes in the bulk of the phospholipids do not effect transhydrogenase and ATPase activities, and that there is no evidence that the bulk of the phospholipid is involved in the activity of these enzymes. However, we cannot exclude the possibility that a limited amount of lipid in immediate contact with the enzyme protein is essential for its activity.     

Immunological Studies on ATPases in Mung Bean Hypocotyl Plasma Membrane: Proposal of the Presence of Two Molecular Species of ATPase

ATPase in a highly purified plasma membrane fraction from mungbean hypocotyls was solubilized by lysolecithin and fractionatedby glycerol density gradient centrifugation. Lysolecithin activatedATPase activity in the lower but not in the upper half of theactivity peak after glycerol density gradient centrifugation.Antibody against maize root plasma membrane ATPase [Nagao etal. (1987) Plant Cell Physiol. 28: 1181] reacted to a 100-kDapolypeptide which was localized only at the lower half of theactivity peak. Antibody against a 67-kDa polypeptide, whichwas proposed to be a subunit of a new type of ATPase in mungbean hypocotyl plasma membrane (Mito et al. the preceding paper),reacted only to its own antigen which was present mainly inthe upper half of the activity peak. The activity peak fractioncontained a low-molecular-mass polypeptide binding N.N'-dicyclohexylcarbodiimide.We propose the presence in mung bean hypocotyl plasma membraneof two distinct ATPases which differ from each other in polypeptideconstitution and in their response to lysolecithin. (Received September 2, 1987; Accepted May 20, 1988)     

In situ assay of the intramitochondrial enzymes: use of alamethicin for permeabilization of mitochondria

The channel-forming antibiotic alamethicin was used to permeabilize mitochondrial membranes for the low molecular mass hydrophilic substrates NADH and ATP. Alamethicin-treated mitochondria show high rotenone-sensitive NADH oxidase, NADH-quinone reductase, and oligomycin-sensitive and carboxyatractylate-insensitive ATPase activities. Alamethicin does not affect Complex I and ATPase activities in inside-out submitochondrial particles. Permeabilized mitochondria quantitatively retain their aconitase and iso-citrate dehydrogenase activities. Electron microscopy of alamethicin-treated mitochondria reveals no disruption of their outer and inner membranes. From the results obtained it is recommended, that alamethicin be used for the in situ catalytic assay of intramitochondrially located enzymes.     

Membrane potential generation by two reconstituted mitochondrial systems: Liposomes inlayed with cytochrome oxidase or with ATPase

Formation of a membrane potential in two types of liposomes, one inlayed with cytochrome c + cytochrome oxidase, and another, with oligomycin-sensitive ATPase, has been demonstrated. To detect a membrane potential, phenyl dicarbaundecaborane (PCB⁻), a penetrating anion probe, was used.

The first type of liposome was reconstituted from a solution of purified cytochrome oxidase, mitochondrial phospholipids and cytochrome c, the latter being enclosed inside liposomes. Cytochrome c bound to the outer surface of the liposome membrane was removed by washing with NaCl. Such liposomes catalyzed oxidation of ascorbate by oxygen in the presence of phenazine methosulfate or N,N,N′,N′-tetramethyl-p-phenylenediamine. The oxidation was found to support the PCB⁻ uptake by liposomes. The PCB⁻ response was prevented and reversed by cyanide, protonophorous uncouplers and external cytochrome c.

Liposomes of the second type were prepared from a solution of mitochondrial phospholipids, coupling factors F₁and F_c, and the hydrophobic proteins of the oligomycin-sensitive ATPase. These liposomes catalyzed ATP hydrolysis coupled with the PCB⁻ uptake. The latter effect was prevented and reversed by oligomycin and uncouplers.

The conclusion is made that membrane potential can be independently formed by enzymic reactions of two different kinds: (1) redox (e.g. cytochrome c oxidase) and (2) hydrolytic (ATPase).     

Effects of plant flavonoids on Manduca sexta (tobacco hornworm) fifth larval instar midgut and fat body mitochondrial transhydrogenase

The reversible, membrane-associated transhydrogenase that catalyzes hydride-ion transfer between NADP(H) and NAD(H) was evaluated and compared to the corresponding NADH oxidase and succinate dehydrogenase activities in midgut and fat body mitochondria from fifth larval instar Manduca sexta. The developmentally significant NADPH-forming transhydrogenation occurs as a nonenergy- or energy-linked activity with energy for the latter derived from either electron transport-dependent NADH or succinate utilization, or ATP hydrolysis by Mg++-dependent ATPase. In general, the plant flavonoids examined (chyrsin, juglone, morine, quercetin, and myricetin) affected all reactions in a dose-dependent fashion. Differences in the responses to the flavonoids were apparent, with the most notable being inhibition of midgut, but stimulation of fat body transhydrogenase by morin, and myricetin as also noted for NADH oxidase and succinate dehydrogenase. Although quercetin inhibited or stimulated transhydrogenase activity depending on the origin of mitochondria, it was without effect on either midgut or fat body NADH oxidase or succinate dehydrogenase. Observed sonication-dependent increases in flavonoid inhibition may well reflect an alteration in membrane configuration, resulting in increased exposure of the enzyme systems to the flavonoids. The effects of flavonoids on the transhydrogenation, NADH oxidase, and succinate dehydrogenase reactions suggest that compounds of this nature may prove valuable in the control of insect populations by affecting these mitochondrial enzyme components.     

Phospholipid Dependence of the Reversible,Energy-Linked,Mitochondrial Transhydrogenase in <Emphasis Type="Italic">Manduca sexta</Emphasis>

Midgut mitochondria from fifth larval instar Manduca sexta exhibit a membrane-associated transhydrogenase that catalyzes hydride ion transfer between NADP(H) and NAD(H). The NADPH-forming transhydrogenations occur as nonenergy- and energy-linked activities. The energy-linked activities couple with electron transport-dependent utilization of NADH/succinate, or with Mg²⁺-dependent ATPase. These energy-linked transhydrogenations have been shown to be physiologically and developmentally significant with respect to insect larval/pupal maturation. In the present study, isolated mitochondrial membranes were lyophilized and subjected to organic solvent or phospholipase treatments. Acetone extraction and addition of Phospholipase A₂ proved to be effective inhibitors of the insect transhydrogenase. Liberation of phospholipids was reflected by measured phosphorous release. Addition of phospholipids to organic solvent- and phospholipase-treated membranes was without effect. Employing a partially lipid-depleted preparation, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine were reintroduced and transhydrogenase activity assessed. Of the phospholipids tested, only phosphatidylcholine significantly stimulated transhydrogenase activity. The results of this study suggest a phospholipid dependence of the M. sexta mitochondrial transhydrogenase.