Differential inhibition of respiration and its dependent H+ extrusion by fluorescamine in rat liver mitochondria

Rat liver mitochondria were treated with varying amounts of fluorescamine ranging from 0 to 30 nmol/mg of protein. The biochemical activities of the modified mitochondria were analyzed. It was found that the respiration rate in the absence of ADP was not significantly affected, but that the state 3 respiration rate and the accompanying $\text{P}\text{O}$ ratio decreased as the labeling extent increased. It was also observed that the treatment inhibited the stimulation of respiration induced by the presence of uncouplers. However, the modification has no effect on the discharging rate of proton gradient by uncouplers. The intrinsic activities of NADH-cytochrome c reductase, succinate-cytochrome c reductase, and cytochrome oxidase of the inner membrane were not affected by the modification. Measurement of the respiration-dependent proton extrusion (in the presence of valinomycin and potassium ion) with secondary ion movements inhibited, showed that the initial extrusion rate was reduced progressively. However, the observed amounts of proton extruded (ΔH⁺) and Δμ_H + were not affected. The observed reduction of the oxygen consumption rate was much less than that of the proton extrusion rate with increased labeling. These results suggest that some fluorescamine titratable primary amino groups may be involved in the controlling of the proton extrusion process. The implications on the mechanism of coupling in respirationdependent proton extrusion are discussed.     

Effects of borohydride-treated oligomycins on processes of energy transduction in mitochondria

Sodium borohydride in ethanol solution under mild conditions brings about the stepwise reduction of the 7-keto and the 11-keto groups of rutamycin and the oligomycins to the corresponding hydroxyl groups without further alterations of the macrocyclic lactone structure or other features of the molecule. The reduced compounds, as well as the parent antibiotics, inhibit the ADP-dependent (state 3) respiration, and the Pi formation and proton extrusion that are linked to ATP hydrolysis, but have no effect on other respiration-linked activities in intact rat liver mitochondria. Analogous inhibitory effects of borohydride-treated antibiotics are also observed in rat-liver submitochondrial particles. The reduced compounds are less potent inhibitors than the parent antibiotics. The reduced compounds are more efficient as inhibitors of Pi formation stimulated by conventional uncouplers (e.g. 2,4-dinitrophenol), than of Pi formation stimulated by certain amine-fluorescamine modifiers (e.g.) the benzylamine-fluorescamine compound. In contrast, the parent antibiotics are unable to discriminate between uncoupler-stimulated and modifier-stimulated Pi formation. It is suggested that rutamycin and the oligomycins bind to H+-ATPase as a result of hydrogen bonding to, at least, the 7-keto and/or the 11-keto groups of the antibiotics. When these keto groups are reduced to hydroxyl groups the hydrogen-bonding is less efficient due to the pronounced directional characteristic of hydrogen-bonding to keto groups.     

Purification and characterization of intact cytochrome b5 from yeast microsomes

The inhibition of an oligomycin sensitive ATPase prepared from bovine heart submitochondrial particles (J.A. Berden and M.M. Voorn-Brouwer, 1978, Biochim. Biophys. Acta 501, 424-439) by a number of cationic dyes has been compared in order to develop a structure-function relationship. Two generalizations emerge from this comparison. First, the most effective dyes have net positive charge at neutral pH; and second, those dyes containing alkyl substituted secondary and tertiary amino groups are more effective than analogs with primary aromatic amino groups. Some of the cationic dyes exhibit uncoupling activity when added to intact rat liver mitochondria, stimulating both State 4 respiration and the latent ATPase activity. The order of effectiveness and concentrations for maximal stimulation of respiration are: coriphosphine (0.3 microM), Nile blue A (0.5 microM), pyronin Y (0.8 microM), and acridine orange (10 microM). Atypically, oligomycin inhibits the stimulation of respiration by these cationic acid uncouplers. The order of effectiveness and concentrations for maximal stimulation of the latent ATPase are: Nile blue A (2 microM), pyronin Y (8 microM), acridine orange (25 microM), and coriphosphine (75 microM). At concentrations greater than those shown for maximal stimulation, the uncoupling dyes inhibited respiration and the latent ATPase. The cationic dyes tested that were not uncouplers are inhibitors of respiration and the latent ATPase of intact mitochondria at all concentrations tested.     

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.     

Spegazzinine,a new inhibitor of mitochondrial oxidative phosphorylation

The effects of spegazzinine, a dihydroindole alkaloid, on mitochondrial oxidative phosphorylation were studied.Spegazzinine inhibited coupled respiration and phosphorylation in rat liver mitochondria. The I₅₀ was 120 μM. Uncouplers released the inhibition of coupled respiration. Arsenate-stimulated mitochondrial respiration was partially inhibited by spegazzinine. The stimulation of mitochondrial respiration by Ca²⁺ and the proton ejection associated with the ATP-dependent Ca²⁺ uptake were not affected by the alkaloid.Oxidative phosphorylation and the P_i-ATP exchange reaction of phosphorylating beef heart submitochondrial particles were strongly inhibited by spegazzinine (I₅₀, 50 μM) while the ATP-dependent reactions, reduction of NAD⁺ by succinate and the pyridine nucleotides transhydrogenase were less sensitive (I₅₀, 125 μM). Oxygen uptake by submitochondrial particles was not affected.The 2,4-dinitrophenol-stimulated ATPase activity of rat liver mitochondria was not affected by 300 μM spegazzinine, a concentration of alkaloid that completely inhibited phosphorylation. However, higher concentrations of spegazzinine did partially inhibit it. The ATPase activities of submitochondrial particles, insoluble and soluble ATPases were also partially inhibited by high concentrations of spegazzinine.The inhibitory properties of spegazzinine on energy transfer reactions are compared with those of oligomycin, aurovertin and dicyclohexylcarbodiimide. It is concluded that spegazzinine effects are very similar to the effects of aurovertin and that its site of action may be the same or near the site of aurovertin.     

The Role of Reactive Oxygen Species in Mitochondrial Permeability Transition

We have provided evidence that mitochondrial membrane permeability transition induced by inorganic phosphate, uncouplers or prooxidants such as t-butyl hydroperoxide and diamide is caused by a Ca²⁺-stimulated production of reactive oxygen species (ROS) by the respiratory chain, at the level of the coenzyme Q. The ROS attack to membrane protein thiols produces cross-linkage reactions, that may open membrane pores upon Ca²⁺ binding. Studies with submitochondrial particles have demonstrated that the binding of Ca²⁺ to these particles (possibly to cardiolipin) induces lipid lateral phase separation detected by electron paramagnetic resonance experiments exploying stearic acids spin labels. This condition leads to a disorganization of respiratory chain components, favoring ROS production and consequent protein and lipid oxidation.     

Electron transport chains and bioenergetics of respiratory nitrogen metabolism in Wolinella succinogenes and other Epsilonproteobacteria

Recent phylogenetic analyses have established that the Epsilonproteobacteria form a globally ubiquitous group of ecologically significant organisms that comprises a diverse range of free-living bacteria as well as host-associated organisms like Wolinella succinogenes and pathogenic Campylobacter and Helicobacter species. Many Epsilonproteobacteria reduce nitrate and nitrite and perform either respiratory nitrate ammonification or denitrification. The inventory of epsilonproteobacterial genomes from 21 different species was analysed with respect to key enzymes involved in respiratory nitrogen metabolism. Most ammonifying Epsilonproteobacteria employ two enzymic electron transport systems named Nap (periplasmic nitrate reductase) and Nrf (periplasmic cytochrome c nitrite reductase). The current knowledge on the architecture and function of the corresponding proton motive force-generating respiratory chains using low-potential electron donors are reviewed in this article and the role of membrane-bound quinone/quinol-reactive proteins (NapH and NrfH) that are representative of widespread bacterial electron transport modules is highlighted. Notably, all Epsilonproteobacteria lack a napC gene in their nap gene clusters. Possible roles of the Nap and Nrf systems in anabolism and nitrosative stress defence are also discussed. Free-living denitrifying Epsilonproteobacteria lack the Nrf system but encode cytochrome cd₁ nitrite reductase, at least one nitric oxide reductase and a characteristic cytochrome c nitrous oxide reductase system (cNosZ). Interestingly, cNosZ is also found in some ammonifying Epsilonproteobacteria and enables nitrous oxide respiration in W. succinogenes.     

Inactivation of the mitochondrial ATPase inhibitor protein by chemical modification with diethylpyrocarbonate

Modification of histidine residue(s) by diethylpyrocarbonate treatment of submitochondrial particles obtained by sonication results in inhibition of ATPase activity and stimulation of oligomycin-sensitive H+ conduction. The inhibition of the ATPase (EC 3.6.1.3) activity persisted in F1 isolated from diethylpyrocarbonate-treated submitochondrial particles, which exhibited the absorbance spectrum of modified histidine. Thus the inhibition of the ATPase activity results from histidine modification in F1 subunits. Removal of the natural inhibitor protein from submitochondrial particles resulted in stimulation of proton conduction. After removal of F1 inhibitor protein from the particles the stimulatory effect exerted by diethylpyrocarbonate treatment on proton conduction was lost. Reconstitution experiments showed that purified F1 inhibitor protein lost, after histidine modification, its capacity to inhibit the ATPase activity and proton conduction. These observations show that the stimulation of proton conduction by the ATPase complex effected by diethylpyrocarbonate treatment results from histidine modification in F1 inhibitor protein.     

Flow-force relationships during energy transfer between mitochondrial proton pumps.

The effect of inhibitors of proton pumps, of uncouplers and of permeant ions on the relationship between input force, delta mu H+, and output flows of the ATPase, redox and transhydrogenase H(+)-pumps in submitochondrial particles was investigated. It is concluded that: (1) The decrease of output flow of the transhydrogenase proton pump, defined as the rate of reduction of NADP+ by NADH, is linearily correlated with the decrease of input force, delta mu H+, in an extended range of delta mu H+, independently of whether the H(+)-generating pump is the ATPase or a redox pump, or whether delta mu H+ is depressed by inhibitors of the H(+)-generating pump such as oligomycin or malonate, or by uncouplers. (2) The output flows of the ATPase and of the site I redox H(+)-pumps exhibit a steep dependence on delta mu H+. The flow-force relationships differ depending on whether the depression of delta mu H+ is induced by inhibitors of the H(+)-generating pump, by uncouplers or by lipophilic anions. (3) With the ATPase as H(+)-consuming pump, at equivalent delta mu H+ values, the output flow is more markedly inhibited by malonate than by uncouplers; the latter, however, are more inhibitory than lipophilic anions such as ClO4-. With redox site I as proton-consuming pump, at equivalent delta mu H+ values, the output flow is more markedly inhibited by oligomycin than by uncouplers; again, uncouplers are more inhibitory than ClO4-. (4) The results provide further support for a delocalized interaction of transhydrogenase with other H(+)-pumps.     

Isolation of totally inverted submitochondrial particles by sonication of beef heart mitochondria

A novel procedure for isolating totally inverted preparations of submitochondrial particles by sonication of beef heart mitochondria is described. The procedure involves only differential centrifugation in 0.25 M sucrose containing 0.15 M KCl. The submitochondrial particles have 96% of their cytoplasmic face cytochromec-binding sites sequestered within the particles. Mild sonication exposes cytochromec-binding sites to the medium. The oligomycin-sensitive ATPase of sonic-derived submitochondrial particles, like that of electron transport particles, is inhibited 98% by exogenous isolated ATPase inhibitor protein. NADH oxidase activity in these particles is inhibited by oligomycin. The respiratory control index (uncoupled rate/oligomycin-inhibited rate) is approximately 3.4 and can be increased by washing the particles with medium containing bovine serum albumin.     

Phosphate-dependent Substrate Transport into Mitochondria: Oxidative Studies

The oxidation of malate and citrate by isolated plant mitochondria can be stimulated by the addition of inorganic phosphate. This stimulation (a) is not inhibited by oligomycin or uncouplers; (b) can not be duplicated by addition of adenine nucleotide; (c) is inhibited by 2-n-butylmalonate; and (d) is not evident in detergent-treated mitochondria. Phosphate was required to elicit uncoupler-stimulated respiration. It is concluded that these effects of phosphate are attributable to a stimulated rate of substrate penetration into the mitochondria, and do not involve the oxidative phosphorylation process.     

Photoinhibition by flash and continuous light of oxygen uptake by intact photosynthetic bacteria

The inhibition of respiration by continuous or flashing light has been studied in intact cells of different species of photosynthetic bacteria. For Rhodopseudomonas palustris, Rhodopseudomonas sphaeroides and Rhodopseudomonas capsulata, the inhibition by short actinic flashes shows a remarkable periodicity of two: each flash induces an inhibition of respiration, but a stimulation is observed after an even number of flashes. On the other hand, no oscillation is observed for Rhodospirillum rubrum and Rhodopseudomonas viridis cells. These different behaviours are explained by a difference in the redox state of the secondary electron acceptor as shown by the effect of ortho-phenanthroline on the amperometric signal. Addition of uncouplers (carbonyl cyanide m-chlorophenylhydrazone) or of an ATPase inhibitor (tri-N-butyl tin), has little effect on the oscillatory pattern induced by flash excitation. However, inhibition of respiration by continuous light is suppressed in the presence of carbonyl cyanide m-chlorophenylhydrazone. In the presence of tri-N-butyl tin the steady-state level is reached more rapidly than in the control experiment for a given light intensity. These results are interpreted as evidence of two modes of light inhibition of respiration in photosynthetic bacteria. A first type of inhibition, clearly shown under flash excitation, is due to interaction between respiratory and photosynthetic chains at the level of electron carriers. After each flash, an electron is diverted from the respiratory chain to the photooxidized reaction center. Because of the gating mechanism at the level of the secondary acceptor, the respiration is stimulated after an even number of flashes. The second mode of inhibition prevails under continuous illumination. Under these conditions, the rate of respiration is controlled essentially by the photoinduced proton electrochemical gradient.     

Proton translation in submitochondrial vesicles

An investigation of proton translocation in submitochondrial vesicles from rat liver has been made under simple experimental conditions. Choline chloride was used both as the oxidizable substrate and the ionic medium for the measurement of activity during oxygen pulse experiments:

1. The passive permeability measured from the decay of proton efflux after an oxygen pulse could be described by a first-order equation. An H⁺/O ratio of 2·5 was obtained for choline oxidation in the presence of oligomycin and/or MgCl₂. Oligomycin decreased the passive proton permeability and respiration, concomitant with an increase in proton uptake. Respiratory control was directly related to the passive proton permeability and inversely related to the magnitude of the proton gradient. The decreased respiration and passive permeability reflecting respiratory control is most evident in the pH rang 5·8–7·5.
2. Preparation of submitochondrial vesicles in the presence of EDTA resulted in proton production during an oxygen pulse given at alkaline pH. Cytochromec enhanced proton uptake by approximately 1 H⁺/cytochromec, but only in the presence of Triton X-100. These results are indicative of the asymmetric behavior of the coupling membrane and provide direct evidence of the participation of electron transport components in proton translocation.

     

Inhibitors of the slow stage of proton transfer in the link connecting respiration with mitochondrial phosphorylation]

Electrophilic agents--derivatives of carbonic acids--are found to inhibit respiration, ATP synthesis and reverse electrone transport in intact mitochondria. The inhibition of respiration and ATPase was observed in intact mitochondria at 3 and 3u states (by Chance). Inhibitors concentrations, which caused 50% inhibition, were approximately the same. Sharp decrease of the effect of electrophilic inhibitors on respiration and ATPase activity in mitochondria and submitochondrial particles with substantially impaired coupling system was observed. The following conclusions are drawn on the basis of the data obtained: 1) electrophilic inhibitor attack the coupling site of respiration and ATP synthesis in mitochondria; 2) the reaction of the proton transport from the respiration proton pump to ATP synthetase is one of the slowest steps of the process of ATP-synthesis in mitochondria. A scheme of working the coupling system is suggested which includes the step of proton lateral diffusion.     

The divalent cation requirement of the mitochondrial glycerol-3-phosphate dehydrogenase

Coupled respiration by blowfly mitochondria has been utilized to demonstrate an absolute divalent cation requirement for glycerol 3-phosphate respiration. With ADP, phosphate and EGTA, the respiration rate (state 3) decreases as a function of the amount of oxygen reduced, to approximately 15% of its maximum value, even at 40 mM dl-glycerol 3-phosphate; it can be increased to its maximum value by the addition of Ca²⁺, Sr²⁺ or Mn²⁺. The decline in state 3 rate is not due to the removal of membrane-bound calcium into the matrix by the calcium carrier, since it occurs in the presence of ruthenium red. The effect is energy-dependent since the state 3 respiration does not decrease in the presence of uncouplers. The increase in respiration upon the addition of calcium is not due to the energy-dependent calcium transport since it is sensitive to oligomycin and insensitive to ruthenium red.The divalent cation effector site is located on the glycerol-3-phosphate dehydrogense, since state 3 (or state 4) pyruvate-proline respiration (NAD-linked) is not affected by EGTA. Yet the state 3 pyruvate-proline respiration removes calcium so effectively from the glycerol-3-phosphate dehydrogenase in the presence of EGTA, that added calcium stimulates glycerol 3-phosphate (26.4 mM) respiration about 22-fold.Since uncouplers stimulate the inhibited glycerol 3-phosphate respiration only to a very small extent, a calcium stimulation of the rate of phenazine methosulfate reduction by glycerol 3-phosphate (26.4 mM) which bypasses all phosphorylation sites, should be detectable. Only a 3-fold stimulation was observed.The present experiments suggest that upon complete removal of divalent cations from the dehydrogenase, glycerol 3-phosphate does not act as a homotropic effector in the coenzyme Q reductase reaction.     

Differential effects on energy transduction processes by fluorescamine derivatives in rat liver mitochondria

Intact rat liver mitochondria were treated with compounds derived from the reaction of fluorescamine with various types of primary amines, including the mycosamine-containing antibiotics amphotericin B and nystatin. The effect of varying amounts of these compounds on ATPase-linked inorganic phosphate (Pi) formation on oxygen consumption, and on MgATP-linked and succinate-linked proton movements was examined. The antibiotic-fluorescamine compounds did not affect the Pi formation rate but strongly inhibited both the ATPase-linked and the succinate-linked H+ extrusion rates to approximately the same extent. The antibiotic derivatives decreased the oxygen consumption rate, but this effect was much smaller than the decrease in the respiration-dependent proton extrusion rate. The benzylamine-fluorescamine compound significantly increased the Pi formation rate, in contrast to the antibiotic analogues. The benzylamine derivative, like the antibiotic derivatives, inhibited both types of proton extrusion rates. The slight decrease in the oxygen consumption rate caused by the benzylamine derivative was significantly smaller than the corresponding decrease observed with the antibiotic derivatives. These studies, in which fluorescamine derivatives bind reversibly to mitochondria, are compared with previous studies in which fluorescamine itself binds irreversibly to mitochondria and results in a Pi formation rate increase and MgATP- and succinate-linked proton extrusion rate inhibition but has no effect on the oxygen consumption rate.     

Interaction of a synthetic polyanion with rat liver mitochondria

The effect of a polyanion (a copolymer of methacrylate, malaete and styrene in a 1:2:3 proportion with an average molecular weight of 10 000) on respiration, ATPase activity and ADP/ATP exchange activity of rat liver mitochondria and submitochondrial particles has been studied.The polyanion (at 17–150 μg/ml concentration, 100 μg polyanion corresponding to 0.83 μequiv. of carboxylic groups) inhibits the oxidation of succinate and NAD-linked substrates in state 3 in a concentration-dependent manner. The extent of this inhibition can be decreased by elevating the concentration of ADP. State 4 respiration is not affected by the polyanion. It has also a slight inhibitory effect on the oxidation of the above mentioned substrates in the uncoupled state (a maximum inhibition of 37% at 166 μg/ml polyanion concentration), which is unaffected by ADP. The strong inhibition of state 3 respiration can be relieved by 2,4-dinitrophenol to the low level observed in the uncoupled state. Ascorbate+TMPD oxidation is slightly inhibited in state 3, while it is not inhibited at all in the uncoupled state.The polyanion, depending on its concentration, strongly inhibits also the DNP-activated ATPase activity of mitochondria (50% inhibition at 40 μg/ml polyanion concentration).The ATPase activity of sonic submitochondrial particles is also inhibited. However, this inhibition is incomplete (reaching a maximum of 65%) and higher concentrations of the polyanion are required than to inhibit the ATPase activity of intact mitochondria.The polyanion inhibits the ADP/ATP translocator activity of mitochondria, measured by the “back exchange” of [2-³H]ADP. After a short preincubation of the mitochondria with the polyanion, the concentration dependence of the inhibition by the polyanion corresponds to that of the DNP-activated ATPase activity of intact mitochondria.It is concluded that, in intact mitochondria, the polyanion has at least a dual effect, i.e. it partially inhibits the respiratory chain between cytochrome b and cytochrome c, and strongly oxidative phosphorylation by blocking the ADP/ATP translocator.     

Effects of fusaric acid on respiration in maize root mitochondria

The effects of fusaric acid, a phytotoxin produced byFusarium pathogens, on the metabolism of isolated maize root mitochondria and on maize seed germination and seedling growth were investigated. The phytotoxin inhibited basal and coupled respiration when succinate and α-ketoglutarate were the substrates. Coupled respiration dependent on NADH was inhibited, but basal respiration was not. Consistently, succinate cytochromec oxidoreductase activity was decreased whereas NADH cytochromec oxidoreductase was not affected. The ATPase activities of carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone stimulated mitochondria and of freeze-thawing disrupted mitochondria were inhibited. These results indicate that the phytotoxin impairs the respiratory activity of maize mitochondria by at least three mechanisms: (1) it inhibits the flow of electrons between succinate dehydrogenase and coenzyme Q, (2) it inhibits ATPase/ATP-synthase activity and (3) it possibly inhibits α-ketoglutarate dehydrogenase. Seed germination and seedling growth were also affected by fusaric acid with the most pronounced effect on root development. These effects can possibly contribute to the diseases ofFusarium- infected plants     

Energy-dependent dissociation of ATP from high affinity catalytic sites of beef heart mitochondrial adenosine triphosphatase

Incubation of [gamma-32P]ATP with a molar excess of the membrane-bound form of mitochondrial ATPase (F1) results in binding of the bulk of the radioactive nucleotide in high affinity catalytic sites (Ka = 10(12) M-1). Subsequent initiation of respiration by addition of succinate or NADH is accompanied by a profound decrease in the affinity for ATP. About one-third of the bound radioactive ATP appears to dissociate, that is, the [gamma-32P]ATP becomes accessible to hexokinase. The NADH-stimulated dissociation of [gamma-32P]ATP is energy-dependent since the stimulation is inhibited by uncouplers of oxidative phosphorylation and is prevented by respiratory chain inhibitors. The rate of the energy-dependent dissociation of ATP that occurs in the presence of NADH, ADP, and Pi is commensurate with the measured initial rate of ATP synthesis in NADH-supported oxidative phosphorylation catalyzed by the same submitochondrial particles. Thus, the rate of dissociation of ATP from the high affinity catalytic site of submitochondrial particles meets the criterion of kinetic competency under the conditions of oxidative phosphorylation. These experiments provide evidence in support of the argument that energy conserved during the oxidation of substrates by the respiratory chain can be utilized to reduce the very tight binding of product ATP in high affinity catalytic sites and to promote dissociation of the nucleotide.     

Localization of a proton-translocating ATPase on sucrose gradients

Ionophore-stimulated ATPase activity and ATP-dependent quinacrine quench were enriched in parallel when microsomal vesicles were prepared from corn (Crow Single Cross Hybrid WF9-Mo17) roots and collected on a cushion of 10% dextran. Activities were highest in the apical 1.5 centimeters of the roots. Vesicles collected on the dextran cushion also contained NADH cytochrome c reductase (enriched in the apical 0.5 cm of the root) and nucleoside diphosphatase (distributed throughout the first four cm). On continuous sucrose gradients, ATP-dependent proton transport and ionophore-stimulated ATPase activity coincided in a broad band extending from 1.08 to 1.15 grams per cubic centimeter with maximum activity at 1.10 to 1.12 grams per cubic centimeter. Large portions of the proton-translocating ATPase activity and ionophore-stimulated ATPase activity were clearly separable from mitochondrial membranes containing cytochrome c oxidase activity and azide-sensitive, pH 8.5 ATPase activity and from membranes bearing β-glucan synthetase I and II. The vesicles coincided with a minor portion of the NADH-cytochrome c reductase and nucleoside diphosphatase activities. It is suggested that the vesicles are of tonoplast origin.