Several crystal forms of the Bacillus stearothermophilus 50 S ribosomal particles

A kinetic analysis is presented of proton translocation, TMPD+ formation and oxidation of endogenous respiratory carriers during oxygen pulses of TMPD supplemented rat-liver mitochondria. The results show that antimycin-insensitive proton ejection observed under coupled conditions derives from oxidation of endogenous respiratory carriers and re-reduction of TMPD+ by hydrogenated donors and not from proton pumping by cytochrome oxidase as claimed by other investigators. The observations presented provide an example of certain interpretative difficulties in the use of redox mediators and of the methodological approaches that can be used to avoid these.     

Tetramethyl-p-phenylenediamine oxidase reaction in Azotobacter vinelandii.

It was possible to quantitate the tetramethyl-p-phenylenediamine (TMPD) oxidase reaction in Azotobacter vinelandii strain O using turbidimetrically standarized resting cell suspensions. The Q(O2) value obtained for whole cell oxidation of ascorbate-TMPD appeared to reflect the full measure of the high respiratory oxidative capability usually exhibited by this genera of organisms. The Q(O2) value for the TMPD oxidase reaction ranged from 1,700 to 2,000 and this value was equivalent to that obtained for the oxidation of the growth substrate, e.g., acetate. The kinetic analyses for TMPD oxidation by whole cells was similar to that obtained for the "particulate" A. vinelandii electron transport particle, that fraction which TMPD oxidase activity is exclusively associated with. Under the conditions used, there appeared to be no permeability problems; TMPD (reduced by ascorbate) readily penetrated the cell and oxidized at a rate comparable to that of the growth substrate. This, however, was not true for the oxidation of another electron donor, 2,6-dichloroindophenol, whose whole cell Q(O2) values, under comparable conditions, were twofold lower. The TMPD oxidase activity in A. vinelandii whole cells was found to be affected by the physiological growth conditions, and resting cells obtained from cells grown on sucrose, either under nitrogen-fixing conditions or on nitrate as the combined nitrogen source, exhibited low TMPD oxidase rates. Such low TMPD oxidase rates were also noted for chemically induced pleomorphic A. vinelandii cells, which suggests that modified growth conditions can (i) alter the nature of the intracellular terminal oxidase formed (or induced), or (ii) alter surface permeability, depending upon the growth conditions used. Preliminary studies on the quantitative TMPD oxidation reaction in mutant whole cells of both Azotobacter and a well-known Mucor bacilliformis strain AY1, deficient in cytochrome oxidase activity, showed this assay can be very useful for detecting respiratory deficiencies in the metabolism of whole cells.     

The effect of some sulfhydryl reagents on the respiratory chain activity

The observation that in cation transport experiments N-ethylmaleimide (NEM) behaves as uncoupler and as a respiratory inhibitor at the same time, the effect of sulfhydryl reagents on the redox state of respiratory chain, has been studied. Spectra of mitochondrial suspension in the span 300-630 nm have revealed that NEM promotes the oxidation of all the respiratory intermediates, cytochrome a included. Azide completely reverses the oxidation effect of NEM, suggesting that it cannot be ascribed to an irreversible damage of mitochondrial intactness. Mersalyl, which shares the highly sensitive SH reagent and specific inhibitor of Pi transport properties of NEM, gives completely different results. It is proposed that, besides the generally accepted inhibitory effect on primary dehydrogenases reacting with their SH groups, NEM may also behave as an oxidizing agent which can promote the release of reducing equivalents from the respiratory chain.     

Mitochondrial regulation of caspase activation by cytochrome oxidase and tetramethylphenylenediamine via cytosolic cytochrome c redox state

Cytochrome c release from mitochondria induces caspase activation in cytosols; however, it is unclear whether the redox state of cytosolic cytochrome c can regulate caspase activation. By using cytosol isolated from mammalian cells, we find that oxidation of cytochrome c by added cytochrome oxidase stimulates caspase activation, whereas reduction of cytochrome c by added tetramethylphenylenediamine (TMPD) or yeast lactate dehydrogenase/cytochrome c reductase blocks caspase activation. Scrape-loading of cells with this reductase inhibited caspase activation induced by staurosporine. Similarly, incubating intact cells with ascorbate plus TMPD to reduce intracellular cytochrome c strongly inhibited staurosporine-induced cell death, apoptosis, and caspase activation but not cytochrome c release, indicating that cytochrome c redox state can regulate caspase activation. In homogenates from healthy cells cytochrome c was rapidly reduced, whereas in homogenates from apoptotic cells added cytochrome c was rapidly oxidized by some endogenous process. This oxidation was prevented if mitochondria were removed from the homogenate or if cytochrome oxidase was inhibited by azide. This suggests that permeabilization of the outer mitochondrial membrane during apoptosis functions not just to release cytochrome c but also to maintain it oxidized via cytochrome oxidase, thus maximizing caspase activation. However, this activation can be blocked by adding TMPD, which may have some therapeutic potential.     

Routes of electron transfer in beef heart cytochrome c oxidase: is there a unique pathway used by all reductants?

Cytochrome c oxidase oxidizes several hydrogen donors, including TMPD (N,N,N',N'-tetramethyl-p-phenyl-enediamine) and DMPT (2-amino-6,7-dimethyl-5,6,7,8-tetrahydropterine), in the absence of the physiological substrate cytochrome c. Maximal enzyme turnovers with TMPD and DMPT alone are rather less than with cytochrome c, but much greater than previously reported if extrapolated to high reductant levels and (or) to 100% reduction of cytochrome a in the steady state. The presence of cytochrome c is, therefore, not necessary for substantial intramolecular electron transfer to occur in the oxidase. A direct bimolecular reduction of cytochrome a by TMPD is sufficient to account for the turnover of the enzyme. CuA may not be an essential component of the TMPD oxidase pathway. DMPT oxidation seems to occur more rapidly than the DMPT--cytochrome a reduction rate and may therefore imply mediation of CuA. Both "resting" and "pulsed" oxidases contain rapid-turnover and slow-turnover species, as determined by aerobic steady-state reduction of cytochrome a by TMPD. Only the "rapid" fraction (approximately 70% of the total with resting and approximately 85% of the total with pulsed) is involved in turnover. We conclude that electron transfer to the a3CuB binuclear centre can occur either from cytochrome a or CuA, depending upon the redox state of the binuclear centre. Under steady-state conditions, cytochrome a and CuA may not always be in rapid equilibrium. Rapid enzyme turnover by either natural or artificial substrates may require reduction of both and two pathways of electron transfer to the a3CuB centre.     

Cytochromes and hydrogen-oxidizing activity in the thermophilic hydrogen-oxidizing bacteria related to the genus Hydrogenobacter

The highly thermophilic, hydrogen-oxidizing aerobic bacteria related to Hydrogenobacter possess a respiratory chain comprising a quinone and b-type (alpha band at 556 nm and 562 nm) and c-type (alpha band at 552 nm) cytochromes. They have no aa₃-type cytochromes and their terminal oxidase is an o-type cytochrome. A polarographic method with an oxygen electrode was used for the measurement of the hydrogen-oxidizing activity. This activity was strongly inhibited by HQNO (2-N-heptyl-4-hydroxyquinoline N-oxide), an inhibitor of the respiratory chain in the quinone-cytochrome b region, and by KCN, an inhibitor of the terminal cytochrome oxidase. This study shows that the electrons released from hydrogen oxidation by the membrane-bound hydrogenase probably enter the respiratory chain at the level of the quinone-cytochrome b region.Abbreviations HQNO 2-N-heptyl-4-hydroxyquinoline N-oxide - TMPD N,N,N',N'-tetramethyl-p-phenylenediamine - DW dry weight     

Respiratory electron transfer activity in an asolectin-isooctane reverse micellar system.

Bovine heart submitochondrial particles (SMP) were solubilized in an asolectin isooctane reverse micellar system and the functionality of the respiratory chain was tested by spectroscopic and amperometric techniques. Electron transfer rate supported by NADH was very slow as evidenced by the low cytochrome reduction levels attained over long incubation periods. In the presence of KCN, NADH caused 34% and 12.5% reduction of the cytochromes aa3 and c, respectively, and negligible reduction of cytochrome b. Supplementation of the system with menadione rose the NADH-dependent reduction of all the cytochromes to levels that were close to the total content. However, no measurable O2 uptake activity took place in the presence of NADH plus menadione, or with ascorbate (or NADH) plus TMPD reducing systems. Therefore, it is suggested that in the organic medium, electron transfer from NADH to O2 is arrested at the terminal oxidase step. Cytochrome oxidase reduced by ascorbate (or NADH) plus TMPD seems to be trapped in its half reduced state (ie, a2+ a3(3+)). Although it is poorly reactive with O2, it can transfer electrons back to cytochrome c and TMPD. The electron transfer block to O2 was overcome when PMS was used instead of TMPD. This seems to be due to the recognized capacity of PMSH2 to carry out simultaneous reduction of both a CuA and a3 CuB redox centers of cytochrome oxidase. The cytochrome oxidase reaction in the organic solvent was highly sensitive to KCN (Ki 1.9 microM) and showed bell-shaped kinetics towards the PMS concentration and a sigmoidal response to water concentration, reaching its maximal turnover number (18 s-1) at 4 mM PMS and 1.1% (v/v) water.(ABSTRACT TRUNCATED AT 250 WORDS)     

Is supercomplex organization of the respiratory chain required for optimal electron transfer activity?

The supra-molecular assembly of the main respiratory chain enzymatic complexes in the form of "super-complexes" has been proved by structural and functional experimental evidence. This evidence strongly contrasts the previously accepted Random Diffusion Model stating that the complexes are functionally connected by lateral diffusion of small redox molecules (i.e. Coenzyme Q and cytochrome c). This review critically examines the available evidence and provides an analysis of the functional consequences of the intermolecular association of the respiratory complexes pointing out the role of Coenzyme Q and of cytochrome c as channeled or as freely diffusing intermediates in the electron transfer activity of their partner enzymes.     

N-ethylmaleimide as oxidizing agent in biological and non-biological systems

The hypothesis that N-ethylmaleimide (NEM) may function as an oxidizing agent in biological and non-biological systems, has been tested. Spectrophotometric determination of cytochrome a-redox-state have revealed that NEM promotes the transition of this respiratory chain component in a more oxidated state. To overcome the possibility that the NEM effect may be determined by the inhibition on primary dehydrogenase, duroquinol (QH2) has been used as substrate in the presence of rotenone and malonate. The stimulation of cytochrome a-oxidation is correlated to the one on the QH2 oxidation determined by following the formation of duroquinone. In the absence of any biological system, spontaneous oxidation of ferrocyanide and TMPD is greatly increased by NEM. The differential stimulation induced by maleimide and succinimide indicate that the oxidizing effect of NEM may be considered a chemico-physical property of its molecule mainly due to the presence of a double bond. It is proposed that besides a sulfhydryl reagent, NEM behaves as an oxidizing agent with an interacting site in the region QH2-cytochrome a of the respiratory chain.     

Modulation of cytochrome c-mediated extramitochondrial NADH oxidation by contact site density.

Data presented in previous reports suggest that in rat liver mitochondria a "bi-trans-membrane" electron transport pathway is present which promotes the transfer of reducing equivalents directly from cytosolic NADH to molecular oxygen inside the mitochondria. Here we show that the oxidation of external NADH is stimulated by atractylate + ADP and greatly inhibited by glycerol. These two conditions have been documented to promote the increase and the decrease respectively of the frequency of "contact sites" between the two mitochondrial membranes. NADH oxidation is not affected at all by glycerol and atractylate + ADP when TMPD and endogenous cytochrome c are utilized as electron carriers. The results obtained are consistent with the proposal that the bi-trans-membrane electron transport chain might be localized at the level of respiratory contact sites having the function of promoting the oxidation of the surplus amount of cytosolic NADH. This electron transport pathway has been suggested to play a decisive role in the early stages of apoptosis [Biochem. Biophys. Res. Commun. 246, 556-561, 1998].     

Mitochondrial respiration at low levels of oxygen and cytochrome c

In the intracellular microenvironment of active muscle tissue, high rates of respiration are maintained at near-limiting oxygen concentrations. The respiration of isolated heart mitochondria is a hyperbolic function of oxygen concentration and half-maximal rates were obtained at 0.4 and 0.7 microM O(2) with substrates for the respiratory chain (succinate) and cytochrome c oxidase [N,N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)+ascorbate] respectively at 30 degrees C and with maximum ADP stimulation (State 3). The respiratory response of cytochrome c-depleted mitoplasts to external cytochrome c was biphasic with TMPD, but showed a monophasic hyperbolic function with succinate. Half-maximal stimulation of respiration was obtained at 0.4 microM cytochrome c, which was nearly identical to the high-affinity K(')(m) for cytochrome c of cytochrome c oxidase supplied with TMPD. The capacity of cytochrome c oxidase in the presence of TMPD was 2-fold higher than the capacity of the respiratory chain with succinate, measured at environmental normoxic levels. This apparent excess capacity, however, is significantly decreased under physiological intracellular oxygen conditions and declines steeply under hypoxic conditions. Similarly, the excess capacity of cytochrome c oxidase declines with progressive cytochrome c depletion. The flux control coefficient of cytochrome c oxidase, therefore, increases as a function of substrate limitation of oxygen and cytochrome c, which suggests a direct functional role for the apparent excess capacity of cytochrome c oxidase in hypoxia and under conditions of intracellular accumulation of cytochrome c after its release from mitochondria.     

Pathways of cytochrome c oxidation by soluble and membrane-bound cytochrome aa3

In media of low ionic strength, membraneous cytochrome c oxidase, isolated cytochrome c oxidase, and proteoliposomal cytochrome c oxidase each bind cytochrome c at two sites, one of low affinity (1 microM greater than Kd' greater than 0.2 microM) and readily reversible and the other of high affinity (0.01 microM greater than Kd) and weakly reversible. When cytochrome c occupies both sites, including the low affinity site, the maximal turnover measured polarographically with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) is independent of TMPD concentration, and lies between 250 and 400 s-1 (30 degrees C, pH 7.4) for fully activated systems. The apparent affinity of the enzyme for cytochrome c is, however, TMPD dependent. When cytochrome c occupies only the high-affinity site, the maximal turnover is closely dependent upon the concentration of TMPD, which, unlike ascorbate, can reduce bound cytochrome c. As TMPD concentration is increased, the maximal turnover approaches that seen when both sites as occupied. The lower activity of isolated cytochrome aa3 is due to the presence of inactive or inaccessible enzyme molecules. Incorporation of isolated enzyme into phospholipid vesicles restores full activity to all the subsequently accessible cytochrome aa3 molecules. Negatively charged (asolectin) vesicles show a higher cytochrome c affinity at the low-affinity sites than do the other enzyme preparations. A model for the cytochrome c-cytochrome aa3 complexes is put forward in which both sites, when occupied, are fully catalytically competent, but in which occupation of the "tight" site by a catalytically functional cytochrome c molecule is required for overall oxidation of cytochrome c via the "loose" site.     

Oxidation and reduction of exogenous cytochrome c by the activity of the respiratory chain.

Oxidation of exogenous NADH by isolated rat liver mitochondria is generally accepted to be mediated by endogenous cytochrome c which shuttles electrons from the outer to the inner mitochondrial membrane. More recently it has been suggested that, in the presence of added cytochrome c, NADH oxidation is carried out exclusively by the cytochrome oxidase of broken or damaged mitochondria. Here we show that electrons can be transferred in and out of intact mitochondria. It is proposed that at the contact sites between the inner and the outer membrane, a "bi-trans-membrane" electron transport chain is present. The pathway, consisting of Complex III, NADH-b5 reductase, exogenous cytochrome c and cytochrome oxidase, can channel electrons from the external face of the outer membrane to the matrix face of the inner membrane and viceversa. The activity of the pathway is strictly dependent on both the activity of the respiratory chain and mitochondrion integrity.     

Post-mortem changes in structure and function of ox muscle mitochondria. 1. Electron microscopic and polarographic investigations

Electron microscopy shows that intact mitochondria can be isolated from neck-muscle stored at 144h post-mortem at 4°. Isolated mitochondria, all in the condensed configuration, have clearly defined outer and inner membranes, outer compartments and intracristal spaces; a larger proportion of swollen ones was isolated from the 144h than from the 120 h post-mortem tissue.Mitochondria from 96 h tissue still retained the following % of the initial values for the ADP/O ratio, respiratory control index (RCI) and state 3 respiratory rate observed for 0–5h tissue: malate+pyruvate, 100, 72 and 53; succinate, 80, 30 and 74; ascorbate+ tetramethyl-p-phenylencdiamine (TMPD), 92, 88 and 72.Both the succinate and ascorbate-TMPD oxidase systems appear to have a critical storage time of about 70 h, whereas the malate+pyruvate system has one of about 96 h. Asharp decline of the ADP/O ratio, RCI and the state 3 respiratory rate occurred after this time, but these three parameters were better preserved in the ascorbate-TMPD oxidase system.The oxidation of the citric acid cycle intermediates in the neck-muscle mitochondria thus shows a higher sensitivity to post-mortem ageing with respect to cytochrome oxidase activity. This is probably due to post-mortem muscle acidification.     

Terminal branching of the respiratory electron transport chain in Neisseria meningitidis

The respiratory components of the envelope membrane preparation of Neisseria meningitidis were investigated. Oxidase activities were demonstrated in this fraction in the presence of succinic acid, reduced nicotinamide adenine dinucleotide, and ascorbate-N,N,N',N'-tetramethyl-p-phenylene-diamine (TMPD). Differences in the kinetics of inhibition by terminal oxidase inhibitors on the three oxidase activities indicated that ascorbate-TMPD oxidation involved only an azide-sensitive oxidase, whereas oxidation of the physiological substrates involved two oxidases, one of which was relatively azide resistant. Spectrophotometric studies revealed that ascorbate-TMPD donated its electrons exclusively to cytochrome o, whereas the physiological substrates were oxidized via both cytochromes o and a. The effects of class II inhibitors on the oxidases suggest terminal branching of the electron transport chain at the cytochrome b level. A model of the respiratory system in N. meningitidis is proposed.     

Inhibition of respiratory oxidation of elemental sulfur (S0) and thiosulfate in Thiobacillus versutus and another sulfur-oxidizing bacterium

Abstract The rates of thiosulfate, elemental sulfur (S⁰) and sulfite oxidation were measured respirometrically with an oxygen electrode using young cells of Thiobacillus versutus growing chemolithoautotrophically on thiosulfate under normal air pressure. Myxothiazol, an inhibitor of the cytochrome b−c₁ segment, and HQNO (2-N-heptyl-4-hydroxyquiniline N-oxide), acting in the quinone-cytochrome b region, both significantly inhibited the thiosulfate oxidation rate. The effect on the oxidation rate of S⁰ was even stronger. The oxidation of sulfite or ascorbate + TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) (substrates releasing electrons at the level of cytochrome c) was not inhibited by myxothiazol and HQNO. Thiosulfate, S⁰, sulfite and ascorbate + TMPD oxidations were strongly inhibited by KCN. These respiratory activities were almost completely eliminated by cell breakage. The reduction of b-type cytochrome was observed in thiosulfate-reduced minus sulfite-reduced difference spectra. This study confirms that S⁰ is an important intermediate of thiosulfate oxidation in Thiobacillus versutus , and that electrons released by S⁰ oxidation enter the respiratory chain in the quinone-cytochrome b region. This would allow an increased gain of energy, while less energy would probably be required for pyridine-nucleotide reduction.     

Effects of oxygen on the metabolism of nitroxide spin labels in cells

The products of the reduction of nitroxides in cells are the corresponding hydroxylamines, which cells can oxidize back to the nitroxides in the presence of oxygen. Both the reduction of nitroxides and the oxidation of hydroxylamines are enzyme-mediated processes. For lipid-soluble nitroxides, the rates of reduction are strongly dependent on the intracellular concentration of oxygen; severely hypoxic cells reduce nitroxides more rapidly than cells supplied with oxygen. In contrast, the rates of oxidation of hydroxylamines increase smoothly with increasing intracellular oxygen concentration up to 150 microM. In order to separate the effects on the rates of metabolism of nitroxides due directly to oxygen from effects due to the redox state of enzymes, we studied the cells under conditions in which each of these variables could be changed independently. Oxygen affects the metabolism of these nitroxides primarily by interacting with cytochrome c oxidase to change the redox state of the enzymes in the respiratory chain. Our results are consistent with the conclusions that in these cells reduction of lipophilic nitroxides occurs at the level of ubiquinone in the respiratory chain in mitochondria, and oxidation of the corresponding hydroxylamines occurs at the level of cytochrome c oxidase.     

Multiphasic oxidation-reduction of cytochrome b in the succinate-cytochrome c reductase

The triphasic course previously reported for the reduction of cytochrome b in the succinate-cytochrome c reductase by either succinate or duroquinol has been shown to be dependent on the redox state of the enzyme preparation. Prior reduction with increasing concentrations of ascorbate leads to partial reduction of cytochrome c1, and a gradual decrease in the magnitude of the oxidation phase of cytochrome b. At an ascorbate concentration sufficient to reduce cytochrome c1 almost completely, the reduction of cytochrome b by either succinate or duroquinol becomes monophasic. Owing to the presence of a trace amount of cytochrome oxidase in the reductase preparation employed, the addition of cytochrome c makes electron flow from substrate to oxygen possible. Under such circumstances, the addition of a limited amount of either succinate or duroquinol leads to a multiphasic reduction and oxidation of cytochrome b. After the initial three phases as described previously, cytochrome b becomes oxidized before cytochrome c1 when the limited amount of added substrate is being used up. However, at the end of the reaction when cytochrome c1 is being rapidly oxidized, cytochrome b becomes again reduced. The above observations support a cyclic scheme of electron flow in which the reduction of cytochrome b proceeds by two different routes and its oxidation controlled by the redox state of a component of the respiratory chain.     

Energy transduction in photosynthetic bacteria. X. Composition and function of the branched oxidase system in wild type and respiration deficient mutants of Rhodopseudomonas capsulata.

The respiratory chain of Rhodopseudomonas capsulata, strain St. Louis and of two respiration deficient mutants (M6 and M7) has been investigated by examining the redox and spectral characteristics of the cytochromes and their response to substrates and to specific respiratory inhibitors. Since the specific lesions of M6 and M7 have been localized on two different branches of the multiple oxidase system of the wild type strain, the capability for aerobic growth of these mutants can be considered as a proof of the physiological significance of both branched systems "in vivo". Using M6 and M7 mutants the response of the branched chain to respiratory inhibitors could be established. Cytochrome oxidase activity, a specific function of an high potential cytochrome b (E'0 = +413 mV) is sensitive to low concentrations of KCN (5-10(-5) M); CO is a specific inhibitor of an alternative oxidase, which is also inhibited by high concentrations of KCN (10(-3) M). Antimycin A inhibits preferentially the branch of the chain affected by low concentrations of cyanide. Redox titrations and spectral data indicate the presence in the membrane of three cytochromes of b type (E'0 = +413, +260, +47 vM) and two cytochromes of c type (E'0 = +342, +94 mV). A clear indication of the involvement in respiration of cytochrome b413, cytochrome c342 and cytochrome b47 has been obtained. Only 50% of the dithionite reducible cytochrome b can be reduced by respiratory substrates also in the presence of high concentrations of KCN or in anaerobiosis. The presence and function of quinones in the respiratory electron transport system has been clearly demonstrated. Quinones, which are reducible by NADH and succinate to about the same extent can be reoxidized through both branches of the respiratory chain, as shown by the response of their redox state to KCN. The possible site of the branching of the electron transport chain has been investigated comparing the per cent level of reduction of quinones and of cytochromes b and c as a function of KCN concentrations in membranes from wild type and M6 mutants cells. The site of the branching has been localized at the level of quinones-cytochrome b47. A tentative scheme of the respiratory chains operating in Rhodopseudomonas capsulata, St. Louis and in the two respiration deficient mutants, M6 and M7 is presented.     

INFLUENCE OF POTASSIUM ON THE STEADY-STATE REDOX POTENTIAL OF THE ELECTRON TRANSPORT CHAIN IN SLICES OF RAT CEREBRAL CORTEX AND THE EFFECT OF OUABAIN

Abstract— The concentration dependence of the modifications by potassium of the respiratory intermediates in incubated slices of rat cerebral cortex has been examined in the presence and absence of calcium. In addition to the immediate increase in respiration and the concomitant oxidation of the respiratory intermediates, longer term increases in the steady-state redox potential were observed at higher potassium concentrations. Addition of calcium to the system did not appreciably alter the immediate effects of potassium, but shifted the redox state of the respiratory intermediates; these changes involved a decrease in reduced intermediate at low concentrations of potassium and a relatively higher level of reduced carriers at high concentrations of potassium. Ouabain (50 μ m ) inhibited both the initial responses to added potassium and modified the changes in steady-state levels of reduced intermediate in the absence of calcium. In the presence of calcium, ouabain (50 μ m ) inhibited the initial oxidation of NAD(P)H observed upon addition of potassium but had no effect on the respiratory response to the addition of low concentrations of potassium. The disassociation of these responses resulted in a large decrease in the steady-state levels of reduced cytochrome. At 30 m m potassium an oxidation of NAD(P)H was observed which accompanied by an increase in levels of reduced cytochromes. These changes in redox state of the respiratory carriers have been discussed in relation to previous reports dealing with the effects of potassium on aerobic glycolysis and oxygen consumption by brain slices.