Effects of chronic ethanol treatment of mitochondrial functions damage to coupling site I

Chronic ethanol feeding to rats produces changes in hepatic mitochondria which persist in the absence of ethanol metabolism. The integrity of isolated mitochondria is well preserved, as evidenced by unchanged activities of latent, Mg²⁺- and dinitrophenol-stimulated ATPase activity, and unaltered permeability to NADH. With succinate or ascorbate as substrates, oxygen uptake by mitochondria from ethanol-fed rats was decreased compared to pair-fed controls. The decrease was comparable under state 4 or state 3 conditions, or in the presence of an uncoupler. However, with the NAD⁺-dependent substrates, ADP-stimulated oxygen consumption (state 3) was decreased to a greater extent than state 4 or uncoupler-stimulated oxygen consumption in mitochondria from ethanol-fed rats. This suggests that the decrease in energy-dependent oxygen consumption at site I may be superimposed upon damage to the respiratory chain. Using NAD⁺-dependent substrates (glutamate, α-ketoglutarate or β-hydroxybutyrate) the respiratory control ratio and the $\text{P}\text{O}$ ratio of oxidative phosphorylation were significantly decreased in mitochondria isolated from the livers of rats fed ethanol. By contrast, when succinate or ascorbate served as the electron donor these functions were unchanged. The rate of phosphorylation is decreased 70% with the NAD⁺-dependent substrates because of a decreased flux of electrons, as well as a lower efficiency of oxidative phosphorylation. With succinate and ascorbate as substrates, the rate of phosphorylation is decreased 20–30%, owing to a decreased flux of electrons. These data suggest the possibility that, in addition to effects on the respiratory chain, energy-coupling site I may be damaged by ethanol feeding. Energy-dependent Ca²⁺ uptake, supported by either substrate oxidation or ATP hydrolysis, was inhibited by chronic ethanol feeding.Concentrations of acetaldehyde (1–3 mm) which inhibited phosphorylation associated with the oxidation of NAD⁺-dependent substrates had no effect on that of succinate or ascorbate. Many of the effects of chronic ethanol feeding on mitochondrial functions are similar to those produced by acetaldehyde in vitro.     

A re-evaluation of conditions required for an accurate estimation of the extramitochondrial ATPADP ratio in isolated rat-liver mitochondria

The values reported in the literature for the extramitochondrial $\text{ATP}\text{ADP}$ ratio in resting rat-liver mitochondria (State 4) vary widely. The conditions required for an accurate determination of this parameter were therefore investigated. (1) In experiments with rat-liver mitochondria incubated under State-4 conditions, it was found that the extramitochondrial $\text{ATP}\text{ADP}$ ratio, as calculated from the values measured in neutralised perchloric acid extracts, was lower than that estimated from the concentrations of creatine and creatine phosphate, using the metabolite indicator method. The discrepancy is due to hydrolysis of ATP occurring in the presence of perchloric acid. (2) Conditions are described for minimising ATP hydrolysis in the presence of perchloric acid, and include the use of low concentrations of perchloric acid, short times of exposure to the acid before neutralisation, low temperatures and the presence of excess EDTA. Under these conditions, the values obtained for the extramitochondrial $\text{ATP}\text{ADP}$ ratio agreed with those calculated by the metabolite indicator method, provided ratios do not exceed the value of 100. (3) In cases where the extramitochondrial $\text{ATP}\text{ADP}$ does exceed 100, phenol/chloroform/isoamyl alcohol must be used to quench the reactions, as described by Slater et al. (Slater, E.C., Rosing, J. and Mol, A. (1973) Biochim. Biophys. Acta 292, 534–553). With this method, the extramitochondrial $\text{ATP}\text{ADP}$ ratio was found to have a value of more than 1000 in rat-liver mitochondria incubated with succinate + rotenone in the resting state (pH 7.0; T = 37°C), in agreement with Slater et al.     

The inactivation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria.

The influence of fatty acid on the interconversion of the pyruvate dehydrogenase complex (PDH) between its active (dephospho-) and inactive (phospho-) forms and on the intramitochondrial $\text{ATP}\text{ADP}$, $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ and $\text{acetyl-CoA}\text{CoASH}$ ratios was studied in isolated rat liver mitochondria. Conditions were found in which the PDH activity was inversely correlated only with the $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio. Under other conditions the PDH activity was inversely correlated solely with the $\text{acetyl-CoA}\text{CoASH}$ ratio. These experiments suggest that the activity of the regulatory enzymes involved in the inactivation and reactivation of the pyruvate dehydrogenase multienzyme complex may be controlled by both the intramitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ and $\text{acetyl-CoA}\text{CoASH}$ ratios.     

Cyclic AMP inhibition of reactions of glyceraldehyde 3-phosphate dehydrogenase

Adenosine 3′,5′-monophosphate (cyclic AMP) is an inhibitor of the reaction of d-glyceraldehyde 3-phosphate dehydrogenase with glyceraldehyde 3-phosphate and benzaldehyde. Inhibition appears to be competitive toward glyceraldehyde 3-phosphate and of a mixed type toward NAD⁺. In the absence of arsenate a plot of $\text{1}\text{V}$ vs (I) is sigmoidal at constant concentrations of glyceraldehyde 3-phosphate and NAD⁺ and linear at constant concentrations of benzaldehyde and NAD⁺. Thus, sigmoidal inhibition plots are dependent on the nature of the aldehyde substrate as was found previously to be the case with inhibition of these reactions by highly branched acyl phosphates. In the presence of 0.013 m arsenate the plots of $\text{1}\text{V}$ vs [I] are linear.     

Control of the pyridine nucleotide-linked Ca release from mitochondria by respiratory substrates

Oxidation of mitochondrial pyridine nucleotides followed by their hydrolysis promotes Ca²⁺ release from intact liver mitochondria. In most of the previous studies oxidation was achieved with pro-oxidants which were added to mitochondria respiring on succinate in the presence of rotenone, a site I-specific inhibitor of the respiratory chain. Here we investigate pro-oxidant dependent and independent Ca²⁺ release from mitochondria when respiration is supported either by the NAD⁺-linked substrate β-hydroxybutyrate, or by succinate. In the presence, as well as in the absence, of the pro-oxidant t-butylhydroperoxide mitochondria retain Ca²⁺ much better with succinate than with β-hydroxybutyrate, as respiratory substrate. When Ca²⁺ release is induced by t-butylhydroperoxide succinate-supported Ca²⁺ retention is impeded by rotenone. Ca²⁺ release (pro-oxidant dependent or independent) is paralleled by oxidation and hydrolysis of intramitochondrial pyridine nucleotides, and Ca²⁺ retention is paralleled by reduction of pyridine nucleotides. It is concluded that the pyridine nucleotide-linked Ca²⁺ release from mitochondria can be controlled by respiratory substrates which regulate the intramitochondrial hydrolysis of oxidized pyridine nucleotides.     

Control of mitochondrial respiration: a quantitative evaluation of the roles of cytochrome c and oxygen.

The dependence of the mitochondrial respiratory rate on the reduction of cytochrome c has been measured as a function of the exogenous $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ ratio and pH. The respiratory rate at $\text{[}\text{ADP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ values of less than 10^-1m^-1 is proportional to the reduction of cytochrome c and independent of pH from pH 6.5 to pH 8.O. The maximal turnover number (at 100% reduction) for cytochrome c is approximately 70 s^?1. As the $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ ratio is increased from 10^?1m^?1 to 10⁴m^?1, the respiration at any given level of reduction of cytochrome c is progressively inhibited. Greater inhibition is observed at more oxidized levels of cytochorme c with respiratory control values for oxidation of reduced cytochrome c exceeding 10. The behavior of mitochondrial respiratory control is shown to be quantitatively consistent with a proposed mechanism in which the regulation occurs in the reaction of oxygen with cytochrome oxidase. A steady-state rate expression is derived which fits the mitochondrial respiratory rate dependence on (i) the extramitochondrial $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ ratio; (ii) the level of reduction of cytochrome c (or the intramitochondrial $\text{[}\text{NAD}{}^{\mathrm{+}}\text{]}\text{[NADH])}$ at different $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ values; (iii) the pH of the suspending medium. This rate expression appears to correctly predict the relationships of the cytoplasmic $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ ratio, the mitochondrial $\text{[}\text{NAD}{}^{\mathrm{+}}\text{]}\text{[}\text{NADH}\text{]}$ ratio, and the mitochondrial respiratory rate in intact cells as well as suspensions of isolated mitochondria.     

On the energy-coupling sites inParacoccus denitrificans

Cell-free preparations fromParacoccus denitrificans grown aerobically under autotrophic and heterotrophic conditions catalyzed succinate- and ascorbate-linked ATP-dependent NAD⁺ reduction. The succinate-linked NAD⁺ reduction was sensitive to rotenone and indicated the participation of coupling site 1. The ascorbate-linked, energy-driven, reversed electron flow was sensitive to antimycin A or 2-n-heptyl-4-hydroxyquinoline-N-oxide and rotenone, thus indicating the mediation of coupling sites 2 and 1. The aerobic oxidation of three ascorbate equivalents was required to reduce one NAD⁺ equivalent in the autotrophic system, while no NAD⁺ reduction coupled to ascorbate oxidation occurred in the heterotrophic system unlessN,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) was present. In the later case, the oxidation of 24 equivalents of reduced TMPD was required to support the reduction of each equivalent of NAD⁺ These results indicated that, while there was an efficient energy-coupling at site 3 in the autotrophic system, the heterotrophic system appeared to posses a very weak or inefficient terminal energy-coupling site.     

Multiple forms of cyclic nucleotide phosphodiesterases in normal and goitrous rat thyroid

The stoichiometry of H⁺ ejection coupled to electron flow from succinate to ferricyanide in the electron transport chain of mitochondria from Ehrlich ascites tumor and AS30-D hepatoma cells was determined. Values close to 4.0 for the $\text{H}{}^{\mathrm{+}}\text{2e}{}^{\mathrm{?}}$ ejection ratio were found in both cell lines, with either Ca²⁺ or K⁺ (+ valinomycin) as charge-compensating permeant cation. The 4 H⁺ ejected were compensated by outward movement of two negative charges to reduce 2 Fe(CN)6^3? to 2 Fe(CN)6^4?, and the uptake of two positive charges in the form of the permeant cation. Experiments on (a) omission of rotenone (b) the effect of antimycin A and (c) depletion of endogenous NAD(P)-linked substrates showed that no significant endogenous electron flow or H⁺ ejection occurred, thus eliminating possible overestimation of the H⁺/2e^? ratio from endogenous substrates. These data on mitochondria from two tumor cell lines are fully consistent with earlier measurements of the H⁺/O stoichiometry for succinate and NADH oxidation in tumor mitochondria and with the $\text{H}{}^{\mathrm{+}}\text{2e}{}^{\mathrm{?}}$ stoichiometry for site 2 in normal rat liver mitochondria.     

Metabolic regulation of malic enzyme activity from Paracoccus denitrificans by glyoxylate and acetylCoA.

$\text{Paracoccus denitrificans}$ contains both NAD⁺- and NADP⁺-linked malic enzyme activities when grown on malate/nitrate. The enzyme is inactive in the absence of NH₄⁺. AcetylCoA inhibits both activities competitively with respect to L-malate. Glyoxylate (0.5 mM) causes 60% inhibition of the NADP⁺-linked activity but has little effect on the NAD⁺-linked activity. Citrate, aspartate, AMP, ADP, and ATP, at 0.5mM, have little effect on either of the two activities. The results are discussed with regards to the control of malic enzyme activity within the cell.     

Relations between extramitochondrial and intramitochondrial adenine nucleotide systems

The control of oxidative phosphorylation by the extramitochondrial $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{]}$ ratio and [P_i] was investigated by incubations of isolated mitochondria with an ADP regenerating system and by a new perifusion technique using glass filters for immobilization of mitochondria. With mitochondria from different sources oxidizing different substrates and with both techniques, similar results were obtained. Changes of the extramitochondrial $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{]}$ ratio from about 100 to 5 transfer mitochondria from the resting state (state 4) to the fully active state (state 3). The importance of the adenine nucleotide translocator in this transition was demonstrated by the influence of its specific inhibitor carboxyatractyloside. The sensitivity to the inhibitor was more pronounced in states with high $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{]}$ ratios than in the fully active state. In the hexokinase-glucose system the action of the inhibitor caused a transition to a new steady state, where a decreased $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{]}$ ratio overcomes the inhibition. Thus, a partial inhibition of the translocator shifted the control characteristics to lower $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{]}$ ratios. When the concentration of inorganic phosphate was decreased, the main effect was a reduction of the maximum rate of oxidative phosphorylation (i.e., in state 3), whereas the $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{]}$ sensitive range was not altered. This effect is caused by changes in the intramitochondrial phosphorylation potential. Furthermore, this indicates that the kinetic properties of the adenine nucleotide translocator prevent a simple equilibration of the phosphorylation potential across the inner membrane. This is also demonstrated by the fact that the extramitochondrial formation of glucose-6-phosphate and the intramitochondrial synthesis of citrulline compete for ATP.     

Effect of temperature on the pathways of NADH-oxidation in broad-bean mitochondria

1. Respiration rates of broad-bean (Vicia faba) mitochondria were studied as a function of temperature. Arrhenius plots of all membrane-bound enzymes, as obtained with saturating substrate concentrations, revealed a break in the lower temperature range. That break was considered to indicate a phase transition of membrane phospholipids, characteristic for chilling-sensitive plants. A second discontinuity at 30°C occurred only with activities linked to energy conservation. — 2. The activation energies for the oxidation of NAD⁺-linked substrates differ between states 3 and 4. State 3 respiration of NAD⁺-linked substrates is the result a superimposition of two branches of electron transport, which can be separated by different sensibilities to rotenone. A characteristic temperature dependency of the respiratory control, as well as a shift of the low temperature break in the Arrhenius plot toward a higher temperature after state 4 to state 3 transition, are calculated to be caused by the superimposition of the two branches. — 3. The temperature dependency of the oxidation of extra-mitochondrial NADH and of succinate differs remarkably from that of the oxidation of matrix-NADH. It has been concluded that the rotenone-resistant oxidation of matrix-NADH and the oxidation of external NADH are mediated via different pathways with individual regulation sites.Abbreviations BSA bovine serum albumin - CCCP carbonylcyanide-m-chlorophenylhydrazone - TPP thiaminepyrophosphate     

Role of substrates in Sr2+-induced oscillations of ionic fluxes in rat liver mitochondria

(1) The reason for substrate specificity of Sr²⁺-induced oscillating cation fluxes in isolated rat liver mitochondria was investigated. (2) With succinate as substrate, rotenone prevented oscillation. In this case the mitochondria were only partially able to take up added Sr²⁺ and did not take up any of the released K⁺. Addition of substances decreasing the mitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio (oxaloacetate or acetoacetate) restored the ability for reuptake of K⁺ and for complete uptake of Sr²⁺ and, therefore, oscillation. (3) Inhibition of substrate-level phosphorylation by arsenite or uncoupling of substrate-level phosphorylation by arsenate in the presence of oligomycin also suppressed the reuptake of cations. This effect of inhibition of substrate-level phosphorylation on oscillation could be circumvented by addition of ATP in the presence of oligomycin. (4) Prevention of the intramitochondrial regeneration of 2-oxoglutarate from acetyl-CoA and oxaloacetate by fluorocitrate or from endogenous glutamate by aminoxyacetate shortened the time during which oscillation with succinate as substrate could be observed. (5) From the key role of substrate level phosphorylation it is concluded that for the reuptake of K⁺ and Sr²⁺ during oscillation, sufficient GTP generation by the succinyl thiokinase (EC 6.2.1.4) reaction is essential. Therefore substrate level phosphorylation seems to be a necessary energy source additional to the respiratory chain. Since the latter process drives the active cation movements, the former may be required for the restoration of a sufficiently low proton conductance of the mitochondrial inner membrane. Oscillation in the absence of exogenous ATP therefore demands 2-oxoglutarate as substrate or the intramitochondrial generation of 2-oxoglutarate for the maintenance of a sufficient GTP production for a longer time.     

The regulation of pyruvate dehydrogenase by fatty acids in isolated rabbit heart mitochondria.

The rate of pyruvate oxidation by isolated rabbit heart mitochondria was inhibited by fatty acylcarnitine derivatives. The extent of inhibition by pyruvate oxidation in State 3 was greatest with palmitylcarnitine and only a minimal inhibition was observed with acetylcarnitine, while octanoylcarnitine or octanoate caused an intermediate extent of inhibition. Analyses of the intramitochondrial $\text{ATP}\text{ADP}$ and $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratios under the different conditions of incubation indicated that it is unlikely that changes in either or both of these parameters were the primary negative effectors of the rate of pyruvate oxidation. A positive correlation between the decrease in the rate of pyruvate oxidation and the decrease in the level of free CoASH in the mitochondria was observed. Extraction and assay of the pyruvate dehydrogenase from rabbit heart mitochondria during the time course of the fatty acid-mediated inhibition of pyruvate oxidation indicated that pyruvate dehydrogenase was strongly inactivated when palmitylcarnitine was the fatty acid, while incubation with octanoate and acetylcarnitine resulted in less extensive inactivation of pyruvate dehydrogenase. Measurement of the effects of NADH, NAD⁺, acetyl-CoA, and CoASH on the inactivation of pyruvate dehydrogenase extracted from rabbit heart mitochondria indicated that NADH and acetyl-CoA activated the pyruvate dehydrogenasee kinase while CoASH strongly inhibited the kinase and NAD⁺ was without effect. In addition, palmityl-CoA and octanoyl-CoA had little, if any, effect on the pyruvate dehydrogenase kinase activity. It was observed that palmityl-CoA but not octanoyl-CoA strongly inhibited the activity of the extracted pyruvate dehydrogenase. Hence, it is concluded that (a) decreased mitochondrial CoASH levels, which essentially remove a potent inhibitor of the pyruvate dehydrogenase kinase, (b) possibly a diminished free CoASH supply, which may be utilized as a substrate for the active complex, and (c) direct inhibitory effects of palmityl-CoA on the active form of the pyruvate dehydrogenase complex combine to make palmitylcarnitine a much more potent inhibitor of mitochondrial pyruvate oxidation than shorter chain length acylcarnitine derivatives.     

An adenine nucleotide translocase in the procaryote Methanobacterium thermoautotrophicum

ATP synthesis, ATP hydrolysis and ADP uptake by membrane vesicles of Methanobacterium thermoautotrophicum are inhibited by inhibitors of mitochondrial $\text{ADP}\text{ATP}$ translocases. Atractyloside binds to one of the membrane proteins. These data demonstrate the presence of an eucaryotic type of $\text{ADP}\text{ATP}$ translocase in a procaryotic microorganism and stress the unique position of methanogenic bacteria in evolution.     

Multiple cytochromes b in Mycobacterium phlei

Electron transport particles from $\text{M. phlei}$ contain at least 3 different active forms of cytochrome b, one reduced by NADH, with a λ_max at 563 nm (b_N563), and the other two reduced by either succinate or NADH, with λ_max at 559 and 563 nm (b_S559) and (b_S563). Low temperature λ_max for cytochrome b reduction with NADH or succinate are described. During steady state only b_S563 was observed with succinate. In the presence of ATP, succinate reduced an increased amount of a b563. A branching of the NAD⁺-linked pathway and a convergence at the level of cytochrome c is suggested, with only one branch accessible to succinate.     

Metabolic effects of glucose deprivation and of various sugars in normal and transformed fibrobalst cell lines.

The sugar composition of the growth medium influenced the $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio, pyruvate and lactate production, and ATP levels in both normal and transformed fibroblast cell lines growing in tissue culture. Removal of glucose led to a rapid three- to fourfold rise in the $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio, followed by a slower decline in the content of ATP. However, there was no change in the adenylate energy charge [$\text{(}\text{ATP}\text{+}\text{1}\text{2}\text{ADP}\text{)/(}\text{ATP + ADP + AMP}\text{)}$] over a 2-h period. The $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio was restored to the original level within 10 s of glucose readdition. The $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ratios in cell lines growing on galactose were as high as for those incubated without sugars; growth on mannose or fructose produced intermediate ratios. There was an inverse relationship between the $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio and pyruvate-lactate production for glucose, fructose and galactose. Thus, all cell lines had a high production of pyruvate and lactate but a low $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio when grown on glucose. In contrast, when galactose served as the sugar source, acid production was low, while the ratio was high. All cell lines had comparable hexokinase activity, and glucose was the best substrate, mannose intermediate and fructose poorest. Hexokinase activity did not correlate with the relative degree of utilization of the sugars. These results suggest that the sugar composition of the growth medium affects the metabolic pattern of a cell line, including the $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio, the ATP content and the production of pyruvate and lactate.     

Light scattering and quenching of 9-aminoacridine fluorescence as indicators of the phosphorylation state of the adenylate system in intact spinach chloroplasts

Upon illumination of suspensions of intact chloroplasts, fluorescence of 9-aminoacridine was quenched, light scattering was increased, chlorophyll fluorescence was decreased after an initial increase, and chloroplast $\text{ATP}\text{ADP}$ ratios were increased. The response of 9-aminoacridine fluorescence quenching and light scattering to light intensity, anaerobiosis and inhibition of electron transport by DCMU was similar to that shown by chloroplast $\text{ATP}\text{ADP}$ ratios. It is discussed under what conditions 9-aminoacridine fluorescence quenching or light scattering can be used to monitor changes in the phosphorylation state of the chloroplast adenylate system.     

Transport as the basis of the kok effect. Levels of some photosynthetic intermediates and activation of light-regulated enzymes during photosynthesis of chloroplasts and green leaf protoplasts

Experiments were performed with intact chloroplasts and leaf cell protoplasts isolated from spinach. The light-dependent decrease in (H⁺) in the chloroplast stroma counteracts carbon reduction and is offset at low light intensities by a large decrease in NADP and a significant increase in $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{]}$ ratios. Excess accumulation of NADPH and/or ATP permits 3-phosphogly cerate reduction to occur. With increasing light intensity, NADP levels and $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{]}$ ratios increased. High rates of photosynthesis were observed at high and at low $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{]}$ ratios. Levels of dihydroxyacetone phosphate were dramatically increased in the light. In chloroplasts, this permitted conversion to ribulose bisphosphate which on carboxylation yields 3-phosphoglycerate. The light-dependent alkalization of the chloroplast stroma is known to be responsible for phosphogly cerate retention in the chloroplasts. A high chloroplast ratio of phosphogly cerate to dihydroxyacetone phosphate aids carbon reduction. Measured ratios of dihydroxyacetone phosphate to phosphogly cerate were averages between low chloroplast ratios and high cytosolic ratios. They were far higher, even under low-intensity illumination, than dark ratios. Since cytosolic NADH levels are known to increase much less in the light than cytosolic dihydroxyacetone phosphate levels, the large increase in the ratio of didydroxyacetone phosphate to phosphogly cerate must considerably increase cytosolic phosphorylation potentials even at very low light intensities. It is proposed that this increase is communicated to the mitochondrial adenylate system, and inhibits dark respiratory activity, giving rise to the Kok effect. The extent of stroma alkalization, the efficiency of metabolite shuttles across the chloroplast envelope, and rates of cytosolic ATP consumption are proposed to be factors determining whether and to what extent the Kok effect can be observed. Light activation of chloroplast enzymes was slow at low and fast at high light intensities. This contrasts to low NADP levels at low and usually higher levels at high light intensities. Maximum enzyme activation was observed far below light saturation of photosynthesis, and light activation of enzymes was often less pronounced at very high than at intermediate light intensities.     

Adenine nucleotide control of the rate of oxygen uptake by rat heart mitochondria over a 15- to 20-fold range

Rat heart mitochondria oxidizing pyruvate (in the presence of 20% as much malate) took up nearly the amount of oxygen required for complete oxidation to CO₂. Thus pyruvate, a physiological substrate of the citrate cycle, is oxidized through the entire cycle in these mitochondria, and they seem suitable for study of regulation of integrated mitochondrial energy transduction. By addition of graded amounts of hexokinase or pyruvate kinase to the suspending medium (in the presence of excess glucose or phosphoenolpyruvate), a wide range of steady-state values of the $\text{ATP}\text{ADP}$ concentration ratio was obtained. At a constant concentration of phosphate, the steady-state rate of oxygen uptake by rat heart mitochondria oxidizing pyruvate was a function of the adenylate energy charge or of the $\text{ATP}\text{ADP}$ ratio, and relatively independent of the absolute concentrations of these nucleotides. The oxygen uptake rates typically spanned a range of about 20-fold. At very high values of the $\text{ATP}\text{ADP}$ ratio, the rate of oxygen uptake is much lower than the “state 4” rate seen after added ADP has been phosphorylated. This result suggests that “state 4” respiration, at least in these freshly prepared mitochondria, measures the rate at which ADP is made available by ATPase activity, rather than indicating uncoupling of electron transport from phosphorylation. The concentration of orthophosphate affected the rate of oxygen uptake and the pattern of response to the $\text{ATP}\text{ADP}$ ratio or the energy charge, but the effects did not seem interpretable in terms of the mass-action expression for hydrolysis of ATP, $\text{(ATP}\text{ADP)}$ (P_i.