The oxygen dependence of cellular energy metabolism.

Suspensions of cultured C 1300 neuroblastoma cells, sarcoma 180 ascites tumor cells, and Tetrahymena pyriformis cells were used to study the oxygen dependence of cellular energy metabolism. Cellular respiration was found to be almost independent of oxygen tension to values of less than 20 μm with an apparent K_m for oxygen of less than 1 μm. In contrast, the reduction of mitochondrial cytochrome c was found to be dependent on oxygen tension at all values from 240 μm downward. Oxygen dependence was also observed in terms of cellular energy metabolism expressed as adenosine triphosphate and adenosine diphosphate concentrations. These data provide direct evidence that in intact cells mitochondrial oxidative phosphorylation is oxygen dependent throughout the physiological range of oxygen tension (air saturation and below). The respiratory rate is maintained constant when the oxygen tension is lowered by decreasing values of the cytosolic $\text{[ATP]}\text{[ADP]}\text{[P}{}_{\mathrm{<mtext>i</mtext>}}\text{]}$ and intramitochondrial $\text{[NAD]}{}^{\mathrm{+}}\text{]}\text{[NADH]}$ because these regulatory parameters adjust to maintain a constant rate of ATP synthesis. The lack of oxygen dependence in the respiratory rate means that the rate of cellular ATP utilization is essentially oxygen independent until the mitochondria can no longer synthesize ATP at the required rate and $\text{[ATP]}\text{[ADP]}\text{[P}{}_{\mathrm{<mtext>i</mtext>}}\text{]}$.     

On the mechanism of the glucagon-induced inhibition of hepatic protein synthesis.

The intraperitoneal administration of glucagon (200 μg) to rats produced a transient increase of the hepatic polypeptide chain completion time, the increase being maximum at 5 min returning to control values at 20 min. This inhibitory effect was sustained when glucagon was constantly supplied by continuous infusion. Postmitochondrial supernatants from livers of the control group or rats treated with glucagon for 5 min showed no difference in their protein synthetic activity. After 20 min of intraperitoneal administration of the hormone, that is, when the effect on protein synthesis had vanished, the levels of cAMP were still 40% above those of the control group, and the ribosomal proteins were 110% more phosphorylated. These results suggest that the observed effect of glucagon is not due to its direct action on the protein synthesis machinery. On the other hand, the variations in the hepatic amino acid content brought about by glucagon do not appear to be quantitatively significant to account for the observed inhibition of protein synthesis. The effect of glucagon was always paralleled by a decrease in the $\text{[ATP]}\text{[ADP]}$ ratio which may be responsible for the observed decrease in the rates of elongation and/or termination steps of protein synthesis. Glucagon also produced a rise in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ ratio in both cellular compartments, cytosol and mitochondria, as reflected by the rise in the lactate to pyruvate and the β-hydroxybutyrate to acetoacetate ratios. This shift of the NAD⁺ couple to a more reduced state seems to be the result of an increased mobilization and oxidation of fatty acids brought about by the hormone. It is postulated then that the primary effect of glucagon leading to a decrease in protein synthesis is probably to increase the state of reduction of the hepatic nicotinamide nucleotide system. This point of view is supported by the fact that the nicotinamide and adenine nucleotide systems in rat liver are in equilibrium through cytosolic equilibrium reactions, so that a decrease in the $\text{[ATP]}\text{[ADP]}$ ratio brought about by glucagon may be secondary to the increase in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ ratio. This hypothesis is supported by the fact that glucagon was not effective in inhibiting hepatic protein synthesis in rats pretreated with a drug, 2-benzene-sulfonamido-5-(β-methoxy-ethoxy)pyrimidine, that prevents fatty acid mobilization and the subsequent changes in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ and $\text{[ATP]}\text{[ADP]}$ ratios. Furthermore, the administration of exogenous fatty acid brings about an inhibition of the rate of hepatic protein synthesis accompanied by a decrease in the ATP levels and an increase in the state of reduction of the NAD⁺ system.     

The adenylate energy charge in marine phytoplantkon: The effect of temperature on the physiological state of Skeletonema costatum (Grev.) Cleve

The adenylate energy charge $\text{([ATP] +}\text{1}\text{2}\text{[ADP])}\text{[0ATP+ADP+AMP]}$ was measured in axenic batch cultures of Skeletonema costatum (Grev.) Cleve at 2°, 10°, 15°, 20°, 24° and 30°C. The results suggest that this eurythermal diatom is physiologically capable of adapting to the 28 °C range of temperature with little apparent difference in the potential energy available to the cell. In N-limited continuous cultures at 15 °C, the energy charge values were lower than those observed in batch culture by 0.2, implying nutrient stress may result in decreased intracellular chemical energy. The utilization of the adenylate energy charge as an indicator of physiological state is suggested.     

Regulation of cellular energy metabolism. The Crabtree effect

The Crabtree effect (inhibition of respiration by glycolysis) is observed in cells with approximately equal glycolytic and respiratory capacities for ATP synthesis. Addition of glucose to aerobic suspensions of glucose-starved cells (Sarcoma 180 ascites tumor cells) causes a burst of respiration and lactate production due to ATP utilization for glucose phosphorylation by hexokinase and phosphofructokinase. This burst of activity is followed by inhibition of both respiration and glycolysis, the former to below the value before glucose addition (Crabtree effect). Both the respiratory rate and the glycolytic flux appear to be regulated by the cytosolic $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ albeit by completely different mechanisms. Respiration is regulated by the free energy of hydrolysis of ATP, such that the rate increases as the $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ decreases and decreases as the $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ increases. The regulatory enzymes of glycolysis are activated by ADP (AMP) and P_i and inhibited by ATP. Thus both respiration and glycolysis increase or decrease as the $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ decreases or increases. The parallel regulation of both ATP-producing pathways by this common metabolite ratio is consistent with the cytoplasmic $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ being an important determinant of homeostatic regulation of cellular energy metabolism.     

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.     

Studies of the nucleotide-binding sites on the mitochondrial F1-ATPase through the use of a photoactivable derivative of adenylyl imidodiphosphate

(1) $\text{N-4-}\text{Azido}\text{-2-}\text{nitrophenyl}\text{-γ-[}{}^3\text{H]aminobutyryl-Ado}\text{PP[}\text{NH}\text{]P(}\text{NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P)}$ a photoactivable derivative of 5-adenylyl imidodiphosphate (AdoPP[NH]P), was synthesized. (2) Binding of ³H]NAP₄-AdoPP[NH]P to soluble ATPase from beef heart mitochrondria (F₁) was studied in the absence of photoirradiation, and compared to that of $\text{[}{}^3\text{H]Ado}\text{PP[}\text{NH}\text{]P}$. The photoactivable derivative of AdoPP[NH]P was found to bind to F₁ with high affinity, like AdoPP[NH]P. Once $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ had bound to F₁ in the dark, it could be released by AdoPP[NH]P, ADP and ATP, but not at all by NAP₄ or AMP. Furthermore, preincubation of F₁ with unlabeled AdoPP[NH]P, ADP, or ATP prevented the covalent labeling of the enzyme by $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ upon photoirradiation. (3) Photoirradiation of F₁ by $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ resulted in covalent photolabeling and concomitant inactivation of the enzyme. Full inactivation corresponded to the binding of about 2 mol $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}\text{mol F}{}_1$. Photolabeling by NAP₄-AdoPP[NH]P was much more efficient in the presence than in the absence of MgCl₂. (4) Bound $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ was localized on the α- and β-subunits of F₁. At low concentrations (less than 10 μM), bound $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ was predominantly localized on the α-subunit; at concentrations equal to, or greater than 75 μM, both α- and β-subunits were equally labeled. (5) The extent of inactivation was independent of the nature of the photolabeled subunit (α or β), suggesting that each of the two subunits, α and β, is required for the activity of F₁. (6) The covalently photolabeled F₁ was able to form a complex with aurovertin, as does native F₁. The ADP-induced fluorescence enhancement was more severely inhibited than the fluorescence quenching caused by ATP. The percentage of inactivation of F₁ was virtually the same as the percentage of inhibition of the ATP-induced fluorescence quenching, suggesting that fluorescence quenching is related to the binding of ATP to the catalytic site of F₁.     

Steady-state kinetics of ADP-arsenate and ATP-synthesis in Rhodospirillum rubrum chromatophores

(1) Rates of ATP synthesis and ADP-arsenate synthesis catalyzed by Rhodospirillum rubrum chromatophores were determined with the firefly luciferase method and by a coupled enzyme assay involving hexokinase and glucose-6-phosphate dehydrogenase. (2) V_m for ADP-arsenate synthesis was about 2-times lower than V_m for ATP-synthesis. With saturating [ADP], K(As_i) was about 20% higher than K(P_i). With saturating [anion], K(ADP) was during arsenylation about 20% lower than during phosphorylation. (3) Plots of $\text{1}\text{v}$ vs. $\text{1}\text{[}\text{substrate}\text{]}$ were non-linear at low concentrations of the fixed substrate. The non-linearity was such as to suggest a positive cooperativity between sites binding the variable substrate, resulting in an increased $\text{V}{}_{\mathrm{<mtext>m</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ ratio. High concentrations of the fixed substrate cause a similar increase in $\text{V}{}_{\mathrm{<mtext>m</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, but abolish the cooperativity of the sites binding the variable substrate. (4) Low concentrations of inorganic arsenate (As_i) stimulate ATP synthesis supported by low concentrations of P_i and ADP about 2-fold. (5) At high ADP concentrations, the apparent K_i of As_i for inhibition of ATP-synthesis was 2–3-times higher than the apparent K_m of As_i for arsenylation; the apparent K_i of P_i for inhibition of ADP-arsenate synthesis was about 40% lower than the apparent K_m of P_i for ATP synthesis. (6) The results are discussed in terms of a model in which P_i and As_i compete for binding to a catalytic as well as an allosteric site. The interaction between these sites is modulated by the ADP concentration. At high ADP concentrations, interaction between these sites occurs only when they are occupied with different species of anion.     

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.     

Adenine nucleotide extraction from multicellular organisms and beach sand: ATP recovery,energy charge ratios and determination of carbon/ATP ratios

Investigations were conducted comparing the efficiency of adenine nucleotide extraction from bacteria, unicellular algae, invertebrates (copepods, isopods and polychaetes), and beach sand using boiling buffers and cold acid extraction procedures. Cellular levels of ATP, ADP, and AMP obtained by these procedures were used to calculate the adenylate energy charge ratio ($\text{EC}{}_{\mathrm{<mtext>A</mtext>}}\text{= [}\text{ATP}\text{] +}\text{1}\text{2}\text{[}\text{ADP}\text{]/[}\text{ATP}\text{] + [}\text{ADP}\text{] + [}\text{AMP}\text{]}$). Although both extraction procedures efficiently extract ATP from unicellular micro-organisms, the results with multicells and beach sand indicate that the cold acid procedure preserves a greater percentage of the total adenine nucleotides ([A_T] = [ATP] + [ADP] + [AMP]) in the form of ATP, resulting in higher energy charge ratios. There were relatively large losses of ATP when multicellular organisms were extracted in boiling buffers. These data suggest that ATP hydrolysis may be important in certain fluid-solid mixtures, and also adds experimental support to the thermal gradient hypothesis.The C/ATP ratios calculated from these data indicate that multicellular organisms have C/ATP ratios < 100, as compared with the 250 ratio commonly found in micro-organisms. These results are discussed in terms of the proportion of structural (non-living) carbon vs protoplasm (living) carbon within each of these groups of organisms, as well as the relative intracellular levels of non-adenine nucleotide triphosphates. These differences in the C/ATP ratios must be considered whenever ATP measurements are used for biomass determinations.     

Measurement of the intramitochondrial P/O ratio

Measurements of the initial rate of ATP synthesis and the initial rate of oxygen consumption in mitochondria in which transport of ADP, Pi and ATP were inhibited were used to obtain a value for the intramitochondrial $\text{P}\text{O}$ ratio. With succinate as substrate this method yielded a $\text{P}\text{O}$ ratio of 2.8 for the phosphorylation of intramitochondrial ADP.     

Energetics of sodium transport in the kidney: Saturation transfer 31P-NMR

³¹P-NMR has been used to quantify inorganic phosphate (P_i) and high-energy phosphates in the isolated, functioning perfused rat kidney, while monitoring oxygen consumption, glomerular filtration rate and sodium reabsorption. Compared with enzymatic analysis, 100% of ATP, but only 25% of ADP and 27% of P_i are visible to NMR. This is indicative that a large proportion of both ADP and P_i are bound in the intact kidney. NMR is measuring free, and therefore probably cytosolic concentrations of these metabolites. ATP synthesis rate, measured by saturation transfer NMR shows the P:O ratio of 2.45 for the intact kidney. This is close to the theoretical value, suggesting the NMR visible pool is that which is involved in oxidative phosphorylation. The energy cost of Na transport, calculated from the theoretical Na:ATP of 3.0 exceeded the measured rate of ATP synthesis. Instead, Na:ATP for active transport in the perfused kidney was 12. Since the phosphorylation potential ($\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{]×[}\text{P}{}_{\mathrm{i}}\text{]}$) by NMR was 10 000 M^?1, the free-energy of ATP hydrolysis was 52 kJ/mol. Using this figure, the rate of ATP hydrolysis observed could fully account for the observed rate of sodium reabsorption.     

An evaluation of the metabolite indicator method for determining the cytosolic phosphate potential in rat liver cells

The cytosolic phosphate potential was estimated in isolated rat liver parenchymal cells incubated with various gluconeogenic substrates. The value of the cytosolic $\text{[}\text{ATP}\text{]}\text{[}\text{ADP][P}{}_{\mathrm{i}}\text{]}$ ratio was either estimated directly from measurements of ATP, ADP and P_i after digitonin fractionation of the cells, or calculated by the metabolite indicator method. When cells were incubated with lactate, pyruvate or alanine so that net flux through the indicator enzymes was in the gluconeogenic direction, there was excellent agreement between the values obtained by the two methods over a wide range of fluxes. However, when the cells were incubated with substrates that could be converted both to glucose and to lactate so that net flux through the indicator enzymes was in the glycolytic direction, a large difference in the values of the cytosolic $\text{[}\text{ATP}\text{]}\text{([}\text{ADP][P}{}_{\mathrm{i}}\text{]}$) ratio as derived by the two methods was observed. It is concluded that the reaction catalysed by glyceraldehyde-3-phosphate dehydrogenase plus 3-phosphoglycerate kinase is out of equilibrium when flux through the reaction is in the glycolytic direction, and that use of the metabolite indicator method for the calculation of the cytosolic phosphate potential under these conditions leads to erroneous values.     

The invisible copper of cytochrome c oxidase. pH and ATP dependence of its midpoint potential and its role in the oxygen reaction.

Formation of the CO compound has been studied in intact mitochondria, submitochondrial particles and isolated cytochrome oxidase. The reaction requires the prior reduction of both cytochrome a₃ and one other single-electron acceptor. It is inferred that the second acceptor is the “invisible” copper which is undetectable by both optical and spin resonance spectroscopy. The overall process can be viewed as two single electron steps plus a ligand binding reaction. At high concentrations of CO, when titrations are performed at oxidation-reduction potentials significantly above the midpoints of either cytochrome a₃ or “invisible” copper, appearance of the CO compound follows a strict n = 2 (2-electron) relationship. Its midpoint potential is also dependent on the prevailing concentration of CO and is increased by approx. 30 mV for each tenfold increase in the level of CO. At redox potentials approaching the midpoints of cytochrome a₃ or “invisible” copper, significant deviations from n = 2 behavior are apparent which are readily detectable experimentally using low CO concentrations.A mathematical analysis of this model is presented and the oxidation-reduction properties of the CO compound are utilized to determine the midpoint potential of the “invisible” copper. This value is estimated to be 340 ± 10 mV at pH 7.8, independent of pH and the prevailing $\text{sol}\text{[ATP]}\text{[ADP]}\text{× [}\text{P}{}_1\text{]}$ ratio.By analogy with the observations on CO binding, the primary intermediate in the oxidase reaction with oxygen is concluded to be a bridged a₃²⁺-O₂-Cu¹⁺ complex. The initial reduction of molecular oxygen can then proceed via a thermodynamically favorable two-electron step to form a bridged peroxide intermediate. Subsequent reduction to water may later occur by way of two single-electron steps or one two-electron step.     

Modulation of 25-hydroxyvitamin D3-24-hydroxylase by aminophylline: a cytochrome P-450 monooxygenase system.

The enzymatic activity of human erythrocyte pyruvate kinase was found to decrease on incubation of the purified enzyme with red blood cell ghosts, ATP and cAMP. If $\text{[}{}^{32}\text{P]γATP}$ was used radioactivity was found associated with the protein after gel electrophoresis. Various effectors protected the enzyme against phosphorylation. Treatment of the modified enzyme with a protein phosphatase restored enzymatic activity and also caused the loss of the radioactive label. Modification of the pyruvate kinase in this way altered the affinity of the enzyme for one of its substrates (phosphoenolpyruvate), but the binding of the other substrate (ADP) was unaffected.     

ATP/ADP exchange activity of gastric (H+ + K+)-ATPase

The ATP/ADP exchange is shown to be a partial reaction of the $\text{(}\text{H}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to $\text{P}{}^1$, $\text{P}{}^5$ -di(adenosine-5′) pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg²⁺ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration ($\text{K}{}_{0.5}\text{= 116 μ}\text{M}$) required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg²⁺ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K⁺ as a function of pH and Mg²⁺.     

Changes in adenine nucleotide levels and respiration during 1-methyladenine induced maturation of starfish oocytes

The effects of 1-methyladenine on oxygen consumption and adenine nucleotide levels were examined in oocytes of Pisaster ochraceus and Patiria miniata. Oocytes of both genera to which 1-methyladenine was added consumed more oxygen than control oocytes beginning 1 to $\text{1}\text{1}\text{2}\text{h}$ after 1-methyladenine addition. The increase in oxygen consumption was correlated with maturation changes in the oocytes and particularly with germinal vesicle breakdown. Pre-fertilization oxygen consumption of eggs did not differ significantly from post-fertilization oxygen consumption of eggs in either genus for $\text{2}\text{1}\text{2}\text{h}$ after fertilization. ATP and AMP concentrations within the oocytes decreased during 1-methyladenine induced maturation, while the ADP concentration increased. It was suggested that increases in ADP concentration and decreases in ATP concentration within maturing starfish oocytes occurred in response to greater energy demands. The simultaneous increase in oocyte oxygen consumption was interpreted as an indicator of increased oxidative phosphorylation acting to restore initial nucleotide concentrations.     

An enzymatic method for [32P]phosphoenolpyruvate synthesis

A convenient method for the enzymatic synthesis of [³²P]phosphoenolpyruvate from [γ-³²P]ATP using partially pufified phosphoenolpyruvate carboxykinase from Escherichia coli is described. The synthesis was shown to convert essentially all the [γ-³²P]ATP to [³²P]phosphoenolpyruvate, which was subsequently separated from residual [γ-³²P]ATP and $\text{[}{}^{32}\text{P}\text{]P}{}_{\mathrm{<mtext>i</mtext>}}$ by chromatography on AG-1-X8-bicarbonate resin.     

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.     

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.