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.     

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.     

Pyruvate flux into resealed ghosts from human erythrocytes

The kinetics of pyruvate transport across the isolated red blood cell membrane were studied by a simple and precise spectrophotometric method: following the oxidation of NADH via lactate dehydrogenase trapped within resealed ghosts. The initial rate of pyruvate entry was linear. Influx was limited by saturation at high pyruvate concentration. Pyruvate influx was greatly stimulated by increasing ionic strength in the outer but not the inner aqueous compartment. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ ranged from 15.0 mM at $\text{μ = 0.05}$ to 3.7 mM at $\text{μ = 0.01}$, while the $\text{V}$ went from $\text{0.611 · 10}{}^{-15}$ to $\text{0.137 · 10}{}^{-15}\text{mol}\text{·}\text{min}{}^{-1}\text{·}\text{ghost}{}^{-1}$. Ionic strength was shown to affect the translocation step and not pyruvate binding. The energy of activation of pyruvate flux into resealed ghosts was 25 kcal/mol, similar to that found in intact red blood cells. Inhibitors of pyruvate influx included such anions as thiocyanate, chloride, bicarbonate, α-cyanocinnamate, salicylate and ketomalonate (but not acetate); noncompetitive inhibitors were phloretin, 1-fluoro-2,4-dinitrobenzene, 4-acetamido-4′-isothiocyanate-stilbene-2,2′-disulfonic acid and o-phenanthroline/CuSO₄ mixtures. The last reagent, known to induce disulfide links in certain membrane proteins, blocked the ionic strength stimulation of pyruvate influx in this study.     

Equilibrium constants for association of guanidinium and ammonium ions with oxyanions: The effect of changing basicity of the oxyanion

Using guanidinium and n-butylammonium cations (C⁺) as models for the positively charged side chains in arginine and lysine, we have determined the association constants with various oxyanions by potentiometric titration. For a dibasic acid, H₂A, three association complexes may exist: $\text{K}{}_{\mathrm{<mtext>1M</mtext>}}\text{=}\text{[CHA]}\text{[C}{}^{\mathrm{+}}\text{]}\text{[HA}{}^{\mathrm{?}}\text{]}$; $\text{K}{}_{\mathrm{<mtext>1D</mtext>}}\text{=}\text{[CA}{}^{\mathrm{?}}\text{]}\text{[C}{}^{\mathrm{+}}\text{]}\text{[A}{}^{\mathrm{2?}}\text{]}$; $\text{K}{}_{\mathrm{<mtext>2D</mtext>}}\text{=}\text{[C}{}_2\text{A]}\text{[C}{}^{\mathrm{+}}\text{]}\text{[CA}{}^{\mathrm{?}}\text{]}$. For guanidinium ion and phosphate, K_1M = 1.4, K_1D = 2.6, and K_2D = 5.1. The data for carboxylates indicate that the basicity of the oxyanion does not affect the association constant: acetate, pK_a = 4.8, K_1M = 0.37; formate, pK_a = 3.8, K_1M = 0.32; and chloroacetate, pK_a = 2.9, K_1M = 0.43, all with guanidinium ion. Association constants are also reported for carbonate, dimethylphosphinate, benzylphosphonate, and adenylate anions.     

A mathematical model describing the effect of temperature and substrate concentration on the activities of M4 and H4 lactate dehydrogenase from the big brown bat (Eptesicus fuscus)

The effect of temperature on the activities of M₄ and H₄ lactate dehydrogenases (LDH, EC 1.1.1.27) isolated from the big brown bat (Eptesicus fuscus) was examined. Temperature effects were dependent on the concentrations of all four LDH substrates, pyruvate, lactate, NADH, and NAD. Arrhenius plots of In v_i vs reciprocal of absolute temperature were linear for all but the lowest substrate concentrations. The slopes of these Arrhenius plots were used to calculate the temperature effect parameter (μ). Substrate-dependent temperature effects for M₄ and H₄ LDH were described by an equation for a rectangular hyperbola, $\text{μ =}\text{[E}{}_{\mathrm{\beta }}\text{S + E}{}_{\mathrm{\alpha }}\text{K}{}_{\mathrm{t}}\text{]}\text{[K}{}_{\mathrm{t}}\text{+ S]}$ proposed by G. R. Harbison and J. R. Fisher (1974, Comp. Biochem. Physiol.47B, 27–32) for adenosine deaminase. The parameters E_α (μ at infinitely low substrate concentration), E_β (μ at infinitely high substrate concentration), and K_t (the concentration of substrate when $\text{μ =}\text{[E}{}_{\mathrm{\alpha }}\text{+ E}{}_{\mathrm{\beta }}\text{]}\text{2}$) can be used to describe the temperature dependence of LDH activity at any substrate concentration and to compare the substrate-dependent temperature effects on the two isoenzymes. Significantly different E_β and K_t values for pyruvate-dependent temperature effects and different E_β, E_α, K_t, and E_β ? E_α (the range of possible μ values) for lactate-dependent temperature effects were found between M₄ and H₄ LDH isoenzymes. High lactate concentrations inhibited bat H₄ LDH activity to a greater degree at low temperatures than at high temperatures. Thus substrate inhibition plays an important role in the effect of temperature on the activity of H-type LDH at high lactate concentrations. Substrate-dependent temperature effects on bat LDH activity were the result of temperature effects on the apparent K_m value of the respective substrate. Since both the apparent K_m for pyruvate and the K_i for the competitive inhibitor oxamate decreased with decreasing temperature, the substrate-dependent temperature effects observed for pyruvate probably resulted from an increased affinity between pyruvate and the LDH-NADH complex with decreasing temperature.     

Equilibrium constants under physiological conditions for the reactions of L-phosphoserine phosphatase and pyrophosphate: L-serine phosphotransferase

The observed equilibrium constants (K_obs) for the l-phosphoserine phosphatase reaction [EC 3.1.3.3] have been determined under physiological conditions of temperature (38 °C) and ionic strength (0.25 m) and physiological ranges of pH and free [Mg²⁺]. Using Σ and square brackets to indicate total concentrations $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{Σ L-serine][Σ P}{}_{\mathrm{i}}\text{]}\text{Σ L-phosphoserine]H}{}_2\text{O]}\text{, K =}\text{L-H · serine}{}^{\mathrm{\pm }}\text{]HPO}{}_4{}^{\mathrm{2?}}\text{]}\text{[L-H · phosphoserine}{}^{\mathrm{2?}}\text{]H}{}_2\text{O]}$. The value of K_obs has been found to be relatively sensitive to pH. At 38 °C, K⁺] = 0.2 m and free [Mg²⁺] = 0; K_obs = 80.6 m at pH 6.5, 52.7 m at pH 7.0 [ΔG_obs⁰ = ?10.2 kJ/mol (?2.45 kcal/mol)], and 44.0 m at pH 8.0 ([H₂O] = 1). The effect of the free [Mg²⁺] on K_obs was relatively slight; at pH 7.0 ([K⁺] = 0.2 m) K_obs = 52.0 m at free [Mg²⁺] = 10^?3, m and 47.8 m at free [Mg²⁺] = 10^?2, m. K_obs was insignificantly affected by variations in ionic strength (0.12–1.0 m) or temperature (4–43 °C) at pH 7.0. The value of K at 38 °C and I = 0.25 m has been calculated to be 34.2 ± 0.5 m [ΔG_obs⁰ = ?9.12 kJ/mol (?2.18 kcal/ mol)]([H₂O] = 1). The K for the phosphoserine phosphatase reaction has been combined with the K for the reaction of inorganic pyrophosphatase [EC 3.6.1.1] previously estimated under the same physiological conditions to calculate a value of 2.04 × 10⁴, m [ΔG_obs⁰ = ?28.0 kJ/mol (?6.69 kcal/mol)] for the K of the pyrophosphate:l-serine phosphotransferase [EC 2.7.1.80] reaction. $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{[}\text{Σ L-serine}\text{][}\text{Σ}\text{P}{}_{\mathrm{<mtext>i</mtext>}}\text{]}\text{[}\text{Σ L-phosphoserine}\text{][}\text{H}{}_2\text{O}\text{]}\text{, K =}\text{[L-H · serine}{}^{\mathrm{\pm }}\text{]HPO}{}_4{}^{\mathrm{2?}}\text{]}\text{[L-H · phosphoserine}{}^{\mathrm{2?}}\text{]H}{}_2\text{O}$. Values of K_obs for this reaction at 38 °C, pH 7.0, and I = 0.25 m are very sensitive to the free [Mg²⁺], being calculated to be 668 [ΔG_obs⁰ = ?16.8 kJ/mol (?4.02 kcal/mol)] at free [Mg²⁺] = 0; 111 [ΔG_obs⁰ = ?12.2 kJ/mol (?2.91 kcal/mol)] at free [Mg²⁺] = 10^?3, m; and 9.1 [ΔG_obs⁰ = ?5.7 kJ/mol (?1.4 kcal/mol) at free [Mg²⁺] = 10^?2, m). K_obs for this reaction is also sensitive to pH. At pH 8.0 the corresponding values of K_obs are 4000 [ΔG_obs⁰ = ?21.4 kJ/mol (?5.12 kcal/mol)] at free [Mg²⁺] = 0; and 97.4 [ΔG_obs⁰ = ?11.8 kJ/ mol (?2.83 kcal/mol)] at free [Mg²⁺] = 10^?3, m. Combining K_obs for the l-phosphoserine phosphatase reaction with K_obs for the reactions of d-3-phosphoglycerate dehydrogenase [EC 1.1.1.95] and l-phosphoserine aminotransferase [EC 2.6.1.52] previously determined under the same physiological conditions has allowed the calculation of K_obs for the overall biosynthesis of l-serine from d-3-phosphoglycerate. $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{[}\text{Σ L-serine}\text{][}\text{Σ NADH}\text{][}\text{Σ}\text{P}{}_{\mathrm{<mtext>i</mtext>}}\text{][}\text{Σ}\text{α-}\text{ketoglutarate}\text{]}\text{[Σ d-3-}\text{phosphoglycerate}\text{][Σ NAD}{}^{\mathrm{+}}\text{][Σ L-glutamat0]}$ The value of K_obs for these combined reactions at 38 °C, pH 7.0, and I = 0.25 m (K⁺ as the monovalent cation) is 1.34 × 10^?2, m at free [Mg²⁺] = 0 and 1.27 × 10^?2, m at free [Mg²⁺] = 10^?3, m.     

Homogeneous enzyme immunoassay of chenodeoxycholate conjugates in serum.

It is shown that biased answers are given by the mathematical method used by Stein and his colleagues (Hankin B. L. Hankin, W. R. Lieb, and W. D. Stein (1972)Biochim. Biophys. Acta288, 114–126) to calculate $\text{K}{}_{21}{}^{\mathrm{<mtext>ic</mtext>}}$, the half-saturation concentration for the entry of glucose into erythrocytes in infinite-cis conditions. A method for calculating $\text{K}{}_{21}{}^{\mathrm{<mtext>ic</mtext>}}$ accurately is described and tested. The published estimates of $\text{K}{}_{21}{}^{\mathrm{<mtext>ic</mtext>}}$ are low; nevertheless, even when they are revised upwards, the asymmetrical carrier model of glucose transport still fails to satisfy the “rejection criteria” of Hankin et al. (1972).     

Selective inhibition of pyruvate and lactate metabolism in bovine epididymal spermatozoa by dinitrophenol and alpha-cyano-3-hydroxycinnamate

The disparity between the effects of the uncouplers, 2,4-dinitrophenol (DNP) and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) on pyruvate metabolism in bovine spermatozoa has been characterized. In bovine epididymal spermatozoa metabolizing pyruvate, the uncouplers of oxidative phosphorylation, DNP (100 μm) and FCCP (0.4 or 5 μm), decreased the intracellular ATP concentration from 30 to ～10 nmol/10⁸ cells. Both uncouplers decreased, but did not abolish, sperm motility. DNP strongly inhibited pyruvate metabolism and stimulated the appearance of free carnitine from the acetylcarnitine pool. In contrast, FCCP enhanced the oxidation of pyruvate, diminished the reduction of pyruvate to lactate, and permitted the maintenance of the normal amount of acetylcarnitine. The effects of DNP and FCCP on mitochondrial pyruvate metabolism were examined in spermatozoa treated with filipin, which renders the plasma membrane permeable to small molecules. In these cells, DNP inhibited metabolism and respiration with pyruvate or lactate, but did not affect respiration supported by acetylcarnitine. Similarly, the pyruvate translocase inhibitor, α-cyano-3-hydroxycinnamate, markedly decreased the rate of metabolism of both pyruvate and lactate. With maximally inhibitory concentrations of DNP or α-cyano-3-hydroxycinnamate, the rates of pyruvate use and lactate use were the same. Metabolism of both lactate and pyruvate and production of ATP were inhibited by similar concentrations of DNP ($\text{I}{}_{50}\text{? 7}\text{μM}$). A common mitochondrial translocase for pyruvate and lactate in bovine spermatozoa is posited. This translocase is inhibited by minimally effective uncoupling concentrations of DNP.     

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.     

Anaerobic stimulation of sugar transport in avian erythrocytes

The kinetic parameters of the sugar transport in avian erythrocytes were evaluated under aerobic and anaerobic conditions. In anaerobic cells, transport measurements with $\text{3-O-[}{}^{14}\text{C}\text{]}$ methylglucose resulted in a set of similar dissociation-like constants. Thus the Michaelis constants of $\text{3-O-[}{}^{14}\text{C}\text{]}$ methylglucose entry and exit, $\text{K}{}_{\mathrm{<mtext>so</mtext>}}$ and $\text{K}{}_{\mathrm{<mtext>si</mtext>}}$, were 8 and 7 mM, respectively. The equilibrium exchange constant, $\text{B}{}_{\mathrm{<mtext>s</mtext>}}$, and the counterflow constant, $\text{R}{}_{\mathrm{<mtext>s</mtext>}}$, were 9 and 11 mM, respectively. The activity constant for $\text{3-O-}\text{methylglucose}$ transport, $\text{F}{}_{\mathrm{<mtext>s</mtext>}}$, defined as $\text{V/K}{}_{\mathrm{<mtext>m</mtext>}}$, was 4 ml/h per g. This set of kinetic constants was compatible with a symmetrical mobile-carrier model. In contrast, the Michaelis constant for glucose entry, $\text{K}{}_{\mathrm{<mtext>go</mtext>}}$, was 2 mM and less than the counterflow constant, $\text{R}{}_{\mathrm{<mtext>g</mtext>}}$ (8 mM). This result could be accounted for by slower movement of the glucose-carrier complex than the free carrier. The activity constant for glucose transport, $\text{F}{}_{\mathrm{<mtext>g</mtext>}}$, was 5 ml/h perg.Under aerobic conditions, two of the dissociation-like constants ($\text{K}{}_{\mathrm{<mtext>si</mtext>}}$ and $\text{B}{}_{\mathrm{<mtext>s</mtext>}}$) for $\text{3-O-}\text{methylglucose}$ transport were significantly larger than those obtained in anaerobic cells, but the remaining two ($\text{K}{}_{\mathrm{<mtext>so</mtext>}}$ and $\text{R}{}_{\mathrm{<mtext>s</mtext>}}$) remained unchanged. The values, for $\text{K}{}_{\mathrm{<mtext>so</mtext>}}\text{, K}{}_{\mathrm{<mtext>si</mtext>}}\text{, B}{}_{\mathrm{<mtext>s</mtext><mtext>\; and</mtext>}}\text{R}{}_{\mathrm{<mtext>s</mtext>}}$ were 8, 26, 20 and 8 mM, respectively. The activity constant, $\text{F}{}_{\mathrm{<mtext>s</mtext>}}$, decreased to 2 ml/h per g. These changes in kinetic constants were consistent with the hypothesis that anoxia accelerated sugar transport by releasing free carrier that was previously sequestered on the inside of the cell membrane.     

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 $\text{[ATP]}\text{[ADP]}\text{[P}{}_{\mathrm{<mtext>i</mtext>}}\text{]}$.