A possible role of inorganic phosphate as a regulator of oxidative phosphorylation in combined urea synthesis and gluconeogenesis in perfused rat liver. A phosphorus magnetic resonance spectroscopy study

Metabolic control of oxidative metabolism was studied in perfused rat liver by means of phosphorus magnetic resonance spectroscopy. Oxygen consumption, ATP, and Pi were measured with different rates of gluconeogenesis and urea synthesis by varying concentrations of the substrates in the perfusate. Five levels of oxygen consumption (VO2) were obtained: an average control value of 1.94 +/- 0.14 and 2.93 +/- 0.25, 3.29 +/- 0.46, 3.85 +/- 0.26, and 4.18 +/- 0.56 mumol/min/g liver (mean +/- S.D., n = 6). The corresponding ATP concentrations were 2.51 +/- 0.20, 2.39 +/- 0.08, 2.24 +/- 0.09, 2.13 +/- 0.12, and 1.91 +/- 0.13 mM. Pi increased stoichiometrically with the decrease in ATP. Free Pi (Pif) was calculated as NMR-visible Pi in control plus -delta ATP (1.94 mM + (-delta ATP]. The kinetic relationship of oxidative phosphorylation as a function of Pif followed a Michaelis-Menten type of equation: VO2 = 5.55/(1 + 0.24/[( Pif] - 1.81]. The observed Km value for Pi of 0.24 mM approximates the reported Km value in isolated mitochondria of 1 mM. The free Pi concentration of 1.94 mM is in the range of the Km value, while the free ADP concentration of 200 microM exceeds the Km value of 20 microM. Therefore, it is suggested that Pi play a major role in the regulation of mitochondrial oxidative phosphorylation in combined urea synthesis and gluconeogenesis.     

31P NMR studies of ATP synthesis and hydrolysis kinetics in the intact myocardium

The origin of the nuclear magnetic resonance (NMR)-measurable ATP in equilibrium Pi exchange and whether it can be used to determine net oxidative ATP synthesis rates in the intact myocardium were examined by detailed measurements of ATP in equilibrium Pi exchange rates in both directions as a function of the myocardial oxygen consumption rate (MVO2) in (1) glucose-perfused, isovolumic rat hearts with normal glycolytic activity and (2) pyruvate-perfused hearts where glycolytic activity was reduced or eliminated either by depletion of their endogenous glycogen or by use of the inhibitor iodoacetate. In glucose-perfused hearts, the Pi----ATP rate measured by the conventional two-site saturation transfer (CST) technique remained constant while MVO2 was increased approximately 2-fold. When the glycolytic activity was reduced, the Pi----ATP rate decreased significantly, demonstrating the existence of a significant glycolytic contribution. Upon elimination of the glycolytic component, the measured Pi----ATP rates displayed a linear dependence on MVO (micromoles of O consumption rate) with a slope of 2.36 +/- 0.15 (N = 8, standard error of the mean). This linear relationship is expected if the rate determined by CST is the net rate of ATP synthesis by the oxidative phosphorylation process, in which case the slope must equal the P:O ratio. The ATP----Pi rates and rate:MVO ratios measured by the multiple-site saturation transfer method at two MVO2 levels were equal to the corresponding Pi----ATP rates and rate:MVO ratios obtained in the absence of a glycolytic contribution. The following conclusions are drawn from these studies: (1) unless the glycolytic contribution to the ATP in equilibrium Pi exchange is inhibited or is specifically shown not to exist, the myocardial Pi in equilibrium ATP exchange due to oxidative phosphorylation cannot be studied by NMR; (2) at moderate MVO2 levels, the reaction catalyzed by the two glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase is near equilibrium; (3) the ATP synthesis by the mitochondrial H+-ATPase occurs unidirectionally (i.e., the reaction is far out of equilibrium); (4) the "operative" P:O ratio in the intact myocardium under our conditions is significantly less than the canonically accepted value of 3.     

The synthesis of hippurate from benzoate and glycine by rat liver mitochondria. Submitochondrial localization and kinetics.

1. Rat liver mitochondria make hippurate at up to 4 nmol/min per mg of protein. The rate of synthesis supported by oxidation of glutamate with exogenous Pi present is identical with that supported by ATP plus oligomycin. Lower rates were obtained with other respiratory substrates, and when glutamate was used without Pi. 2. A matrix localization for hippurate synthesis is indicated by the latency of benzoyl-CoA synthetase and glycine N-acyltransferase to their extramitochondrial substrates, failure of exogenous benzoyl-CoA to inhibit incorporation of [14C]hippurate and inhibition of hippurate synthesis supported by ATP, but not glutamate, by carboxyatractyloside. 3. The relative activities of the individual enzymes and the mitochondrial content of benzoyl-CoA in the presence and absence of glycine suggest that hippurate synthesis is rate-limited by formation of benzoyl-CoA. 4. The increases in rates of ATP hydrolysis and of O2 consumption on the addition of benzoate and glycine were in good agreement with those required to support hippurate synthesis. The increase in respiration indicates that State-4 respiration [Chance & Williams (1957) Adv. Enzymol 17, 65-134] is not used, with these conditions, for ATP synthesis.     

Characteristics of α-glucan phosphorylase from Chlorella vulgaris

α-Glucan phosphorylase from Chlorella vulgaris has been partially purified. In the direction of glucan phosphorolysis the apparent K_m for Pi was ca 2.4 mM at pH 7.1. In the direction of glucan synthesis the K_m for G1P was ca 0.12 mM at pH 6.2. The enzymic activity was inhibited by physiological concentrations of ADP, ATP, ADPG and UDPG. In the direction of starch degradation in the presence of 2.4 mM Pi the I_0.5 values for ADP and ATP were ca 1.6 and 2.9 mM, respectively, while in the direction of synthesis in the presence of 0.12 mM G1P the values were ca 0.23 and 1.4 mM, respectively. The Hill plots for starch degradation showed n values of 2.2 for ADP and 2.2 for ATP and values of 1.5 and 1.2, respectively, for starch synthesis. Both ADPG and UDPG were linear competitive inhibitors either with respect to Pi or with respect to GIP. The K_i values for ADPG and UDPG in the direction of phosphorolysis were shown to be ca 0.11 and 0.51 mM, respectively, and those in the direction of synthesis 0.033 and 0.15 mM, respectively.     

Relationships between the rate of synthesis of ATP and the concentrations of reactants and products of ATP hydrolysis in maize root tips, determined by 31P nuclear magnetic resonance

Using 31P NMR spectroscopy, we have measured the rate of ATP synthesis, and the free concentrations of ATP, ADP, cytoplasmic Pi, and H+ in maize root tips under a wide range of conditions. We show that the ratio [ATP]/[ADP] in normoxic root tips is greater than 25. We found no simple relationship between the concentration of ATP and the rate of ATP synthesis: when the rate of ATP synthesis decreases in response to different treatments, the concentration of ATP can increase, decrease, or remain unchanged. Clear relationships were obtained, however, when the rate of synthesis of ATP was plotted against the logarithm of the ratio psi, defined as [ATP]/[ADP][Pi][H+]. Two curves were obtained, depending on which of two situations pertained. First, if mitochondrial ATP synthesis was inhibited, e.g., by KCN or hypoxia, ln psi decreased monotonically as rates of ATP synthesis decreased. The decrease in ln psi may account for decreases in the rates of biosynthetic reactions dependent on ATP, such as protein synthesis, as they approach equilibrium. Second, if consumption of ATP for biosynthetic reactions was inhibited, by treatment with succinate, ln psi increased as rates of ATP synthesis decreased. The increase in ln psi may account for decreases in the rate of ATP synthesis, as oxidative phosphorylation approaches equilibrium.     

A phosphorus-31 nuclear magnetic resonance study of elicitor-mediated metabolic changes in Catharanthus roseus suspension cultures

Summary The induction of metabolic changes in suspension cultured cells of Catharanthus roseus upon elicitation has been investigated. Addition of a yeast glucan preparation to the growth medium resulted in induction of phenylalanine ammonia lyase. Phosphate uptake and metabolism of elicited cells was followed by ³¹P nuclear magnetic resonance. The uptake rate of Pi from the medium by oxygenated cells of C. roseus was reduced immediately after elicitation. Despite this reduced Pi uptake elicited cells had significantly increased amounts of ATP (twofold increase within 6 h). Cytoplasmic levels of Pi, phosphomonoesters, and Uridine Diphasphate glucose (UDP-Glc) were unaffected by eliciation. Furthermore, the cytoplasmic and vacuolar pH remained constant after addition of elicitor.     

Kinetic modalities of ATP synthesis. Regulation by the mitochondrial respiratory chain

Two interconvertible kinetic modes are described for ATP synthesis by bovine heart submitochondrial particles. One mode is characterized by low apparent Km values for ADP (6-10 microM) and Pi (less than or equal to 0.25 mM), and a limited capacity for ATP synthesis (apparent Vmax approximately 500 nmol ATP.min-1.mg of protein-1). ATP synthesis occurs predominantly in this mode when the coupled activity of the respiratory chain relative to the number of functional ATP synthase complexes is low. The second kinetic mode is characterized by high apparent Km values for ADP (50-100 microM) and Pi (approximately 2.0 mM) and a high capacity for ATP synthesis (Vmax greater than 1800 nmol ATP.min-1.mg of protein-1). This mode of ATP synthesis predominates when the available free energy relative to the number of functional ATP synthase units is high. These results suggest that energy pressure in mitochondria might regulate ATP synthesis such that at low levels of energy the ATP synthase operates economically (low substrate Km values, low turnover capacity for ATP synthesis), while at high levels of energy these kinetic constraints are relaxed (high substrate Km values, high turnover capacity for ATP synthesis). The implications of these findings are discussed in relation to the cooperative-type kinetics of ATP synthesis and hydrolysis, the differential effects of a number of F0-F1 inhibitors on the rates of ATP synthesis and hydrolysis, and the controversy as to whether protonic energy in mitochondria is localized or delocalized.     

Coupling of ATP synthesis to reversal of rat liver microsomal Ca2+-ATPase

The reversal of the rat liver microsomal Ca2+-ATPase transport cycle was studied. Microsomes were loaded with 45Ca2+ (approximately 30 nmol/mg of protein) in an ATP-dependent process, and the time dependency of the microsomal 45Ca2+ efflux was determined with various ADP and inorganic phosphate (Pi) concentrations. Pseudo-first-order rate constants (K'e) for 45Ca2+ efflux were determined. Although there was considerable 45Ca2+ efflux in the absence of added ADP or Pi, the addition of ADP or Pi alone had minimal effects upon the K'e; in contrast, a 2.5-fold increase in the K'e was observed in the presence of both ADP and Pi. The apparent Km values for ADP and Pi were 4 microM and 0.22 mM, respectively. Stimulation of 45Ca2+ efflux by ADP and Pi was associated with ATP synthesis. The calcium ionophore A23187 prevented ATP synthesis, which indicates that the Ca2+ gradient facilitates the coupling of ATP synthesis to Ca2+ efflux.     

Fructose protects rat hepatocytes from anoxic injury. Effect on intracellular ATP, Ca2+i, Mg2+i, Na+i, and pHi.

The effects of fructose on the intracellular ionic changes evoked by anoxia were studied in freshly isolated rat hepatocytes maintained in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer (KHB). Cytosolic free calcium (Ca2+i) was measured with aequorin, intracellular sodium (Na+i) with sodium-binding benzofuran isophthalate, intracellular pH (pHi) with 2'-7'-bis(carboxyethyl)-5,6-carboxyfluorescein, lactic dehydrogenase (LDH) by the increase in NADH absorbance during lactate oxidation to pyruvate, and viability by trypan blue exclusion. ATP, Pi, phosphomonoesters, and the cell phosphorylation potential assessed by the reciprocal of the Pi/ATP ratio were measured by 31P NMR spectroscopy in real time. Intracellular free Mg2+ (Mg2+i) was calculated from the chemical shift of beta-ATP relative to alpha-ATP in the NMR spectra. Anoxia was induced by perfusing the cells with KHB saturated with 95% N2, 5% CO2. When the perfusate contained 5 mM glucose as substrate, anoxia caused a fall in ATP, a rise in Pi, and in the Pi/ATP ratio, a biphasic increase in Ca2+i that reached 1.45 +/- 0.42 microM and a 6-fold increase in LDH. When 15 mM fructose was used as substrate during the anoxic period, intracellular ATP decreased much faster than with glucose, Pi did not increase, and the concentration of phosphomonoesters increased 2.5-fold. During the first hour of anoxia, the Pi/ATP ratio was higher in the fructose than in the glucose group indicating that the hepatocyte phosphorylation potential and ATP decreased faster and to lower levels with fructose than with glucose. On the other hand, ATP and the phosphorylation potential of the fructose group increased during the second hour of anoxia, in contrast to their continuous decline in the glucose group. The major surge in Ca2+i was depressed 52% when glucose was replaced by fructose: Ca2+i reached only 0.7 +/- 0.2 microM instead of 1.45 +/- 0.42 microM (p less than 0.01). Anoxia also caused an increase in Na+i and an intracellular acidosis. The rise in Na+i was significantly greater with fructose than with glucose. Na+i rose from a control value of 15.9 +/- 2.4 to 32.2 +/- 0.4 mM with glucose and to 48.7 +/- 0.7 mM with fructose (p less than 0.001). The decrease in pHi from a control value of 7.43 +/- 0.03 was consistently greater and faster with fructose than with glucose: 6.59 +/- 0.03 and 7.04 +/- 0.01, respectively. At the same time, fructose completely suppressed LDH release and reduced the loss of viability produced by anoxia from 27.7 +/- 2.9 to 14 +/- 3.1% (p less than 0.05).     

水稻叶片蔗糖磷酸合成酶的一些特性

水稻叶片粗提液经硫酸铰分部沉淀、DE 52纤维素及 Sephadex G—200柱层析,得到较纯的蔗糖磷酸合成酶。该酶的最适 PH约7.0;UTP,UDP,ATP能明显地抑制其酶活;UTP是该酶UDPG的竞争性抑制剂,Mg~( )对它有促进作用;G6P则无影响。酶的两个底物F6P及UDPG的饱和动力学曲线分别为双曲线型和S型;K_m(F6P)=0.93 mmol/L;K_m(UDPG)=20.0 mmol/L;V_m(F6P)=83.3 nmol Suc mg~(-1)Protein min~(-1);V_m(UDPG)=333 nmol Suc mg~(-1)protein min~(-1);Hill(F6P)=1.0,Hill(UDPG)=1.4。水稻叶片蔗糖磷酸合成酶的活性受 ATP,UTP,UDP,UDPG等因素的调节。水稻叶片中蔗糖合成酶的总活力大于或等于蔗糖磷酸合成酶。     

Kinetic control of mitochondrial ATP synthesis

In order to gain a clearer understanding of the kinetic control of ATP synthesis, rat liver and rat heart mitochondria were incubated under conditions that resulted in various rates of net ATP synthesis or ATP hydrolysis. Radiolabeled phosphate was included in the incubation media, and exchange rates between phosphate and ATP were determined as a function of rates of net ATP synthesis. Since ATP synthase is a highly reversible enzyme, the catalyzed reaction was expected to approach equilibrium especially at low rates of respiration and net ATP synthesis. Thus ADP + Pi V1 in equilibrium V2 ATP. If V1 is the rate of incorporation of radiolabeled phosphate into ATP, then net ATP synthesis (or hydrolysis) is V1 - V2. Since V1 and V1 - V2 could be measured, it was possible to calculate V2. V1 doubled in the transition from zero to maximal net ATP synthesis, whereas V2 decreased by over 90% when the rate of ATP synthesis was high due to high-media ADP. In heart mitochondria at 37 degrees C when respiration increased from 104 +/- 10 to 842 +/- 51 nanoatoms of O2/(min X mg), incorporation of [33P]phosphate into ATP (V1) increased from 1,100 +/- 60 to 1,978 +/- 121 and V2 decreased from 1,100 to near zero. These data demonstrate that mitochondrial ATP synthesis does not occur near equilibrium under physiological conditions and relatively high rates of ATP synthesis. A reaction with a high ratio of forward to reverse flux is obviously not near equilibrium. The important most sensitively controlled reaction appears to be V2, ATP hydrolysis. Possible mechanisms of kinetic control of V2 are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of proteins showing differential changes during ATP oscillations in chondrogenesis

Prechondrogenic condensation is a critical step for skeletal pattern formation. Our previous study showed that ATP oscillations play an essential role in prechondrogenic condensation because they induce oscillatory secretion. However, the molecular mechanisms that underlie ATP oscillations remain poorly understood. We examined how differential changes in proteins are implicated in ATP oscillations during chondrogenesis by using liquid chromatography/mass spectrometry. Our analysis showed that a number of proteins involved in ATP synthesis/consumption, catabolic/anabolic processes, actin dynamics, cell migration and adhesion were detected at either the peak or the trough of ATP oscillations, which implies that these proteins have oscillatory expression patterns that are coupled to ATP oscillations. On the basis of the results, we suggest that (1) the oscillatory expression of proteins involved in ATP synthesis/consumption and catabolic/anabolic processes can contribute to the generation or maintenance of ATP oscillations and that (2) the oscillatory expression of proteins involved in actin dynamics, cell migration and adhesion plays key roles in prechondrogenic condensation by inducing collective adhesion and migration in cooperation with ATP oscillations. Copyright © 2014 John Wiley & Sons, Ltd.     

Role of intramitochondrial pH in the energetics and regulation of mitochondrial oxidative phosphorylation.

The dependence of ATP synthesis coupled to electron transfer from 3-hydroxy-butyrate (3-OH-B) to cytochrome c on the intramitochondrial pH (pHi) was investigated. Suspensions of isolated rat liver mitochondria were incubated at constant extramitochondrial pH (pHe) with ATP, ADP, Pi, 3-OH-B, and acetoacetate (acac) (the last two were varied to maintain [3-OH-B]/[acac] constant), with or without sodium propionate to change the intramitochondrial pH. Measurements were made of the steady-state water volume of the mitochondrial matrix, transmembrane pH difference, level of cytochrome c reduction, concentration of metabolites and rate of oxygen consumption. For each experiment, conditions were used for which transmembrane pH was near maximal and minimal values and the measured extramitochondrial [ATP], [ADP], and [Pi] were used to calculate log[ATP]/[ADP][Pi]. When [3-OH-B]/[acac] and [cyt c2+]/[cyt c3+] were constant, and pHi was decreased from approx. 7.7 to 7.2, log [ATP]/[ADP][Pi] at high pHi was significantly (P less than 0.02) greater than at low pHi. The mean slope (delta log [ATP]/[ADP][Pi] divided by the change in pHi) was 1.08 +/- 0.15 (mean +/- S.E.). This agrees with the slope of 1.0 predicted if the energy available for ATP synthesis is dependent upon the pH at which 3-hydroxybutyrate dehydrogenase operates, that is, on the pH of the matrix space. The steady-state respiratory rate and reduction of cytochrome c were measured at different pHi and pHe values. Plots of respiratory rate vs.% cytochrome c reduction at different intra- and extramitochondrial pH values indicated that the respiratory rate is dependent upon pHi and not on pHe. This implies that the matrix space is the source of protons involved in the reduction of oxygen to water in coupled mitochondria.     

Phosphate Uptake by Excised Maize Root Tips Studied by in VivoP Nuclear Magnetic Resonance Spectroscopy

The extent of phosphate uptake measured by the relative changes in cytoplasmic Pi, vacuolar Pi, ATP, glucose-6-phosphate, and UDPG was determined using in vivo³¹P nuclear magnetic resonance spectroscopy. Maize (Zea mays) root tips were perfused with a solution containing 0.5 or 1.0 millimolar phosphate at pH ~6.5 under different conditions. In the aerated state, phosphate uptake resulted in a significant increase (>80%) in vacuolar Pi, but cytoplasmic Pi only transiently increased by 10%. Under N₂, the cytoplasmic Pi increased ~150% which could be attributed to a large extent to the breakdown of ATP, sugar phosphates and UDPG. Vacuolar Pi increased but only to the extent of ~10% of that seen under aerobic conditions. 2-deoxyglucose pretreatment was utilized to decrease the level of cytoplasmic Pi. When pretreated with the 2-deoxyglucose, the excised maize roots absorbed phosphate from the perfusate with a significant increase in the cytoplasmic Pi. The increase could only be traced to external phosphate since the concentrations of other phosphorus containing species remained constant during the uptake period. With 2-deoxyglucose pretreatment, phosphate uptake under anaerobic conditions was substantially inhibited with only the vacuolar phosphate showing a slight increase. When roots were treated with carbonyl cyanide m-chlorophenyl hydrazone, no detectable Pi uptake was found. These results were used to propose a H⁺-ATPase related transport mechanism for phosphate uptake and compartmentation in corn root cells.     

Phosphorus-31 nuclear-magnetic-resonance study of phosphorylated metabolites compartmentation, intracellular pH and phosphorylation state during normoxia, hypoxia and ethanol perfusion, in the perfused rat liver

A quantitative analysis of the phosphorus-31 NMR spectra of excised perfused rat liver has been carried out at 80.9 MHz using a 30-mm sample cell. The results indicate that in liver from fed rats, all intracellular ATP is detected by NMR. In contrast, only the cytosolic fractions of Pi and ADP can be observed as indicated by careful analysis of spectra obtained from perchloric acid liver extracts and intact liver under valinomycin perfusion. In well-oxygenated perfused liver the ATP concentration is 7.4 mM. Values of 5.3 mM and 0.9 mM are found respectively for Pi and ADP concentrations in the cytosolic compartment. Cytosolic pH value (pHi) is 7.25 +/- 0.05 and free magnesium concentration 0.5 mM. Addition of 70 mM (0.4%) ethanol to the perfusate of a fed rat liver induces 25% and 38% reduction of ATP and Pi levels, respectively. A large amount of sn-glycerol 3-phosphate is synthesized (up to 11 mM) in the cytosol. After ethanol withdrawal, a large overshoot in cytosolic Pi is observed, which is indicative of a net uptake of Pi across the plasma membrane that occurred during ethanol oxidation. No significant pH variation is observed during ethanol infusion. In perfused liver of rats subjected to 48-h fasts, the concentrations of cytosolic phosphorylated metabolites are 5.3 mM, 0.8 mM and 11.5 mM for ATP, ADP and Pi, respectively. The perfusion of the liver with 70 mM ethanol does not change the adenine nucleotide levels, while the Pi content is decreased by 10%. During a 4-min hypoxia, induced by reducing the perfusion flow rate from 12 ml to 3 ml min-1 (100 g body weight)-1, ATP concentration decreases to 5.8 mM in the fed rat liver. Cytosolic Pi and ADP increase to 8.7 mM and 1.6 mM, respectively. The cytosolic pH evolves to more acidic values and reaches 7.02 +/- 0.05 at the end of the 4-min hypoxic period.     

Is adenosine a second metabolic substrate for human red blood cells?

Adenosine is present in the micromolar range in human plasma. In this study, metabolism of adenosine, which was maintained between 0.62 +/- 0.03 and 2.92 +/- 0.43 microM by means of a continuous infusion using a Harvard infusion pump, was investigated in human red blood cells. It was found that lactate production increases linearly as the adenosine concentration was raised. Cells infused with an average adenosine concentration of 2 microM produced lactate comparable to that produced by 5 mM glucose. The extent to which ATP concentration is maintained by adenosine also depends on its concentration. After a 4 h infusion with an average adenosine concentration of 0.7 microM, ATP content amounts to 75% of the glucose control. Raising the adenosine infusion concentration to 1.5 microM results in a full maintenance of ATP levels and at concentrations higher than 1.5 microM, adenosine produces a net synthesis of ATP. A net synthesis of ATP also occurs with adenosine concentration below 1.5 microM, if supplemented with glucose. In contrast, inosine infusion provides only a partial support of ATP and fails to produce a net synthesis of ATP in the presence of glucose. In addition, the presence of purine nucleoside and glucose together influence the metabolism of each other, depending on inorganic phosphate content (Pi). At a Pi concentration of 1 mM, the glucose consumption rate is reduced by approx. 25% by purine nucleoside infusion and vice versa. In sharp contrast, glucose consumption at 16 mM Pi is potentiated by adenosine. These findings suggest that plasma adenosine contributes significantly to human red cell energetics, even though it is present at a concentration several orders of magnitude lower than glucose.     

31P NMR saturation transfer measurements of phosphorus exchange reactions in rat heart and kidney in situ

31P NMR spectra of rat kidney and heart, in situ, were obtained at 97.2 MHz by using chronically implanted radio-frequency coils. Previous investigators have used magnetization transfer techniques to study phosphorus exchange in perfused kidney and heart. In the current experiments, saturation transfer techniques were used to measure the steady-state rate of exchange between inorganic phosphate (Pi) and the gamma-phosphate of ATP (gamma ATP) in kidney, and between phosphocreatine (PCr) and gamma ATP, catalyzed by creatine kinase, in heart. The rate constant for the exchange detected between Pi and gamma ATP in kidney, presumably catalyzed by oxidative phosphorylation, was 0.12 +/- 0.03 s-1. This corresponds to an ATP synthesis rate of 12 mumol min-1 (g wet weight)-1. Comparison of previously published O2 consumption and Na+ reabsorption rates for the intact kidney with the NMR-derived rate for ATP synthesis gave flux ratios of JATP/JO2 = 1.6-3.3 and JNa+/JATP = 4-10. The rate constants for the creatine kinase reaction, assuming a simple two-site exchange, were found to be 0.57 +/- 0.12 s-1 for the forward direction (PCr----ATP) and 0.50 +/- 0.16 s-1 for the reverse direction (ATP----PCr). The forward rate (0.78 +/- 0.18 intensity unit/s) was significantly larger (p less than 0.05) than the reverse rate (0.50 +/- 0.16 intensity unit/s). This difference between the forward and reverse rates of creatine kinase has been previously noted in the perfused heart. The difference has been attributed to participation of ATP in other reactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the sidedness of membrane phosphorylation by Pi and ATP synthesis during reversal of the Ca2+ pump of sarcoplasmic reticulum vesicles.

The membrane sidedness of Pi interaction in reactions which characterize reversal of the Ca2+ pump of sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle was investigated. Vesicles previously loaded with calcium [32P]phosphate were incubated with 0.1 mM ADP and different concentrations of nonradioactive Pi. Alternatively, vesicles loaded with nonradioactive calcium phosphate were incubated in a medium containing 32Pi. The rates of Ca2+ efflux and ATP synthesis were siginficantly activated only when Pi was included in the assay medium. Although the Pi contained by the vesicles crosses the membrane at a rate proportional to the Ca2+ efflux, [gamma-32P]ATP was synthesized only when 32Pi interacted with the outer surface of the membrane. Similarly, ATP in equilibrium 32Pi or ITP in equilibrium 32Pi exchange could be measured only when the external pool of Pi was labeled. Both for ATP synthesis and for the ITP in equilibrium Pi exchange reaction, membrane phosphorylation by 32Pi was negligible unless the external pool of Pi was labeled. The ionophore X-537 A increased the rate of Ca2+ efflux but inhibited the synthesis of ATP. During reversal of the Ca2+ pump, Pi apparently interacts with the membrane only at the outer surface, and at a site different from that where Ca2+ crosses the membrane.     

Inorganic Phosphate (Pi) Enhancement of Dark Respiration in the Pi-Limited Green Alga Selenastrum minutum (Interactions between H+/Pi Cotransport,the Plasmalemma H+-ATPase,and Dark Respiratory Carbon Flow)

Inorganic phosphate (Pi) enrichment of the Pi-limited green alga Selenastrum minutum in the dark caused a 2.5-fold increase in the rate of O2 consumption. Alkalization of the media during Pi assimilation was consistent with a H+/Pi cotransport mechanism with a stoichiometry of at least 2 H+ cotransported per Pi. Dark O2 consumption remained enhanced beyond the period of Pi assimilation and did not recover until the medium was reacidified. This result, coupled with an immediate decrease in adenylate energy charge following Pi enrichment, suggested that respiration is regulated by the ATP requirements of a plasmalemma H+-ATPase that is activated to maintain intracellular pH and provide proton motive force to power Pi uptake. Concentrations of tricarboxylic acid cycle intermediates decreased following Pi enrichment and respiratory CO2 efflux increased, indicating that the tricarboxylic acid cycle was activated to supply reductant to the mitochondrial electron transport chain. These results are consistent with direct inhibition of electron transport by ADP limitation. Enhanced rates of starch breakdown and increases in glycolytic metabolites indicated that respiratory carbon flow was activated to supply reductant to the electron transport chain and to rapidly assimilate Pi into metabolic intermediates. The mechanism that initiates glycolytic carbon flow could not be clearly identified by product:substrate ratios due to the complex nature of Pi assimilation. High levels of triose-P and low levels of phosphoenolpyruvate were the primary regulators of pyruvate kinase and phosphofructokinase, respectively.     

ATP-Mg/Pi carrier activity in rat liver mitochondria.

The ATP-Mg/Pi carrier in liver mitochondria is activated by micromolar Ca2+ and mediates net adenine nucleotide transport into and out of the mitochondrial matrix. The purpose of this study was to characterize certain features of ATP-Mg/Pi carrier activity that are essential for understanding how the mitochondrial adenine nucleotide content is regulated. The relative importance of ATP and ADP as transport substrates was investigated using specific trap assays to measure their separate rates of carrier-mediated efflux with Pi as the external counterion. Under energized conditions ATP efflux accounted for 88% of total ATP+ADP efflux. With oligomycin present to lower the matrix ATP/ADP ratio, ATP efflux was eliminated and ADP efflux was relatively unaffected. Mg2+ was stoichiometrically required for ATP influx and is probably transported simultaneously with ATP. Ca2+ and Mn2+ could substitute for the stoichiometric Mg2+ requirement. ADP influx and Pi-induced adenine nucleotide efflux were unaffected by external Mg2+. Experiments with Pi analogues suggested that Pi is transported as the divalent anion, HPO4(2-). The results show that ATP-Mg and divalent Pi are the major transport substrates; the most probable transport mechanism for the ATP-Mg/Pi carrier is an electroneutral exchange. The results are consistent with the hypothesis that the direction and magnitude of net adenine nucleotide movements are determined mainly by the (ATP-Mg)2- and HPO4(2-) concentration gradients across the inner mitochondrial membrane.