A 31P-nuclear magnetic resonance study of skeletal muscle metabolism in rats depleted of creatine with the analogue β-guanidinopropionic acid

Rats were fed a diet containing 1% β-guanidinopropionic acid (GPA) for 6–10 weeks to deplete their skeletal muscle of creatine. ³¹P-NMR was used to monitor metabolic changes in the gastrocnemius muscle at rest, during stimulated steady-state isometric contraction at 4 Hz and during recovery from stimulation. In resting muscles, the [creatine phosphate] was reduced to 10% (2.8 μmol·g^?1) and the [ATP] to 50% (3.3 μmol·g^?1) of those found in rats fed a control diet. The concentration of the phosphorylated form of the analogue (PGPA) was 23 μmol·g^?1. There was no significant difference in muscle performance or in the relative changes in the [ATP] during stimulation. Intracellular pH decreased rapidly on stimulation and recovered during the stimulation period to near resting values in both groups. In control rats, the initial decrease in pH was greater and the time to recovery was longer than in GPA-fed rats. The rate at which PGPA supplied energy to the contracting muscle (0.027 mM·s^?1) was insignificant relative to the minimum estimated rate of ATP turnover (1 mM·s^?1). The rate of PGPA resynthesis during recovery (0.018 mM·s^?1) is enzyme-limited and provides an independent estimate of creatine kinase flux during this period (18.9 mM·s^?1). The creatine kinase flux (creatine phosphate → ATP) in the resting muscle of GPA-fed rats was 12-fold less than in control animals, 1.3 vs. 15.7 mM·s^?1. These results demonstrate that neither the [creatine phosphate] nor the activity of creatine kinase is critical for aerobic metabolism. Skeletal muscle appears to adapt to a diminished creatine pool by enhancing its aerobic capacity.     

Detrimental cerebrometabolic effects of hyperoxia in newborn rats

To investigate the pathogenesis of oxygen toxicity in the newborn brain, we exposed one-day-old Sprague-Dawley albino rats to 100% O₂ and measured whole-brain high-energy phosphates, glucose, lactate, and free fatty acids (FFA) after 0, 15, 30, 60 and 120 min. Whole-brain adenosine triphosphate and creatine phosphate fell significantly from about 4.5 to 2.5 μmol-mg⁻¹ protein. Brain lactate remained at about 0.3 μmol·mg⁻¹ protein in hyperoxic rats, but increased in normoxic rats, from 0.3 to 1.3 μmol·mg⁻¹ protein at 120 min. Total FFA decreased from 30 to 15 nmol·mg⁻¹ protein during normoxia, but increased to 40 nmol·mg⁻¹ protein during hyperoxia. Undetectable in normoxic rats, arachidonic acid increased to between 4 and 6 nmol·mg⁻¹ protein during hyperoxia while oleic acid increased by two to threefold. In normoxia, palmitate decreased by 70% from 12 to 4 nmol·mg⁻¹ protein whereas in hyperoxia it remained at 10 nmol·mg⁻¹ protein. Normobaric 100% O₂ has detrimental metabolic effects on the neonatal brain which cannot be attributed to cerebral vasospasm or seizure-induced cerebral anoxia because lactic acidosis was not observed. FFA changes suggest that a likely explanation is membrane lipid peroxidation from O₂-induced free radicals.     

Use of cytosolic metabolite patterns to estimate free magnesium in normoxic myocardium

Cytosolic free magnesium (Mg_f) is considered relatively constant. To test this concept, Mg_f was estimated during hyperkalemic ventricular akinesis, normal and maximum adrenergic stimulation, and sulfate loading of the normoxic perfused guinea-pig heart. The Mg_f estimates utilized a new sliding scale derived from the Mg²⁺-dependence of glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase (GAPDH/PGK). The pseudo constant K_GAPDH′·K_PGK′ was measured as ([creatine phosphate][3-phosphoglycerate][lactate]K_LDH/([creatine][P_i[glyceraldehyde 3-phosphate][pyruvate]K_CK), which varied with magnesium due to K_CK (CK, LDH = creatine kinase, lactate dehydrogenase). However, the correct magnesium dependencies of the true constants K_GAPDH·K_PGK and K_CK were taken from the literature. The [Mg²⁺] at which pseudo K_GAPDH′·K_PGK′ equalled true K_GAPDH·K_PGK was the best estimate of Mg_f. Mg_f fell to ≈0.13 mM in hyperkalemic arrest from a control of ≈0.6 mM, rising to ≈0.85 mM only during maximum adrenergic stress. Mg_f increased further to ≈1.3 mM during sulfate loading which induced ATP catabolism. Mg_f and ATP were reciprocally related. Thus; (1) myocardial free [Mg²⁺] judged from GAPDH/PGK mass-action relations changed appreciably only under extreme physiological states; (2) ATP was a major chelator of Mg²⁺ in perfused myocardium, i.e., acute ATP pool size reduction may be associated with increments in Mg_f.     

31P NUCLEAR MAGNETIC RESONANCE STUDY OF INTRACELLULAR PHOSPHATE POOLS AND POLYPHOSPHATE METABOLISM IN HETEROSIGMA AKASHIWO (HADA) HADA (RAPHIDOPHYCEAE)1

The effects of starvation and subsequent addition of phosphate-containing medium on the phosphate metabolic intermediates were studied by ³¹P-NMR spectroscope of perchloric acid extracts and intact cells of Heterosigma akashiwo (Hada) Hada. When orthophosphate in the medium was completely depleted the medium was enriched with orthophosphate (4.5 μM). In the phosphate starved condition, the P cell quota was 76 fmol·cell⁻¹ and the major components of phosphate intermediates were phosphodiester, sugar phosphate and orthophosphate (P_i). After addition of P_i, rapid uptake of P_i was observed and the P cell quota increased to 108 fmol·cell⁻¹ in 2 h, 134 fmol·cell⁻¹ in 5 h and 222 fmol·cell⁻¹ in 1 day after addition of phosphate. The ³¹P-NMR spectrum indicated that a major portion of P was stored as polyphosphate, in which the average chain length of polyphosphate increased from 10 to 20 phosphate residues in one day after addition of P_i.     

Light and CO2 limitation of photosynthesis and states of the reactions regenerating ribulose 1,5-bisphosphate or reducing 3-phosphoglycerate

Spinach leaves were illuminated at various temperatures or CO₂ concentrations until steady-state photosynthesis could be measured. Subsequently, they were frozen rapidly in liquid nitrogen and freeze-dried. From the dry material, chloroplasts were isolated in a mixture of organic solvents in which polar metabolites are insoluble. Metabolite levels were determined in the chloroplast fraction. From measured levels of dihydroxyacetone phosphate, fructose 6-phosphate (Fru-6-P), ribulose 1,5-bisphosphate (Rbu-1,5-P₂), ATP and ADP, mass-action ratios of the reaction dihydroxyacetone phosphate + 2 glyceraldehyde 3-phosphate + 3 ATP + Fru-6-P → 3 Rbu-1,5-P₂ + 3 ADP + P_i were computed. They increased at constant light intensity with increasing CO₂ concentration or increasing temperature as photosynthetic flux increased. Surprisingly, however, mass action ratios decreased as flux increased with increasing light intensities. Moreover, mass-action ratios were linearly correlated to light-limitation coefficients which were obtained by computing the light limitation of photosynthesis from the slopes of light and CO₂ response curves and multiplying obtained values with that increment of photosynthesis which was measured on increasing the light intensity to saturation. The results are interpreted to indicate tight enzymic control of the formation of ribulose bisphosphate by light. As light intensities are increased, light-regulated enzymes are activated to an extent which permits a decrease in the mass action ratios instead of the increase expected to drive increased carbon flux. Since the reactions catalyzed by phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase are close to thermodynamic equilibrium even when photosynthetic fluxes are large, ratios of dihydroxyacetone phosphate to 3-phosphoglycerate indicated the state of chloroplast phosphorylation potentials and the redox state of NADP which together form the assimilatory power [ATP] · [ADP]⁻¹ · [P_i]⁻¹ · [NADPH] · [NADP⁺]⁻¹. Assimilatory power decreased as carbon flux increased with increasing light intensity and increasing CO₂ concentration, but increased as carbon flux increased with increasing temperature. Again this indicates a decrease in the flow resistance of the carbon cycle as light or CO₂ is increased. The decrease in the flow resistance is attributed to enzyme activation when light is increased, or to increased carboxylation when CO₂ is increased.     

Glutamine synthetase activity and free amino acid pools of eelgrass (Zostera marina L.) roots

Activity of the enzyme glutamine synthetase (GS, EC 6.3.1.2) was determined in vitro for roots of the marine angiosperm Zostera marina L. (eelgrass) collected from a population in Great Harbor, Woods Hole, Massachusetts, U.S.A. The GS synthetase activity was lowest in roots of plants collected from the shallow region of the eelgrass bed (12.0 μmol·g⁻¹ (fresh wt)· h⁻¹) and increased in the mid (3.0 m, 40.3 μmol·g⁻¹ (fresh wt)·h⁻¹) and deep (5.0 m, 72.3 μmol·g⁻¹ (fresh wt)·h⁻¹) plant collection depths. GS transferase activity increased with collection depth in a similar manner: shallow, 28.6 μmol·g⁻¹ (fresh wt)·h⁻¹; mid, 52.0 μmol·g⁻¹ (fresh wt)·h⁻¹; deep, 92.8 μmol·g⁻¹ (fresh wt)·h⁻¹. When sediment-embedded plants were held in continuous darkness for 2 days to create extended root anoxia, root GS activities nearly doubled. In contrast, in vivo incorporation of ¹⁴C-glutamate into glutamine and protein residue remained constant or declined under short-term hypoxia and anoxia. During aerobic recovery from anoxia, root labelling of glutamine and protein increased markedly. Free amino acid patterns of eelgrass roots growing in situ were determined over a diurnal cycle. Total free amino acid content was maximal at dawn and decreased 50% by noon. In contrast, the proportion of glutamine was lowest at dawn and maximal at noon for both shallow and deep-growing plants. Despite differences in depth-specific plant sizes, root/rhizome/shoot ratios, and relative growth rates, the daily whole plant nitrogen demand of shallow and deep growing plants were equivalent. When corrected for assay temperature response, the enzyme synthetase activities measured in vitro suggest that all of the plant nitrogen assimilation requirements can be met within daylight hours during the period of peak summer biomass.     

Smooth muscle and NMR review: An overview of smooth muscle metabolism

Nuclear magnetic resonance (NMR) is a non-invasive technique which allows us to examine the biochemical, physiological and metabolic events occurring inside living tissue; such as vascular and other smooth muscles.It has been found that the smooth muscle metabolism is compartmented such that mitochondrial function fuels contraction and that much glycolytic ATP production is used for membrane pumps. Using NMR we have been able to observe the ATP and phosphocreatine (PCr) concentrations and estimate the ADP concentration, as well as flux through the creatine kinase (CK) system. It has also been found that the smooth muscle metabolism is able to maintain ATP concentration in the absence of mitochondrial function (cyanide inhibition). Therefore, the vessels are able to adapt to metabolic demands as necessary.NMR is versatile in the information it can provide because it has also yielded important contributions with regard to the intracellular pH and ionic status. For example, the intracellular free Mg²⁺ ([Mg²⁺]_i) can be measured with NMR simultaneously with ATP concentrations and NMR has shown us that the [Mg²⁺]_i is highly protected in the muscle (within confined range), but also responds to the environment around it.In this review we conclude that NMR measurements of smooth muscle research is a useful technique for assessing chronic and acute changes that occur in the tissue and during diseases.     

Role of phosphocreatine in energy transport in skeletal muscle of bullfrog studied by 31P-NMR

To evaluate the energy-shuttle hypothesis of the phosphocreatine/creatine kinase system, diffusion rates for ATP, phosphocreatine and flux through the creatine kinase reaction were determined by 31P-NMR in resting bullfrog biceps muscle. The diffusion coefficient of phosphocreatine measured by 31P-pulsed gradient NMR was 1.4-times larger than ATP in the muscle, indicating the advantage of phosphocreatine molecules for the intracellular energy transport. The flux of the creatine kinase reaction measured by 31P-saturation transfer NMR was 3.6 mmol/kg wet wt. per s in the resting muscle. The flux is equal to the turnover rate of ATP, ADP, phosphocreatine and creatine molecules, therefore, the life-times of these substrates and the average distance traversed after the life-times by the diffusing molecules were calculated using the diffusion coefficients obtained by 31P-NMR. The mean square length of one-dimensional diffusion was 22 microns in ATP molecules and the minimum diffusion length was 1.8 microns in ADP molecules. The latter was calculated using free ADP concentration, 30 mumol/kg wet wt., obtained from the equilibrium constant of the creatine kinase reaction and the diffusion coefficient assumed to be the same of ATP in muscle. Similar diffusion lengths of ADP were calculated using the reported values for the flux of the creatine kinase reaction in heart and smooth-muscle. The diffusion lengths of all substrates involved in the creatine kinase reaction were larger than the radii of myofibrils. Therefore, in the muscles with an alternating arrangement of mitochondria and myofibrils, such as heart and certain skeletal muscles, ATP and ADP molecules can move freely between myofibrils and mitochondria without the aid of the creatine kinase reaction; thus, we conclude that the energy-shuttle hypothesis is not obligatory for energy transport between the mitochondria and the myofibrils.     

Active creatine kinase is present in matrix vesicles isolated from femurs of chicken embryo: Implications for bone mineralization

Proteomic analysis of matrix vesicles (MVs) isolated from 17-day-old chicken embryo femurs revealed the presence of creatine kinase. In this report we identified the enzyme functionally and suggest that the enzyme may participate in the synthesis of ATP from ADP and phosphocreatine within the lumen of these organelles. Then, ATP is converted by nucleotide hydrolyzing enzymes such as Na⁺, K⁺-ATPase, protein kinase C, or alkaline phosphatase to yield inorganic phosphate (P_i), a substrate for mineralization. Alternatively, ATP can be hydrolyzed by a nucleoside triphosphate pyrophosphatase phosphodiesterase 1 producing inorganic pyrophosphate (PP_i), a mineralization inhibitor. In addition, immunochemical evidence indicated that VDAC 2 is present in MVs that may serve as a transporter of nucleotides from the extracellular matrix. We discussed the implications of ATP production and hydrolysis by MVs as regulatory mechanisms for mineralization.     

Reverse NaCa exchange requires internal Ca and/or ATP in squid axons

Classical NaCa exchange models are based on a symmetric carrier system where Na and Ca competing from the same site, can produce net movement of the other against its electrochemical gradient. We have explored this symmetric assumption by studying the Ca_o and Na_o-dependent Na efflux in dialyzed squid axons in which proper control of both external and internal medium was achieved. The results show: (1) In axons dialyzed without Ca_i and ATP, Ca_o-dependent Na efflux cannot be detected even in the absence of Na_o. Under these conditions, the level of Na efflux (1 pmol · cm⁻² · s⁻¹) is close to that predicted by an electrical ‘leak’. (2) In axons dialyzed with Ca_i (100 μM) and without ATP, Na efflux measured in 440 mM Na_o, is about 4–5 pmol · cm⁻² · s⁻¹ and rather insensitive to Ca_o between 0 and 10 mM. However, in the absence of Na_o, a Ca_o-dependent Na efflux is observed similar in magnitude to that found in the presence of external Na. (3) In the presence of both Ca_i and ATP, Na efflux into artificial sea-water (440 mM Na, 10 mM Ca) is 18 pmol · cm⁻² · s⁻¹. In the absence of Na_o the efflux of Na is 7.5 pmol · cm⁻² · s⁻¹. In the absence of both Na_o and Ca_o the efflux is close to ‘leak’. With full Na_o but no Ca_o, the Na efflux average 12.6 pmol · cm⁻² · s⁻¹. These results indicate a marked asymmetry in the modus operandi of the NaCa exchange system with respect to Ca_i and ATP. These two substrates are required from the cis side to promote Ca_o-dependent Na efflux (reversal NaCa exchange).     

Creatine kinase kinetics,ATP turnover,and cardiac performance in hearts depleted of creatine with the substrate analogue β-guanidinopropionic acid

Rats were fed a diet containing 1% of the creatine substrate analogue β-guanidinopropionic acid for 6–10 weeks. ³¹P-NMR investigation of isolated, glucose-perfused working hearts showed a 90% reduction in [phosphocreatine] from 22.2 to 2.5 μmol/g dry wt in guanidinopropionic acid-fed animals but no change in [P_i], [ATP], or intracellular pH. The unidirectional exchange flux in the creatine kinase reaction (direction phosphocreatine → ATP) was measured by saturation transfer NMR in hearts working against a perfusion pressure of 70 cm of water. This exchange was 10 μmol/g dry wt per s in control hearts and decreased 4-fold to 2.5–2.8 μmol/g dry wt per s in hearts from guanidinopropionic acid-fed animals. Oxygen consumption and cardiac performance were measured in parallel experiments at two perfusion pressures, 70 and 140 cm. No significant differences were observed in oxygen uptake or in any of the performance criteria between hearts from control and guanidinopropionic acid-fed rats at either workload. Assuming an ADP:O ratio of 3, the oxygen consumption measurements correspond to ATP turnover rates of 4.2–7.8 μmol/g dry per s. These rates are 1.5–3-times greater than the rate of the phosphocreatine → ATP exchange in hearts from guanidinopropionic acid-fed rats. These data suggest that phosphocreatine cannot be an obligate intermediate of energy transduction in the heart.     

Saturation-transfer studies of ATP-Pi exchange in isolated perfused rat liver

The rate of exchange between inorganic phosphate and ATP was measured in isolated perfused rat livers in the direction of ATP synthesis using ³¹P NMR spectroscopy and the saturation-transfer technique. Measurement of ATP hydrolysis was not observable, even after treatment of rats with 100 μg T₃/day per 100 g body wt. When the perfused livers were treated with iodoacetate in order to inhibit glycolysis, NMR measurable exchange between ATP and P_i was eliminated. It is concluded that the inorganic phosphate → ATP conversion detected by saturation transfer is catalyzed by enzymes of the glycolytic pathway and that the mitochondrial ATPase rate is too slow to contribute to the observed effect.     

Inhibition of photophosphorylation by ATP and the role of magnesium in photophosphorylation

ATP and pyrophosphate at high concentration (> 1 mM) inhibited photophosphorylation of isolated spinach chloroplasts in the normal salt medium and did not cause stimulation of electron transport. The inhibition of photophosphorylation by ATP or pyrophosphate was shown to be abolished by the addition of excess MgCl₂, ADP and phosphate. It has been demonstrated that the rates of photophosphorylation in the absence and presence of ATP or pyrophosphate are determined similarly by the concentrations of magnesium-ADP (Mg · ADP^?) and magnesium-phosphate (Mg · P_i) complexes.It is highly probable that Mg · ADP^? and Mg · P_i, but not free ADP and free phosphate, are the active form of the substrates of photophosphorylation. This is in support of the view that ATP inhibits photophosphorylation by decreasing the concentration of Mg²⁺ which is available for the formation of the complex with ADP and phosphate.     

Exercise and recovery metabolism in the pacific spiny dogfish (<Emphasis Type="Italic">Squalus acanthias</Emphasis>)

We examined the effects of exhaustive exercise and post-exercise recovery on white muscle substrate depletion and metabolite distribution between white muscle and blood plasma in the Pacific spiny dogfish, both in vivo and in an electrically stimulated perfused tail-trunk preparation. Measurements of arterial-venous lactate, total ammonia, -hydroxybutyrate, glucose, and l-alanine concentrations in the perfused tail-trunk assessed white muscle metabolite fluxes. Exhaustive exercise was fuelled primarily by creatine phosphate hydrolysis and glycolysis as indicated by 62, 71, and 85% decreases in ATP, creatine phosphate, and glycogen, respectively. White muscle lactate production during exercise caused a sustained increase (~12 h post-exercise) in plasma lactate load and a short-lived increase (~4 h post-exercise) in plasma metabolic acid load during recovery. Exhaustive exercise and recovery did not affect arterial PO₂, PCO₂, or PNH₃ but the metabolic acidosis caused a decrease in arterial HCO₃^– immediately after exercise and during the first 8 h recovery. During recovery, lactate was retained in the white muscle at higher concentrations than in the plasma despite increased lactate efflux from the muscle. Pyruvate dehydrogenase activity was very low in dogfish white muscle at rest and during recovery (0.53±0.15 nmol g wet tissue^–1 min^–1; n=40) indicating that lactate oxidation is not the major fate of lactate during post-exercise recovery. The lack of change in white muscle free-carnitine and variable changes in short-chain fatty acyl-carnitine suggest that dogfish white muscle does not rely on lipid oxidation to fuel exhaustive exercise or recovery. These findings support the notion that extrahepatic tissues cannot utilize fatty acids as an oxidative fuel. Furthermore, our data strongly suggest that ketone body oxidation is important in fuelling recovery metabolism in dogfish white muscle and at least 20% of the ATP required for recovery could be supplied by uptake and oxidation of -hydroxybutyrate from the plasma.Abbreviations CoA-SH free coenzyme A - CPT-1 carnitine palmitoyltransferase-1 - CrP creatine phosphate - H⁺_m metabolic proton load - Lac lactate load - PDH pyruvate dehydrogenase - PVP polyvinylpyrrolidone - SCFA-carnitine short-chain fatty acyl-carnitine - TAG triacylglycerol - TENS trancutaneous electrical nerve stimulator Communicated by: L.C.-H. Wang     

The steady-state rate of ATP synthesis in the perfused rat heart measured by 31P NMR saturation transfer

The steady-state rate of ATP synthesis in the isolated, Langendorff-perfused rat heart was determined using a ³¹P NMR saturation transfer method. At 37°C and a perfusion pressure of 70 cm H₂O the value is 2.8 ± 0.3 (n=5 ± S.E.M.) μmol.s^?1. (g. dry wt.)^?1. The activity of creatine phosphokinase measured in the same experiments was 14.6 ± 1.0 μ mol.s^?1 .(g. dry wt.)^?1. From the rate of ATP synthesis and the separately measured oxygen consumption we calculated an apparent mitochondrial ADP:O ratio of 3.5 ± 0.8 in the intact tissue.     

31P-NMR saturation transfer study of the in vivo kinetics of arginine kinase in Carcinus crab leg muscle

The kinetics of the reaction catalyzed by arginine kinase have been determined at 9.5 and 23°C for in vivo leg muscle of Carcinus maenas (the common shore crab) using the noninvasive technique of ³¹P-NMR spectroscopy. Concentrations of mobile phosphorus metabolites were the same at both temperatures: 78.7 mM for arginine phosphate, 9.0 mM for adenosine triphosphate (ATP), and 2.6 mM for inorganic phosphate (P_i), as estimated from NMR resonance intensities and literature values for ATP concentration as assayed by traditional biochemical methods. Apparent unidirectional rate constants for formation of ATP from arginine phosphate and ADP were 0.09 s^?1 at 9.5°C and 0.27 s^?1 at 23°C. Pseudo-first-order rate constants for arginine phosphate generation from Arg and ATP were 0.38 and 1.10 s^?1 at 9.5 and 23°C, respectively. In vivo Q₁₀ for the arginine kinase reaction between 9.5 and 23°C was thus 2.2 for both directions. When the kinetic data are analyzed using the Arrhenius equation, activation energies of 126 kJ/mol for ATP formation and 105 kJ/mol for arginine phosphate formation are found. The measured chemical fluxes through arginine kinase in the forward reaction (arginine phosphate hydrolysis) were twice those in the reverse reaction, consistent with either compartmentation of substrates or participation of substrates in alternative metabolic pathways.     

Preservation of phosphagen kinase function during transient hypoxia via enzyme abundance or resistance to oxidative inactivation

Mammalian lactate dehydrogenase and phosphofructokinase are more susceptible in vitro to superoxide and hydroxyl (·OH) radicals than pyruvate kinase and glucose-6-phosphate dehydrogenase, suggesting that differential inactivation of regulatory enzymes contributes to the metabolic disintegration in stenoxic tissues during transient hypoxia. Like-wise, creatine kinase in smooth muscle from porcine ileum is significantly reduced by hypoxia-reoxygenation ex vivo from 300 (±18.2 SE,n=8) to 196 U·g wet wt^-1 (±16.7,P0.001, ANOVA). Conversely, arginine kinase, from the myocardium ofLimulus polyphemus, a species that tolerates anoxia for days was 2.9-fold less susceptible to oxidative inactivation. To examine whether preservation of kinase function is related to euryoxic capacity, a combination of non-invasive³¹P-NMR spectroscopy and enzyme-linked assays was used to follow ATP and phosphagen status during hypoxia-reoxygenation in porcine ileum smooth muscle,L. polyphemus myocardium, and the myocardium ofArgopecten irradians, a scallop species tolerant of hypoxia for only 24 h. Despite wide differences in phylogeny, euryoxic capacity and oxidative vulnerability of the phosphagen kinases, in all three tissues, the phosphagen pool recovered concomitant with ATP during reoxygenation, thereby revealing competent kinase function. In the mammalian tissue, such preservation of kinase function is facilitated by a 2400-fold excess of enzyme activity.     

Requirement of medium ADP for the steady-state hydrolysis of ATP by the proton-translocating Paracoccus denitrificans Fo·F1-ATP synthase

F_o·F₁-ATP synthase in inside-out coupled vesicles derived from Paracoccus denitrificans catalyzes P_i-dependent proton-translocating ATPase reaction if exposed to prior energization that relieves ADP·Mg²⁺-induced inhibition (Zharova, T.V. and Vinogradov, A.D. (2004) J. Biol. Chem.,279, 12319–12324). Here we present evidence that the presence of medium ADP is required for the steady-state energetically self-sustained coupled ATP hydrolysis. The initial rapid ATPase activity is declined to a certain level if the reaction proceeds in the presence of the ADP-consuming, ATP-regenerating system (pyruvate kinase/phosphoenol pyruvate). The rate and extent of the enzyme de-activation are inversely proportional to the steady-state ADP concentration, which is altered by various amounts of pyruvate kinase at constant ATPase level. The half-maximal rate of stationary ATP hydrolysis is reached at an ADP concentration of 8 × 10⁻⁶ M. The kinetic scheme is proposed explaining the requirement of the reaction products (ADP and P_i), the substrates of ATP synthesis, in the medium for proton-translocating ATP hydrolysis by P. denitrificans F_o·F₁-ATP synthase.     

Direct Detection of the Acetate-forming Activity of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily^1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya⁶. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila^7-14. An acetate kinase which can only utilize PP_i but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus^15,16.In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann^17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD⁺ by the enzymes pyruvate kinase and lactate dehydrogenase^21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine²³. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP⁺ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase²⁴.Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PP_i.     

The determination of inorganic orthophosphate in biological systems

A simple and rapid method is described for determining P_i by spectrophotometric measurement of a soluble complex of phosphomolybdic acid and Cirrasol ALN-WF, a non-ionic detergent formerly known as Lubrol W. The measured complex has a molar extinction coefficient of 4.59 · 10³ at 390 nm and little interference is found with relatively high concentrations of chelating agents, salts, and other compounds which interfere with most other P_i assays. Linearity is observed in the range 0–1.2 μmoles P_i and developed assay samples are stable for 8 h at 20 °C or 24 h at 4 °C. The method is suitable for use in the presence of moderate concentrations of protein or ATP.After suitable modification the assay can be used at pH 4.0. Sensitivity is reduced at this pH (ε_{M, 390nm} = 2.79 · 10³) but linearity is maintained up to 1 μmole P_i and the coloured complex is stable for 4 h at 20 °C. The pH-4 procedure is suitable for measurement of P_i in the presence of very labile phosphate esters such as creatine phosphate.The phosphomolybdic acid-Cirrasol complex can be reduced at ambient temperature in both the above systems. A blue complex results with ε_{M, 820nm} of 9.9 · 10³ at pH 4.0, and 1.8 · 10⁴ under more acidic conditions.