Values For ADP Research Articles

Interaction between aurovertin and adenine nucleotide binding sites on mitochondrial F1-ATPase and the isolated beta subunit.

The effect of aurovertin on the binding parameters of ADP and ATP to native F1 from beef heart mitochondria in the presence of EDTA has been explored. Three exchangeable sites per F1 were titrated by ADP and ATP in the absence or presence of aurovertin. Curvilinear Scatchard plots for the binding of both ADP and ATP were obtained in the absence of aurovertin, indicating one high affinity site (Kd for ADP = 0.6-0.8 microM; Kd for ATP = 0.3-0.5 microM) and two lower affinity sites (Kd for ADP = 8-10 microM; Kd for ATP = 7-10 microM). With a saturating concentration of aurovertin capable of filling the three beta subunits of F1, the curvilinearity of the Scatchard plots was decreased for ATP binding and abolished for ADP binding, indicating homogeneity of ADP binding sites in the F1-aurovertin complex (Kd for ADP = 2 microM). When only the high affinity aurovertin site was occupied, maximal enhancement of the fluorescence of the F1-aurovertin complex was attained with 1 mol of ADP bound per mol of F1 and maximal quenching for 1 mol of ATP bound per mol of F1. When the F1-aurovertin complex was incubated with [3H]ADP followed by [14C]ATP, full fluorescence quenching was attained when ATP had displaced the previously bound ADP. In the case of the isolated beta subunit, both ADP and ATP enhanced the fluorescence of the beta subunit-aurovertin complex. The Kd values for ADP and ATP in the presence of EDTA were 0.6 mM and 3.7 mM, respectively; MgCl2 decreased the Kd values to 0.1 mM for both ADP and ATP. It is postulated that native F1 possesses three equivalent interacting nucleotide binding sites and exists in two conformations which are in equilibrium and recognize either ATP (T conformation) or ADP (D conformation). The negative interactions between the nucleotide binding sites of F1 are strongest in the D conformation. Upon addition of aurovertin, the site-site cooperativity between the beta subunits of F1 is decreased or even abolished.

Purification and characterization of glutamine synthetase from the archaebacterium Methanobacterium ivanovi.

Glutamine synthetase (GS) was purified to electrophoretic homogeneity from the obligate anaerobic archaebacterium Methanobacterium ivanovi. The 130-fold-purified enzyme was obtained by heat treatment, ion-exchange chromatography, and gel filtration. Like all other eubacterial GSs known so far, the GS of M. ivanovi was found to be a dodecamer of about 600,000 daltons composed of a single type of subunit. The enzyme was stable at 63 degrees C for 10 min and was not sensitive to oxygen. The isoelectric point was 4.6, and the optimum pH of gamma-glutamyltransferase activity was 8.0. The Km values for hydroxylamine, glutamine, and ADP in the transferase reaction were 6.8, 22.7, and 0.35 mM, respectively. L-Methionine-DL-sulfoximine strongly inhibited the activity. Like the GS from gram-positive bacteria, Anabaena sp., several yeasts, and mammals, the enzyme from M. ivanovi was not regulated by adenylylation as demonstrated by snake venom phosphodiesterase treatment. Inhibition of the transferase activity by L-alanine, glycine, L-histidine, and L-tryptophan was observed. L-Glutamine alone or in the presence of AMP did not inhibit the GS synthetic activity. The GS of Methanobacterium ivanovi did not cross-react with a variety of antisera against GS from Escherichia coli, Anabaena strain 7120, or Bacillus megaterium. Archaebacterial GS appears to be structurally and functionally similar to eubacterial GS in gram-positive bacteria.

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.

Isolation and kinetic properties of 5′-nucleotidase from guinea-pig skeletal muscle

5′-Nucleotidase (EC 3.1.3.5) has been solubilized and purified 1200-fold from guinea-pig skeletal muscle, to a specific activity of 40 U/mg protein. The purified enzyme yields a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Guinea-pig skeletal muscle 5′-nucleotidase is extremely sensitive to inhibition by nucleoside di- and triphosphates. The inhibition is of the competitive type, and can be reversed only by strong excess of Mg 2+. Nucleoside diphosphates are more powerful inhibitors than nucleoside triphosphates. The K i values for ADP and ATP are 0.036 and 0.28 μM, respectively. The purified enzyme does not require exogenous cations for maximal activity and is inhibited by EDTA. This inhibition is reversed by divalent cations. This indicates that the enzyme contains a tightly bound metal cation.

5'-Nucleotidase from rat heart membranes. Inhibition by adenine nucleotides and related compounds.

ADP and ATP and their analogues were evaluated as inhibitors of 5'-nucleotidase purified from heart plasma membrane. ADP analogues are more powerful inhibitors than the corresponding ATP analogues. The most powerful inhibitor found is adenosine 5'-[alpha beta-methylene]diphosphate (AOPCP) for which the enzyme shows a Ki of 5 nM at pH 7.2. Measurements of pKi values for ADP and AOPCP as a function of pH indicate that the major inhibitory species of both nucleotides is the dianion. In the physiological range of pH values, AOPCP is a more powerful inhibitor than ADP principally because a higher percentage of AOPCP exists in the dianion form. The methylenephosphonate analogue of AMP (ACP), though not a substrate, is a moderately effective inhibitor. The corresponding analogues of ADP (ACPOP) and ATP (ACPOPOP) are as good inhibitors as ADP and ATP respectively. The thiophosphate analogues of ADP all inhibit 5'-nucleotidase, although not as powerfully as ADP, the most effective of these analogues being adenosine 5'-O-(1-thiodiphosphate) diastereoisomer B (ADP[alpha S](B)]. Other nucleotides inhibit the enzyme, but none is as effective as AOPCP. Inorganic tripolyphosphate and methylenediphosphonate are better inhibitors of the enzyme than is inorganic pyrophosphate. Inorganic thiophosphate is a better inhibitor than is orthophosphate. Hill plots of the ADP and AOPCP inhibition yield slopes close to 1; Hill plots of the ATP inhibition yield slopes of about 0.6. MgADP- is not an inhibitor, and MgATP2- is at best a very weak inhibitor of the enzyme.

Kinetic studies of fructose 6-phosphate,2-kinase and fructose 2,6-bisphosphatase.

The reactions catalyzed by a bifunctional enzyme, fructose-6-P,2-kinase and fructose 2,6- bisophosphatase , were studied. Fructose-6-P,2-kinase catalyzes the isotope exchange of ADP with ATP in the absence of fructose-6-P at about 3% of the rate of the overall reaction. The Km values for ATP and ADP for the reaction are 120 and 66 microM, respectively. No isotope exchange between fructose-6-P with fructose-2,6-P2 in the absence of adenine nucleotide was observed. The ADP-ATP exchange is not inhibited by the presence of 25 microM (1.6 X Km) fructose-6-P and less than 20% inhibition was observed at higher concentrations. Fructose-6-P,2-kinase catalyzes the reversal of the reaction at a rate approximately one-half that of the forward direction. The fructose 2,6-bisphosphatase reaction is not inhibited by 1 to 20 mM ADP. Plots of the reciprocal of the concentration of either substrate versus the reciprocal of the concentration of the other substrate yield intersecting lines for both forward and reverse reactions. The Michaelis constants for ATP and fructose-6-P are 150 and 16 microM, respectively, while those values for ADP and fructose-2,6-P2 are 62 and 8 microM, respectively. ADP is a competitive inhibitor with respect to ATP with an inhibition constant of 0.6 mM. All other product inhibition patterns including ADP versus fructose-6-P and fructose-2,6-P2 versus ATP and fructose-6-P are noncompetitive. Fructose-6-P,2-kinase also catalyzes the hydrolysis of ATP at about 10% of the rate of the overall reaction. There is no detectable burst of ADP formation upon incubating ATP with a stoichiometric amount of the enzyme. Similarly no burst of fructose-6-P formation is observed by reacting fructose-2,6-P2 with fructose 2,6-bisphosphatase. These results demonstrate that (a) the fructose-6-P,2-kinase reaction is consistent with a sequential mode of substrate addition rather than a "ping-pong" mechanism, (b) phosphorylenzyme , if formed, is not likely a kinetically important intermediate, and (c) no common phosphorylenzyme intermedite between fructose-6-P,2-kinase and fructose 2,6-bisphosphatase seems to exist.

Evidence for a specific phosphoryl binding site in swine kidney phosphofructokinase.

Phosphofructokinase (PFK) from swine kidney was purified by a procedure which included affinity chromatography on Cibacron blue F3GA-Sepharose 4B and ATP-Sepharose 4B columns in order to examine its binding properties. The homogeneous enzyme was purified more than 3000-fold with a yield of 30% and it had a specific activity of 39.8 mumol/min/mg of protein at 25 degrees C. The molecular weight of the native enzyme was 360 000 and it contained 4 identical subunits of molecular weight 88 000. The principal catalytically reacting form of the enzyme had a S20,w of 13.7 S which corresponds to a molecular weight of 360 000 +/- 6 000. The initial velocity patterns in the forward and reverse directions suggested a sequential mechanism for the reaction. The Km values for fructose 6-phosphate, ATP, fructose, 1,6-bisP and ADP were 33 microM, 8.3 microM, 460 microM and 110 microM, respectively. The homogeneous native enzyme binds specifically to phosphoryl groups immobilized in cellulose phosphate columns. ATP and fructose 6-phosphate interacted with the enzyme and decreased its affinity for phosphoryl binding sites. Other metabolites including fructose 1,6-bisP, glucose 6-phosphate and various nucleotides, alone or in various combinations, were ineffective in promoting the dissociation of the enzyme. Allosteric effectors of the enzyme, such as citrate and AMP were also inactive. However, the cooperatively altered the concentration of ATP required to dissociate the enzyme from phosphoryl groups. The bound enzyme was enzymatically inactive. The enzyme was also inactivated when it was treated with pyridoxal 5'-phosphate and reduced with sodium borohydride and the inactive enzyme no longer bound to cellulose phosphate. These effects were not observed when treatment with pyridoxal 5'-phosphate was carried out in the presence of fructose 6-phosphate. These observations and the results of similar studies with swine kidney fructose 1,6-bisphosphatase (FBPase) show that both enzymes share the unique property of binding specifically to phosphoryl groups. FBPase interacts through its allosteric AMP binding site and PFK binds through its fructose 6-P binding site. This specific binding of both enzymes through these sites result in the inactivation of PFK and the desensitization of FBPase to allosteric inhibition by AMP. In the unbound state PFK may be active and FBPase can be inhibited by AMP. Taken collectively, these binding effects could play a role in the reciprocal regulation of these enzymes during gluconeogenesis in kidney.

Characteristics of adenine nucleotide fluxes and transport in human tumor mitochondria

The efflux of adenine nucleotides from three human tumor mitochondria has been investigated with mitochondria prelabeled with radioactive ATP. Uncouplers induce a large efflux of adenine nucleotides from mitochondria from human hepatoma and oat cell carcinoma while efflux from astrocytoma mitochondria is less. This efflux does not require exchangeable anions, i.e., adenine nucleotides or pyrophosphate, in the extramitochondrial medium, and is not sensitive to atractyloside. The efflux is more extensive with dinitrophenol and CCCP than with valinomycin-K +, and may account for the differential effects of the two types of uncouplers on uncoupler-stimulated ATPase of tumor mitochondria previously reported by us. Dinitrophenol and CCCP do not elicit any efflux of adenine nucleotides from normal liver mitochondria. Efflux of orthophosphate from tumor mitochondria is also greater with dinitrophenol and CCCP; however, the more interesting finding is that the concentration of orthophosphate in these mitochondria is unusually high, i.e., 10–40-times greater than the intramitochondrial phosphate concentration of liver mitochondria. Atractyloside sensitive transport of ATP and ADP in human tumor mitochondria has also been determined. V max values for both ADP and ATP transport are lower than those obtained with liver mitochondria, especially with ADP transport. ATP transport in tumor mitochondria is not affected by CCCP in contrast to the 4–5-fold stimulation observed in liver mitochondria.

Nucleoside diphosphate kinase from Artemia embryos

1.1. Nucleoside diphosphate kinase (EC. 2.7.4.6) has been purified 1900-fold with 17% yield from Artemia dormant embryos.2.2. The purified enzyme exhibits a ping-pong mechanism of reaction. The calculated Km values for ADP, GTP, ITP, UTP and CTP are 0.04, 0.17, 0.25, 0.27 and 0.62 mM, respectively.3.3. The role of this enzyme in the conversion of diguanosine tetraphosphate into ATP is also discussed. Maximum velocity of nucleoside diphosphate kinase (50 U/g dry cysts) is well in excess (around thousand-fold) over the enzymes which transform diguanosine tetraphosphate into AMP.

Purification, catalytic and regulatory properties of Rana ridibunda erythrocyte pyruvate kinase

1. 1. Pyruvate kinase of Rana ridibunda erythrocytes is one of the regulatory enzymes of glycolysis. PK was purified about 7800-fold. 2. 2. The purified enzyme showed on SDS-electrophoresis three protein bands with an apparent molecular weight of between 60 and 65 kD. 3. 3. The enzyme is subject to activation by FDP and to inhibition by ATP. 4. 4. It showed K m values for PEP and ADP of 0.095 and 0.98 mM respectively. 5. 5. It was activated by K +, Mg 2+ and Ca 2+ ions whereas it was inhibited by Na + ions. 6. 6. The role of PK of Rana ridibunda erythrocytes, as a key and rate controlling enzyme of the glycolytic flux is discussed.

ATP-AMP phosphotransferase from Paracoccus denitrificans.

Adenylate kinase has been extracted from Paracoccus denitrificans by toluene treatment and purified 370-fold (overall yield of 44%, 554 U/mg, Mr 22 000, pI 4.7; pH optimum 7.5-8.0) by using successive column chromatography on DEAE-cellulose DE-52, Matrex-Blue A and Sephadex G-75. The enzyme was homogeneous by dodecylsulfate gel electrophoresis and constancy of specific activity across the single peak found in Sephadex G-75 chromatography. Crystals in the form of heavy needles have been obtained in poly(ethylene glycol)-400. The enzyme is specific for adenine (deoxyadenine) nucleotides with Km values for ATP, ADP and AMP of 340 microns, 980 microM and 93 microM respectively and dissociation constants, for ATP, MgATP and AMP of 7.3 microM, 7.1 microM and 6.9 microM respectively. The enzyme was noted to have no disulfide bond, two free sulfhydryl groups and a total of 208 residues. Aboe 50 mM KCl the enzyme activity drops off gradually. The enzyme is stable at neutral pH and below a temperature of 44 degrees C. Adenylate kinase from P. denitrificans resembles adenylate kinases from other sources with respect to such properties as the dependence on specific divalent cations, maximal activities at a ratio of Mg2+:ATP of about 1:1 and the ratio of Mg2+:ADP of 1:2, in having MgATP (or MgADP) binding site and another substrate binding site for AMP (or ADP), and in requiring an intact histidine and one or more arginine residues for enzymatic activity. Adenylate kinase from P. denitrificans appears to resemble the so-called muscle-type (cytoplasmic) rather than the mitochondrial-type adenylate kinase by comparisons of the following properties: low molecular weight, having --SH groups and no disulfide bonds, pH-optimum of 8.0 for formation of ADP, positive cross-reaction by ELISA test and in requiring a low concentration of Ap5A to inhibit 95% activity.

Ribonucleotide reductase induced by herpes simplex type 1 virus. Characterization of a distinct enzyme.

The ribonucleotide reductase induced by herpes simplex virus type 1 (HSV-1) was purified in high yield from serum-starved baby hamster kidney (BHK-21) cells infected with HSV-1 (strain H-29). The enzyme preparation was essentially free of both eucaryotic ribonucleotide reductase and contaminating enzymes that could cause significant depletion of substrates. The HSV-1-induced enzyme was assayed in 0.2 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-Na at the pH optimum of 8.1 and the optimal dithiothreitol concentration of 10 mM. Nucleoside diphosphates were the substrates of this enzyme. The HSV-1-induced ribonucleotide reductase was inhibited by anions. EDTA also inhibited the enzyme and this inhibition was not reversed by the addition of FeCl2. Hydroxyurea acted as a noncompetitive inhibitor versus CDP reduction (Kii = 1.3 mM Kis = 2.4 mM). The enzyme was inhibited by either free Mg2+ or free ATP. However, it was neither inhibited nor activated by the ATP X Mg complex. Reduction of either CDP or ADP was only weakly inhibited by dATP, dTTP, dGTP, and dCTP. No activation of this enzyme by these compounds was observed. The V'm values for reduction of CDP, UDP, and ADP were similar, while the GDP V'm was 2-fold greater. The K'm values were 80, 12, 1.2, and 0.65 microM for UDP, ADP, GDP, and CDP, respectively. The K'm values for CDP, GDP, and ADP were the lowest values observed for any ribonucleotide reductase. Each ribonucleoside diphosphate substrate competitively inhibited the reduction of each other substrate. The K'is values obtained for inhibition by a given ribonucleoside diphosphate were similar to the K'm value obtained for that compound as a substrate. Kinetic analysis of the combined rate of product formation when both CDP and ADP were simultaneously present as substrates produced patterns that were consistent with reduction at a common catalytic site. The 2'-deoxynucleoside diphosphate products were also competitive inhibitors versus the substrates. The K'is values versus CDP reduction were 310, 140, 9, and 5 microM for dUDP, dADP, dGDP, and dCDP, respectively. Similar K'is values were obtained when ADP was the substrate. All of these data are most consistent with the hypothesis that the HSV-1-induced ribonucleotide reductase catalyzes the reduction of all substrates at a common site. The apparent lack of significant allosteric modulation of HSV-1-induced ribonucleotide reductase, its kinetic behavior, and its low K'm for CDP, GDP, and ADP clearly differentiate this enzyme from other ribonucleotide reductases.

Synthesis and hydrolysis of ADP-arsenate by beef heart submitochondrial particles.

The kinetic parameters for inorganic phosphate and inorganic arsenate as substrates for the synthesis of ATP and ADP-arsenate, respectively, by beef heart submitochondrial particles have been determined. The Vm and Km values for arsenate and phosphate, as well as the Km values for ADP in the two reactions, are the same within experimental error of the measurements. These data are consistent with covalent bond formation not being the rate-limiting step for either ATP or ADP-arsenate synthesis. The hydrolysis of ATP and of ADP-arsenate was studied under conditions of net synthesis of ATP or ADP-arsenate. The apparent first order rate constant for ATP hydrolysis increased with submitochondrial particle concentration, indicating that hydrolysis of ATP was catalyzed by the submitochondrial particle preparation. Nonenzymic hydrolysis of ATP was negligible compared to enzymic hydrolysis. The apparent first order rate constant for ADP-arsenate hydrolysis did not vary with submitochondrial particle concentration, indicating that ADP-arsenate hydrolysis was nonenzymic. Enzymic hydrolysis of ADP-arsenate was too slow, compared with its rapid nonenzymic hydrolysis, to be detected. The first order rate constant for ADP-arsenate hydrolysis at pH 7.5, 30 degrees C, was determined to be greater than 5 min-1 and was estimated to be 70 min-1. These data confirm previous suggestions that arsenate "uncouples" oxidative phosphorylation by a mechanism involving synthesis of ADP-arsenate, followed by its rapid nonenzymic hydrolysis.

Adenine nucleotide transport in hepatoma mitochondria. Characterization of factors influencing the kinetics of ADP and ATP uptake

Initial velocity measurements of [ 3H]ADP and [ 3H]ATP uptake have been made with mitochondria isolated from Morris hepatomas of differing growth rates, and factors known to influence the rates of nucleotide exchange have been examined in an effort to determine whether the elevated rates of aerobic glycolysis in these tumors can be attributed to altered carrier activity. These studies included the determination of the apparent kinetic constants for nucleotide uptake as a function of the mitochondrial energy state and the dependence of transport rates on temperature. Also included in these studies were measurements of the mitochondrial levels of endogenous inhibitors, divalent cations and internal adenine nucleotides. Results obtained showed that with mitochondria isolated from the various tumor lines, the apparent kinetic constants for nucleotide uptake are different from those of control rat or regenerating liver mitochondria; the apparent V max values for both ADP and ATP uptake are significantly lower. Furthermore, under conditions of a high-energy state, the K m and V max values for ATP uptake are greater than the K m and V max value for ADP uptake but that under uncoupled conditions, the opposite is observed. Comparison of the levels of mitochondrial Ca 2+, Mg 2+, long-chain acyl-CoA ester and adenine nucleotide from the various mitochondria showed that important differences exist between liver and hepatoma mitochondria in the levels of Ca 2+, long-chain acyl-CoA ester and AMP. Mitochondrial Ca 2+ levels are elevated 3–5-fold in all tumor lines, and for Morris 7777 hepatoma (a rapidly growing tumor) by a remarkable 70-fold; whereas the levels of acyl-CoA ester and AMP are significantly lower in the more rapidly growing tumors. Arrhenius plots for nucleotide uptake in mitochondria from liver and hepatoma are characterized as being biphasic, having similar activation energies above and below the break point temperature (28–38 and 6–16 kcal/mol, respectively). However, the transition temperature for mitochondria from the various hepatomas is uniformly 4–5°C lower than mitochondria from control liver. The latter difference may reflect a variation in membrane composition, most probably lipid components. It is concluded that the presence of elevated levels of Ca 2+ and lower levels of AMP in hepatoma mitochondria and difference of membrane compositions may play an important role in limiting adenine nucleotide transport activity in vivo and that the impaired carrier activity may contribute to higher rates of aerobic glycolysis observed in these tumors.

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.

Ammonia utilization by a proposed bacterial pathogen in human periodontal disease, Capnocytophaga ochracea

Capnocytophaga ochracea strain 25 was originally isolated from a patient with severe juvenile periodontitis. An NAD-specific glutamate dehydrogenase (GDH) (EC 1.4.1.2.) was found in cell-free extracts from this organism. The NADH-dependent reductive, or ammonia-assimilating activity (NADH-GDH), of the enzyme was 8–10-fold higher than its NAD-dependent oxidative, or ammonia-releasing activity (NAD-GDH), suggesting that the primary physiological role of the GDH is ammonia-fixing. Capnocytophaga ochracea GDH was purified approximatsly 39-fold by a rapid, single-step purification procedure using DEAE-cellulose (DE52) ion-exchange column chromatography which gave 90 per cent recovery of total enzyme units. Paper chromatography of an NADH-GDH assay mixture containing the partially purified enzyme showed that glutamate was, indeed, a product of the ammonia-assimilating reaction. The pH optimum for the NAD-GDH reaction was 9.0; that for the NADH-GDH reaction was 7.5. Although a number of mono- and divalent cations were tested, none had a large effect on either NAD-GDH or NADH-GDH activity. The NAD-GDH reaction showed a hyperbolic kinetic response to glutamate and NAD and the K m values for glutamate and NAD were 2.44 and 0.083 mM respectively. The kinetic response of the NADH-GDH reaction to NADH, α-ketoglutarate and ammonium chloride also obeyed Michaelis-Menten kinetics and their respective K m values were 0.069, 1.44 and 3.33 mM. Of a number of biologically-active compounds tested for their ability to modulate GDH activity, only ADP and NAD exerted much effect. The NADH-GDH activity showed a negative hyperbolic kinetic response to both ADP and NAD and Dixon plot-analysis of the NAD and ADP saturation data gave K i values for ADP and NAD of 4.0 and 0.46 mM respectively. Both NAD and ADP appeared to exert their negative effects on NADH-GDH activity by completely inhibiting the binding of the reduced coenzyme, NADH, to the enzyme.

Sequential hydrolysis of the γ- and β-phosphate groups of ATP by the ATP diphosphohydrolase from pig pancreas

The ATP dipbosphohydrolase (EC 3.6.1.5) from pig pancreas hydrolyzes triphospho- and diphosphonucleosides. The reaction products of ATP hydrolysis are ADP, AMP and orthophosphate, but AMP accumulates at a faster rate than ADP. A time-course study showed a simultaneous breakdown of ATP and ADP with initial rates for ATP and ADP hydrolysis of 2.1 and 3.8μmol/min per mg protein, respectively. However, the rates reached similar values toward the end of the incubation period. According to double reciprocal plots and Dixon plots, the K m values for ATP and ADP are similar, V max for ADP hydrolysis is twice the V max for ATP hydrolysis and both nucleotides are competitive inhibitors of the other with their K i values similar to their K m. These results are consistent with a sequential hydrolysis of the two diphosphoester bonds of ATP: ATP first binds to the enzyme, its γ-phosphate group is hydrolyzed and released, resulting in an enzyme-ADP complex which either breaks down to free enzyme and ADP or is further processed via hydrolysis of the β-phosphate group, releasing free enzyme, AMP and P i. The experimental data showed that the processing step is favored.

Location and properties of two isoenzymes of cardiac adenylate kinase.

Adenylate kinase catalyses the equilibrium 2ADP = ATP + AMP. There are two isoenzymes of adenylate kinase in bovine ventricular tissue, one cytoplasmic, the other mitochondrial. Mitochondrial subfractionation locates this isoenzyme between the mitochondrial membranes with fatty acid-CoA ligase. The cytoplasmic and mitochondrial isoenzymes are distributed in ratio 3:2, and both forms were purified to homogeneity. They differ principally by charge, Km values for ATP, ADP and AMP, pH-stability and -activity profiles, and susceptibility to the inhibitor adenosine pentaphosphoadenosine. The forward and reverse reactions show similar energies of activation for the cytoplasmic enzyme, but differ for the mitochondrial enzyme. The molecular weights are indistinguishable. An integrated mechanism is formulated whereby one isoenzyme suppresses the activation of fatty acid and the other enhances carbohydrate utilization in hypoxic myocytes.

Nucleotide isotachophoretic assay: Method and application for determination of ATP/ADP ratio in several rat tissues

AbstractSeparation of ATP and ADP was achieved in isotachophoresis by using malonate, inorganic phosphate (Pi), creatine phosphate and lactate as spacers, which isolate these nucleotides from other UV‐absorbing products in rat tissues. By combining isotachophoresis with enzymatic end‐point analysis of several rat tissues, calibration for ATP and ADP was achieved obtaining an operative calibration curve that allows the use of isotachophoresis as the only analytical technique in further assays. ATP and ADP quantities of rat skeletal and smooth muscle, heart, liver, kidney and lung were determined by isotachophoresis. Individual variation was also calculated by analyzing 30 animals in the same control conditions, muscular tissues showing the greatest values for ATP and ADP, whereas kidney and lung show the smaller values. ATP/ADP ratio is discussed as a representative parameter for describing the energy content in biological tissues, there are also experimental reasons which imply that other product concentrations in this parameter not be included. This ATP/ADP ratio is calculated in the rat tissues studied, as well as its individual variation, obtaining the larger values for muscular tissues and the smaller for kidney. A great individual variation is found in kidney, followed by heart and skeletal muscle, and the most constant values are found in liver and smooth muscle. The results obtained in this calibration method for isotachophoresis assay suggest the use of a similar procedure in order to apply this technique for other nucleotide analysis.

Kinetics of ATP Synthesis in Pea Cotyledon Submitochondrial Particles

A kinetic study of oxidative phosphorylation by pea submitochondrial particles gave two K(m) values for ADP, one low, the other high. The high value probably reflected a damaged site or a population of leaky mitochondria. Only the high affinity site with a low K(m) for ADP was involved in ATP synthesis. alpha,beta-Methylene ADP was found to be a competitive inhibitor of ATP synthesis. The inorganic phosphate analog, thiophosphate, decreased the apparent K(m) of ADP while the rate of the reaction remained approximately the same. Adenyl imidodiphosphate, a specific inhibitor of ATP hydrolysis activity, had little effect on oxidative phosphorylation. A slight decrease in the K(m) of the high affinity binding site for ADP was noted. Aurovertin was found to be a potent inhibitor of oxidative phosphorylation in pea submitochondrial particles. The K(m) of the high affinity site was increased 10-fold. Also, the inhibition normally exerted by ADP on ATPase activity was severely reduced by aurovertin. In contrast, increasing the concentration of aurovertin only slightly affected the level of inhibition caused by adenyl imidodiphosphate on ATP hydrolysis.