MgATP Concentration Research Articles

Modulation of Ca2+ channel activity by ATP metabolism and internal Mg2+ in guinea-pig basilar artery smooth muscle cells.

1. Single smooth muscle cells were isolated from the basilar artery of the guinea-pig and, within 10 h, inward currents through voltage-gated Ca2+ channels were recorded using the amphotericin or conventional whole-cell voltage-clamp techniques. 2. In amphotericin whole-cell recordings, bath application of 2,4-dinitrophenol (DNP, an uncoupler of mitochondrial ATP production) induced an initial stimulation (14% increase in 5 of 11 cells) and then pronounced inhibition (50% decrease in 9 of 11 cells within 9.5 min) of voltage-dependent Ca2+ current (I(Ca)) elicited by depolarizing to +10 mV in 1.5 mM extracellular Ca2+ solution. By contrast, inhibition of glycolysis by replacing glucose in the bath with 2-deoxy-D-glucose had no effect. 3. Na+ current through Ca2+ channels (I[(Ca)(Na)]) recorded in the absence of extracellular divalent cations also responded to DNP, again with stimulation followed by inhibition of current. The stimulation of I[(Ca)(Na)] was associated with a leftward shift of the Ca2+ channel activation curve which averaged -9 mV. A combination of 2-deoxy-D-glucose, mannoheptulose and 3-0-methyl-glucose had only minor effects on I[(Ca)(Na)], whereas rotenone had an effect similar to that of DNP in six of eight cells. 4. The amplitude of I[(Ca)(Na)] in conventional whole-cell recordings was not different from that in amphotericin whole-cell recordings, even without ATP in the recording pipette and with metabolic poisons in the bath solution. Furthermore, attempts to dephosphorylate the Ca2+ channels in ATP-free conditions did not prevent I[(Ca)(Na)], and a high concentration of Mg-ATP with or without a phosphorylation-supporting medium in the recording pipette did not increase its amplitude. 5. In the absence of ATP, Mg2+ inhibited whole-cell I[Ca)(Na)] with a K(d) of about 100 mu M at -10 mV and induced a leftward shift of the Ca2+ channel activation curve. When ATP and a phosphorylation-supporting medium were in the recording pipette the blocking effect of free Mg2+ was reduced but the shift in the Ca2+ channel activation curve was unaffected. 6. From these data it is suggested that inhibition of mitochondrial, but not glycolytic, ATP production has stimulatory and inhibitory effects on voltage-gated Ca2+ channels of basilar artery smooth muscle cells. Effects of intracellular Mg2+ on the Ca2+ channels were modulated by ATP and mimicked the effects of metabolic poisoning by DNP. A hypothesis is discussed in which the intracellular free Mg2+ concentration may be a key factor coupling ATP production to Ca2+ channels.

Regulation of Membrane-bound Phospholipase D by Protein Kinase C in HL60 Cells: SYNERGISTIC ACTION OF SMALL GTP-BINDING PROTEIN RhoA

In HL60 cells, the membrane-bound phospholipase D (PLD) was stimulated by 4beta-phorbol 12-myristate 13-acetate (PMA) in the presence of the cytosolic fraction from HL60 cells or rat brain. The cytosolic factor for this PMA-induced PLD activation was subjected to purification from rat brain by sequential chromatographies. The PLD stimulating activity was found in protein kinase C (PKC) fraction containing alpha, betaI, betaII, and gamma isozymes. PKC isozymes were further separated by hydroxylapatite chromatography. PKCalpha and - beta, but not gamma, isozymes were found to activate membrane-bound PLD. PKCalpha was much more effective than PKCbeta for PLD activation. Millimolar concentrations of MgATP were required for the PKC-mediated PLD activation in HL60 membranes. MgATP is utilized to maintain the levels of phosphatidylinositol 4,5-bisphosphate (PIP2) under these assay conditions. The PKC-mediated PLD activation was completely inhibited by neomycin, a high affinity ligand for PIP2, and this suppression was recovered by the addition of exogenous PIP2. Thus, these results suggest that PIP2 is supposed to play a key role in PKC-mediated PLD activity in HL60 membranes. Furthermore, PKCalpha-mediated PLD activation was potentiated by the addition of recombinant RhoA protein in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). The results obtained here indicate that PKCalpha and RhoA (GTP form) exert a synergistic action in the membrane-bound PLD activation in HL60 cells.

Binding and Hydrolysis of TNP-ATP by Escherichia coli F1-ATPase

It had previously been suggested that Vmax hydrolysis rate of 2', 3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) by F1-ATPase required filling of only two catalytic sites on the enzyme (Grubmeyer, C., and Penefsky, H. S. (1981) J. Biol. Chem. 256, 3718-3727), whereas recently it was shown that Vmax rate of ATP hydrolysis requires that all three catalytic sites are filled (Weber, J., Wilke-Mounts, S., Lee, R. S. F., Grell, E., and Senior, A. E. (1993) J. Biol. Chem. 268, 20126-20133). To resolve this apparent discrepancy, we measured equilibrium binding and hydrolysis of MgTNP-ATP under identical conditions, using betaY331W mutant Escherichia coli F1-ATPase, in which the genetically engineered tryptophan provides a direct fluorescent probe of catalytic site occupancy. We found that MgTNP-ATP hydrolysis at Vmax rate did require filling of all three catalytic sites, but in contrast to the situation with MgATP, "bisite hydrolysis" of MgTNP-ATP amounted to a substantial fraction (approximately 40%) of Vmax. Binding of MgTNP-ATP to the three catalytic sites showed strong binding cooperativity (Kd1 < 1 nm, Kd2 = 23 nm, Kd3 = 1.4 microM). Free TNP-ATP (i.e. in presence of EDTA) bound to all three catalytic sites with lower affinity but was not hydrolyzed. These data emphasize that the presence of Mg2+ is critical for cooperativity of substrate binding, formation of the very high affinity first catalytic site, and hydrolytic activity in F1-ATPases and that these three properties are strongly correlated.

Coupling between myosin ATPase cycle and creatinine kinase cycle facilitates cardiac actomyosin sliding in vitro. A clue to mechanical dysfunction during myocardial ischemia.

There is much evidence to support the favorable effects of the phosphocreatine shuttle on myocardial contraction and relaxation. However, experiments in which cardiac muscle fiber or myofibril was used have not elucidated its precise mechanism. Active movements of fluorescently labeled actin filaments on a cardiac myosin layer coimmobilized with creatinine kinase (CK) onto a nitrocellulose-coated glass coverslip were studied under various concentrations of adenine nucleotides. At a constant phosphocreatine concentration (5 mmol/L, pH 7.1), the relation of sliding velocity to MgATP concentration followed Michaelis-Menten kinetics. The apparent Km was significantly smaller in the presence of CK (0.041 +/- 0.001 mmol/L) than in the absence of CK (0.080 +/- 0.001 mmol/L), indicating that coattached CK facilitated the propelling of actin filaments by the myosin ATPase. This phenomenon was also seen under acidic conditions (pH 6.7) as well as in the presence of inorganic phosphate (10 mmol/L. At a constant MgATP concentration (1 mmol/L), the inhibitory effect of MgADP on the actin-myosin interaction was weaker in the presence of CK than in the absence of CK. Another ATP-regenerating system, pyruvate kinase and phospho(enol)pyruvate, while maintaining a low ratio of [MgADP] to [MgATP], did not reduce the Km value (0.156 +/- 0.001 mmol/L), suggesting that the effect of coattached CK was not achieved only by prevention of MgADP accumulation. Coupling between the ATPase cycle and the CK cycle may serve not only to maintain the ATP concentration within the myofibril but also to provide optimal conditions for cardiac actomyosin interaction. Consideration of this coupling will offer a clue to elucidating the systolic or diastolic dysfunction during myocardial ischemia or reperfusion.

ATP is required in platelet serotonin exocytosis for protein phosphorylation and priming of secretory vesicles docked on the plasma membrane.

Calcium-evoked secretion generally requires the presence of millimolar concentrations of Mg-ATP. We investigated the role of Mg-ATP in the secretion of serotonin from electropermeabilized bovine platelets. The secretion of serotonin was lost within 5 minutes when the Mg-ATP concentration was diluted to less than 0.1 mM, but was maintained when ATP-gamma S (adenosine 5'-O-3-thiotriphosphate) was used instead of ATP. Okadaic acid, a potent inhibitor of protein phosphatase, could also maintain the exocytotic activity even when ATP was diluted. Decrease in the secretory activity was paralleled by a decrease in phosphorylation level of four proteins after dilution of ATP, but the activity was maintained when the thiophosphorylation level of these proteins was maintained. Two of these proteins were digested by a protease, calpain, which has been shown to lead to a loss in the exocytotic activity. Electron microscopic studies showed that calcium did not induce the formation of distinct bridge-like structures between the granule membrane and the plasma membrane in Mg-ATP-diluted cells, previously shown as the structure transiently formed prior to fusion of the two membranes. Anchorage of the secretory dense granules to the plasma membrane and the presence of the amorphous structures between the granules and the plasma membrane were unchanged by dilution of ATP. These results indicate that ATP is not required for the anchorage itself, but is required to prime anchored granules for calcium-triggered secretion. Maintenance of the phosphorylated state of proteins by ATP enables the calcium trigger to form the bridge-like structures preceding membrane fusion events.

Steady-state kinetic studies of dithionite utilization, component protein interaction, and the formation of an oxidized iron protein intermediate during Azotobacter vinelandii nitrogenase catalysis.

Steady-state kinetic analysis of the two-component protein system of Azotobacter vinelandii (Av) nitrogenase is reported. A precisely obeyed half-order reaction in dithionite was observed at concentrations up to 21 mM with no indication of saturation by this substrate. This behavior was monitored by optical, amperometric, and manometric kinetic techniques, and the results were mathematically fit to establish the half-order reaction in dithionite. Under conditions where the MgATP and dithionite concentrations remain unchanged, Av2 (the Fe protein component) interacts with Av1 (the MoFe protein component according to the rate law, suggesting a rapid 1:1 Av2-Av1 interaction: [formula: see text]. with [Av2] the free Fe protein concentration, K = 5.9 microM, and Vmax = 2314 nmol of H2 min-1 (mg of Av1)-1. Under dithionite-depleted conditions, Av2 undergoes an Av1-mediated, one-electron oxidation, consistent with its proposed role as a specific, single-electron reductant for Av1. During steady-state turnover as a function of Av2/Av1 ratio, optical spectroscopy demonstrated the presence of 25-30% oxidized Av2 as an enzyme intermediate. Computer-averaged EPR spectra showed that Av1 was > 95% EPR-silent and Av2 was up to 30% oxidized (Av2ox), consistent with the optical measurements. These optical and EPR results show that up to six Av2ox per Av1 can accumulate in the presence of dithionite during catalysis, suggesting that the conversion of Av2ox back into Av2red is a relatively slow process.

Altered Drug-stimulated ATPase Activity in Mutants of the Human Multidrug Resistance Protein

The characteristics of P-glycoprotein (MDR1), an ATP-dependent drug extrusion pump responsible for the multidrug resistance of human cancer, were investigated in an in vitro expression system. The wild-type and several mutants of the human MDR1 cDNA were engineered into recombinant baculoviruses and the mutant proteins were expressed in Sf9 insect cells. In isolated cell membrane preparations of the virus-infected cells the MDR1-dependent drug-stimulated ATPase activity, and 8-azido-ATP binding to the MDR1 protein were studied. We found that when lysines 433 and/or 1076 were replaced by methionines in the ATP-binding domains, all these mutations abolished drug-stimulated ATPase activity independent of the MgATP concentrations applied. Photoaffinity labeling with 8-azido-ATP showed that the double lysine mutant had a decreased ATP-binding affinity. In the MDR1 mutant containing a Gly185 to Val replacement we found no significant alteration in the maximum activity of the MDR1-ATPase or in its activation by verapamil and vinblastine, and this mutation did not modify the MgATP affinity or the 8-azido-ATP binding of the transporter either. However, the Gly185 to Val mutation significantly increased the stimulation of the MDR1-ATPase by colchicine and etoposide, while slightly decreasing its stimulation by vincristine. These shifts closely correspond to the effects of this mutation on the drug-resistance profile, as observed in tumor cells. These data indicate that the Sf9-baculovirus expression system for MDR1 provides an efficient tool for examining structure-function relationships and molecular characteristics of this clinically important enzyme.

Characterization of pp60c-src tyrosine kinase activities using a continuous assay: autoactivation of the enzyme is an intermolecular autophosphorylation process.

A continuous assay for pp60c-src tyrosine kinase (srcTK) was developed. A lag in phosphorylation of the peptide RRLIEDAEYAARG was observed that could be eliminated by preincubation with MgATP. The induction time for this lag was dependent upon MgATP and srcTK concentrations. When autophosphorylation was monitored by 32P incorporation from [gamma-32P]ATP, a lag in the time course was also observed. These results demonstrate that autoactivation is an intermolecular process. The electrospray ionization mass spectrum of the enzyme before and after activation demonstrated an increase in the phosphorylation state of the enzyme after incubation with MgATP. The delta 85-N-terminal mutant protein and a full-length G2A pp60c-src mutant, which removes the myristylation site, used in these studies were partially phosphorylated on Y338 and Y530 as isolated. This is the first report of phosphorylation on Y338, but the significance of this site of phosphorylation is unknown. These phosphorylations were insufficient to active the enzyme for transfer of the gamma-phosphoryl of MgATP to the peptides. The unphosphorylated enzyme initially present was converted to a monophosphorylated species upon treatment with MgATP. Y-419 phosphorylation was evident only after treatment with MgATP. These data are consistent with autophosphorylation on Y-419 as predicted. Intermolecular autophosphorylation is consistent with the ability of srcTK to dimerize, which is analogous to activation of receptor tyrosine kinases such as the EGF receptor kinase in response to growth factors. These results indicate that dimerization leading to activation does not require binding to the membrane or a hydrophobic N-terminus in the case of srcTK.

P-glycoprotein Is Stably Inhibited by Vanadate-induced Trapping of Nucleotide at a Single Catalytic Site

P-glycoprotein (Pgp or multidrug-resistance protein) shows drug-stimulated ATPase activity. The catalytic sites are known to be of low affinity and specificity for nucleotides. From the sequence, two nucleotide sites are predicted per Pgp molecule. Using plasma membranes from a multidrug-resistant Chinese hamster ovary cell line, which are highly enriched in Pgp, we show that vanadate-induced trapping of nucleotide at a single catalytic site produces stably inhibited Pgp, with t 1/2 for reactivation of ATPase activity of 84 min at 37 degrees C and >30 h at 4 degrees C. Reactivation of ATPase correlated with release of trapped nucleotide. Concentrations of MgATP and MgADP required to produce 50% inhibition were 9 and 15 microM, respectively, thus the apparent affinity for nucleotide is greatly increased by vanadate-trapping. The trapped nucleotide species was ADP. Divalent Cation was required, with magnesium, manganese, and cobalt all effective: cobalt yielded a very stable inhibited species, t1/2 at 37 degrees C = 18 h. No photocleavage of Pgp was observed after vanadate trapping with MgATP, nor was UV-induced photolabeling of Pgp by trapped adenine nucleotide observed. Vanadate-trapping with 8-azido-ATP followed by UV irradiation caused permanent inactivation and specific labeling of Pgp. Vanadate-induced inhibition was also shown with pure, reconstituted Pgp, with similar characteristics to those in plasma membranes. Vanadate trapping overcomes technical difficulties posed by lack of high affinity nucleotide-binding site(s) or a covalent enzyme-phosphate catalytic intermediate in Pgp. The finding that vanadate trapping of nucleotide at just one site/Pgp is sufficient to give full inhibition at ATPase activity shows that the two predicted nucleotide sites can not function independently as catalytic sites.

Muscle Creatine Kinase-deficient Mice: I. ALTERATIONS IN MYOFIBRILLAR FUNCTION

The regulation of contractile activity in mice bearing a null mutation of the M-isoform of creatine kinase gene, has been investigated in tissue extracts and Triton X-100-treated preparations of ventricular, soleus, and gastrocnemius muscles of control and transgenic mice. Skinned fiber experiments did not evidence any statistical difference in the maximal force or the calcium sensitivity of either muscle type. Rigor tension development at a low MgATP concentration was greatly influenced by phosphocreatine in control but not in transgenic mice as should be expected. In calcium-activated ventricular preparations, although the force developed by each cross-bridge was the same in control and transgenic animals, the rate constant of tension changes appeared to be markedly slowed in transgenic animals. As the ventricular isomyosin pattern was not altered, we suggested that, in transgenic animals, cross-bridge cycling was hindered by a local decrease in the MgATP to MgADP ratio, due to lack of a local MgATP regenerating system. Myokinase activity was not significantly changed while activities of pyruvate kinase or glyceraldehyde-3-phosphate dehydrogenase were found to be increased in transgenic animals. These results show that no fundamental remodelling occurs in myofibrils of transgenic animals but that important adaptations modify the bioenergetic pathways including glycolytic metabolism.

Effect of Mg2+ and ATP on depolarization-induced Ca2+ release in isolated triads

The effect of different free Mg2+ and ATP concentrations on depolarization-induced Ca2+ release in isolated skeletal muscle triadic vesicles was examined by simultaneously monitoring direct effects on ryanodine receptors from either isolated or coupled terminal cisternae. Free Mg2+ was increased to concentrations of 11-14 microM, 81 microM, 175-181 microM, and 1 mM while total ATP concentration was kept constant or MgATP concentration was kept constant. We observed the following. 1) Increasing MgATP reduces the measurable Ca2+ release from isolated vesicles by stimulating the Ca(2+)-ATPase in the terminal cisternae. 2) Half-maximal inhibition of functionally coupled ryanodine receptors during depolarization-induced Ca2+ release is observed at 1 mM Mg2+, whereas half-maximal inhibition of the nondepolarized ryanodine receptor is seen at 75 microM Mg2+ at the same free ATP and MgATP concentrations. 3) Two separate time constants for Ca2+ release were obtained for nondepolarized ryanodine receptors with free Mg2+ at 14 microM and free ATP at 6.1 mM; this may represent triadic ryanodine receptors vs. isolated terminal cisternae ryanodine receptors.

Enzyme turnover is essential for deactivation of F 0F 1-ATPase in plant mitochondria

In potato tuber mitochondria, ATPase deactivates immediately after treatment with an uncoupler or with polyoxyethylene 9-lauryl ether (Lubrol), a non-ionic detergent. Deactivation was completely prevented by another non-ionic detergent, lauryldimethylamine oxide (LDAO). LDAO also induced slow reactivation of inactive ATPase formed in deenergized mitochondria. Freezing of the active state by LDAO was used to study the process of ATPase deactivation following deenergization in intact mitochondria. Deactivation was slowed down by carboxyatractyloside (CATR), which prevents ATP import into the matrix, and by ATPase inhibitors. ATP hydrolysis was also triggered by Lubrol with CATR-treated mitochondria. The initial rate was close to the capacity for ATP synthesis but rapidly decayed. The rate of decay increased with the concentration of MgATP and no decay was observed in the presence of EDTA. The following conclusions were drawn. (1) Deenergization in itself is not sufficient for ATPase deactivation in plant mitochondria: enzyme turnover is also required. The probability of one enzyme to be deactivated at each turnover is much higher in potato tuber than in pea leaf organelles. (2) Enzyme turnover probably shifts the IF1-F 1 complex from an active to an inactive form; the rate of deactivation indeed does not seem to be controlled by the binding of the inhibitory peptide. (3) The short-term effect (protection) and the long-term effect (reactivation) of LDAO on MF 0MF 1 may tentatively be used to titrate the activated versus total amounts of these enzymes in cells.

Myosin binding-induced cooperative activation of the thin filament in cardiac myocytes and skeletal muscle fibers

Myosin binding-induced activation of the thin filament was examined in isolated cardiac myocytes and single slow and fast skeletal muscle fibers. The number of cross-bridge attachments was increased by stepwise lowering of the [MgATP] in the Ca(2+)-free solution bathing the preparations. The extent of thin filament activation was determined by monitoring steadystate isometric tension at each MgATP concentration. As pMgATP (where pMgATP is -log [MgATP]) was increased from 3.0 to 8.0, isometric tension increased to a peak value in the pMgATP range of 5.0-5.4. The steepness of the tension-pMgATP relationship, between the region of the curve where tension was zero and the peak tension, is hypothesized to be due to myosin-induced cooperative activation of the thin filament. Results showed that the steepness of the tension-pMgATP relationship was markedly greater in cardiac as compared with either slow or fast skeletal muscle fibers. The steeper slope in cardiac myocytes provides evidence of greater myosin binding-induced cooperative activation of the thin filament in cardiac as compared with skeletal muscle, at least under these experimental conditions of nominal free Ca2+. Cooperative activation is also evident in the tension-pCa relation, and is dependent upon thin filament molecular interactions, which require the presence of troponin C. Thus, it was determined whether myosin-based cooperative activation of the thin filament also requires troponin C. Partial extraction of troponin C reduced the steepness of the tension-pMgATP relationship, with the effect being significantly greater in cardiac than in skeletal muscle. After partial extraction of troponin C, muscle type differences in the steepness of the tension-pMgATP relationship were no longer apparent, and reconstitution with purified troponin C restored the muscle lineage differences. These results suggest that, in the absence of Ca2+, myosin-mediated activation of the thin filament is greater in cardiac than in skeletal muscle, and this apparent cooperativity requires the presence of troponin C on thin filament regulatory strands.

Inhibition of cardiac sarcolemma Na(+)-K+ ATPase by oxyradical generating systems.

The Na(+)-K+ ATPase activity and SH group content were decreased whereas malondialdehyde (MDA) content was increased upon treating the porcine cardiac sarcolemma with xanthine plus xanthine oxidase, which is known to generate superoxide and other oxyradicals. Superoxide dismutase either alone or in combination with catalase and mannitol fully prevented changes in SH group content but the xanthine plus xanthine oxidase-induced depression in Na(+)-K+ ATPase activity as well as increase in MDA content were prevented partially. The Lineweaver-Burk plot analysis of the data for Na(+)-K+ ATPase activity in the presence of different concentrations of MgATP or Na+ revealed that the xanthine plus xanthine oxidase-induced depression in the enzyme activity was associated with a decrease in Vmax and an increase in Km for MgATP; however, Ka value for Na+ was decreased. Treatment of sarcolemma with H2O2 plus Fe2+, an hydroxyl and other radical generating system, increased MDA content but decreased both Na(+)-K+ ATPase activity and SH group content; mannitol alone or in combination with catalase prevented changes in SH group content fully but the depression in Na(+)-K+ ATPase activity and increase in MDA content were prevented partially. The depression in the enzyme activity by H2O2 plus Fe2+ was associated with a decrease in Vmax and an increase in Km for MgATP. These results indicate that the depressant effect of xanthine plus xanthine oxidase on sarcolemmal Na(+)-K+ ATPase may be due to the formation of superoxide, hydroxyl and other radicals. Furthermore, the oxyradical-induced depression in Na(+)-K+ ATPase may be due to the formation of superoxide, hydroxyl and other radicals.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation and Characterization of Two Mg 2+-ATPase Activities from the Human Erythrocyte Membrane

Two distinct Mg 2+-ATPase activities were isolated from Triton X-100-solubilized human erythrocyte membranes using a combination of calmodulin-agarose to remove Ca 2+-ATPase and ion exchange chromatography to separate the Mg 2+-ATPase activities. The activity designated Mg 2+-ATPase A was inhibited by low concentrations of vanadate (IC 50 ≍ 2 μM) and by calcium (IC 50 ≍ 1.5 mM), La 3+ (80% inhibition at 0.5 mM), and F − (IC 50 ≍ 1 mM). Inhibition by F − increased in the presence of 10 μM AlCl 3. The activity appeared to be dependent on the concentration of MgATP. Two millimolar deoxyATP supported nearly full activity and 2 mM GTP supported 40% of the activity compared to that seen with 2 mM ATP. The activity was stimicrolated twofold by the addition of 1% (w/v) phosphatidylserine. These properties are consistent with a role of this enzyme in the control of red blood cell shape, possibly through association with the ATP-dependent translocation of phosphatidylserine and phosphatidylethanolamine from the outer to the inner leaflet of the bilayer. In contrast, Mg 2+-ATPase B was slightly stimicrolated by vanadate and by calcium but was unaffected by LaCl 3, and by F − ± AlCl 3. The activity was further stimicrolated by added magnesium in excess of fixed concentrations of ATP. In addition, at a fixed concentration of 10 mM MgCl 2, the activity with increasing ATP was biphasic, with the maximicrom at 1 mM. GTP 2 mM failed to support activity while 2 mM deoxyATP supported only 30% of activity compared to that seen with 2 mM ATP. The activity was not stimicrolated by oxidized glutathione or the glutathione conjugate lithocholic acid, suggesting it is not involved in the ATP-dependent transport of such compounds out of the erythrocyte.

Effects of pyrophosphate and nucleotide analogs suggest a role for ATP hydrolysis in cystic fibrosis transmembrane regulator channel gating.

Single channel analysis of artificial lipid planar bilayers reconstituted with wild-type human cystic fibrosis transmembrane regulator (CFTR) revealed a 10.3 pS Cl- selective channel that was activated upon phosphorylation with protein kinase A. Gating of this channel was described by a simple kinetic model consisting of a single open burst state and two closed states. The open probability of CFTR channels in bilayers increased as a function of increasing Mg-ATP concentration and exhibited negative cooperativity, suggesting the interaction of two or more ATP binding sites in channel gating. Mg-ATP increased channel open probability by decreasing the duration of the long-lived closed state but had no effect on either the mean open time or the fast closed state. ADP inhibited channel opening by precisely antagonizing the effect of ATP, suggesting that ADP inhibits the CFTR channel by competing with ATP for binding. Poorly hydrolyzable ATP analogs such as AMP-PNP and ATP gamma S, polyphosphates such as pyrophosphate (PPi) and tripolyphosphate (PPPi), and orthovanadate failed to support channel activity alone. When applied in the presence of ATP, these compounds all caused the CFTR channel to "lock" into a prolonged open burst state. These data support a model in which hydrolysis of ATP leads to closure of channels that have been opened by ATP.

ADP inhibits the sliding velocity of fluorescent actin filaments on cardiac and skeletal myosins.

We studied the effect of MgADP on the mechanical interaction of actomyosin in cardiac and skeletal muscles using an in vitro motility assay. The sliding velocities of fluorescently labeled actin filaments on rat cardiac and skeletal myosins were measured at various MgATP and MgADP concentrations. The filament velocity depended on MgATP concentration according to classic Michaelis-Menten kinetics with apparent Michaelis constants (Km) of 43 and 137 mumol/L and maximum velocity of 5.6 and 8.6 microns/s for cardiac and skeletal myosins, respectively. The presence of 2 mmol/L MgADP decreased the filament velocity and shifted the substrate concentration dependence of the velocity toward higher MgATP concentrations, yielding the inhibition constants of 194 and 478 mumol/L for cardiac and skeletal myosins, respectively. The activation energies determined by the temperature dependence of the velocity were 61 and 83 kJ/mol for rat V1 and rabbit cardiac myosins, which were similar to those of the dissociation rate constant of actomyosin-ADP complex reported in a solution study. The inhibition of the velocity by MgADP can be explained by the crossbridge scheme in which MgADP competes with MgATP for the substrate site on myosin molecules. In cardiac myosin, addition of a concentration of MgADP as low as 25 mumol/L significantly inhibited the velocity in the presence of 2 mmol/L MgATP, suggesting that increased intracellular MgADP may reduce the rate of crossbridge detachment, resulting in a decreased ATP consumption and an increased economy of force production under ischemic conditions. The present results support the idea that MgADP may be a physiologically important modulator of contraction in cardiac muscle.

Kinetic Characteristics of Phosphofructokinase from Bacillus stearothermophilus: MgATP Nonallosterically Inhibits the Enzyme

The kinetic mechanism of phosphofructokinase from Bacillus sterothermophilus has been investigated using steady-state measurements. The double-reciprocal patterns observed for initial velocity, product inhibition, and mixed alternate substrate studies of the reverse reaction establish that the mechanism involves rapid-equilibrium random binding of substrates and the formation of an abortive complex composed of enzyme, MgADP, and fructose 6-phosphate (E-MgADP-Fru-6P). Initial velocity patterns for the forward reaction show significant nonlinearity and resemble those seen for competitive substrate (MgATP) inhibition of an enzyme that obeys a random mechanism. A mutant BsPFK enzyme (GV212) was used to show that the inhibition is not due to MgATP binding in the effector site. Product and dead-end inhibition studies of the forward reaction are consistent with a random mechanism, after taking into account the effects of substrate inhibition by MgATP. Initial velocity measurements at low MgATP concentration show that the binding of MgATP is not a rapid-equilibrium process; i.e., the rate of catalysis is faster than the rate of substrate binding. It is concluded that the kinetic mechanism of the forward reaction is sequential random, with the rate of MgATP binding slower than the catalytic rate. A model is presented that incorporates these results and proposes that substrate binding proceeds through two alternative pathways, one of which is kinetically disfavored. The observed MgATP substrate inhibition arises from both reaction flux through the disfavored pathway and, to some extent, abortive binding of MgATP in the Fru-6P site.

Varicella-Zoster virus thymidine kinase: Characterization and substrate specificity

The varicella-zoster virus (VZV) thymidine kinase (TK) (EC 2.7.2.21) catalyzes the phosphorylation of many anti-VZV nucleosides. Purified, bacterially expressed VZV TK was characterized with regard to N-terminal amino acid sequence, pI value, pH optimum, metal ion requirement, phosphate donor and acceptor specificity, and inhibition by dTTP. Initial velocities of thymidine phosphorylation with variable MgATP concentrations fit a two-site model with apparent K m values for MgATP of 0.10 and 900 μM. dTTP was a noncompetitive inhibitor of thymidine phosphorylation but was competitive with MgATP. Phosphate donor and acceptor specificities of the bacterially expressed enzyme were indistinguishable from those of VZV TK purified from infected cells. Detailed studies of the nucleoside specificity with the bacterially expressed enzyme showed that, for a given sugar moiety, thymine nucleosides were the most efficient substrates followed by nucleosides of cytosine, uracil, adenine, and with some exceptions, guanine. For a given pyrimidine or purine (except guanine), 2′-deoxyribonucleosides were the most efficient substrates, followed by arabinosides, ribonucleosides, 2′,3′-dideoxyribonucleosides, and the acyclic moiety of acyclovir.

Sulfite both stimulates and inhibits the yeast vacuolar H(+)-ATPase.

The yeast vacuolar H(+)-ATPase (V-ATPase) exhibits nonlinear hydrolysis kinetics, i.e. an initial rapid rate followed by a slower, steadily declining rate. Sulfite (50-100 mM) stimulates the yeast V-ATPase specifically during the latter period. Sulfite activation has been observed for the F-ATPases and archaebacterial ATPases and is thought to be caused by the release of tightly bound nucleotide at the catalytic site. However, turnover-dependent inactivation of the yeast V-ATPase, and sulfite stimulation, were only observed at MgATP concentrations > 1.0 mM. Below 1.0 mM MgATP, the hydrolysis time course was linear, and sulfite was inhibitory. The inhibition during the initial phase and the stimulation during the later phase of the time course could be accounted for by a 5.5-fold sulfite-induced increase in the apparent Km, and a small increase in the apparent Vmax. Sulfite also protected the enzyme against inhibition by cold inactivation and by dicyclohexylcarbodiimide but not by bafilomycin. Sulfite stimulation during the later phase was antagonized by delta mu H+, particularly by delta pH. In contrast to its effects on hydrolysis, sulfite inhibited the formation of a pH gradient at all times and failed to enhance the membrane potential even when delta pH was collapsed by nigericin. These results indicate that sulfite partially uncouples hydrolysis from proton transport in a way that preserves regulation by delta mu H+.