Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mechanism of biosynthetic, transferase, ATPase, and transphosphorylation reactions catalyzed by unadenylylated glutamine synthetase from E. coli was studied. Activation complex(es) involved in the biosynthetic reaction are produced in the presence of either Mg2+ or Mn2+ ; however, with the Mn2+-enzyme inhibition by the product, ADP, is so great that the overall forward biosynthetic reaction cannot be detected with the known assay methods. Binding studies show that substrates (except for NH3 and NH2OH which are not reported here) can bind to the enzyme in a random manner and that binding of the ATP-glutamate, ADP-Pi or ADP-arsenate pairs is strongly synergistic. Inhibition and binding studies show that the same binding site is utilized for glutamate and glutamine in biosynthetic and transferase reactions, respectively, and that a common nucleotide binding site is used for all reactions studied. Studies of the reverse biosynthetic reaction and results of fluorescent titration experiments suggest that both arsenate and orthophosphate bind at a site which overlaps the gamma-phosphate site of nucleoside triphosphate. In the reverse biosynthetic and transferase reactions, ATP serves as a substrate for the Mn2+-enzyme but not for the Mg2+-enzyme. The ATP supported transferase activity of Mn2+-enzyme is probably facilitated by the generation of ADP through ATP hydrolysis. When AMP was the only nucleotide substrate added, it was converted to ATP with concomitant formation of two equivalents of glutamate, under the reverse biosynthetic reaction conditions, and no ADP was detected. The reversibility of 180 transfer between orthophosphate and gamma-acyl group of glutamate was confirmed. ATPase activity of Mg2+ and Mn2+ unadenylylated enzymes is about the same. Both enzymes forms catalyze transphosphorylation reactions between various purine nucleoside triphosphates and nucleoside diphosphates under biosynthetic reaction conditions. The data are consistent with the hypothesis that a single active center is utilized for all reactions studied. Two stepwise mecanisms that could explain the results are discussed.
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PMID:Mechanistic studies of glutamine synthetase from Escherichia coli. An integrated mechanism for biosynthesis, transferase, ATPase reaction. 0 53

This paper demonstrates, by pulse-chase techniques, the binding to rat liver mitochondrial carbamoyl phosphate synthetase of the ATP molecule (ATPB) which transfers its gamma-phosphoryl group to carbamoyl phosphate. This bound APTB can react with NH3, HCO-3 and ATP (see below) to produce carbamoyl phosphate before it exchanges with free ATP. Mg2+ and N-acetylglutamate, but not NH3 or HCO-3, are required for this binding; the amount bound depends on the concentration of ATP (Kapp = 10--30 microns ATP) and the amount of enzyme. At saturation at least one ATPB molecule binds per enzyme dimer. Binding of ATPB follows a slow exponential time course (t1/2 8--16 s, 22 degrees C), independent of ATP concentration and little affected by NH3, NCO-3 or by incubation of the enzyme with unlabelled ATP prior to the pulse of [gamma-32P]ATP. Formation of carbamoyl phosphate from traces of NH3 and HCO-3 when the enzyme is incubated with ATP follows the kinetics expected if it were generated from the bound ATPB, indicating that the latter is a precursor of carbamoyl phosphate ('Cbm-P precursor') in the normal enzyme reaction. This indicates that the site for ATPB is usually inaccessible to ATP in solution but becomes accessible when the enzyme undergoes a periodical conformational change. Bound ATP becomes Cbm-P precursor when the enzyme reverts to the inaccessible conformation. Pulse-chase experiments in the absence of NH3 and HCO-3 (less than 0.2 mM) also demonstrate binding of ATPA (the molecule which yields Pi in the normal enzyme reaction), as shown by a 'burst' in 32Pi production. Therefore, (in accordance with our previous findings) both ATPA and ATPB can bind simultaneously to the enzyme and react with NH3 and HCO-3 in the chase solution before they can exchange with free ATP. However, at low ATP concentration (18 micron) in the pulse incubation, only ATPB binds since ATP is required in the chase (see above). Despite the presence of two ATP binding sites, the bifunctional inhibitor adenosine(5')pentaphospho(5')adenosine(Ap5A) fails to inhibit the enzyme significantly. A more detailed modification of the scheme previously published [Rubio, V. & Grisolia, S. (1977) Biochemistry, 16, 321--329] is proposed; it is suggested that ATPB gains access to the active centre when the products leave the enzyme and the active centre is in an accessible configuration. The transformation from accessible to inaccessible configuration appears to be part of the normal enzyme reaction and may represent to conformational change postulated by others from steady-state kinetics. The properties of the intermediates also indicate that hydrolysis of ATPA must be largely responsible for the HCO-3-dependent ATPase activity of the enzyme. The lack of inhibition of the enzyme by Ap5A indicates substantial differences between the Escherichia coli and the rat liver synthetase.
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PMID:Mechanism of carbamoyl-phosphate synthetase. Binding of ATP by the rat-liver mitochondrial enzyme. 21 11

ESR spectra of nitroxyl derivative labelling with parachlormercury benzoate bound with SH-groups of Ca, Mg-dependent ATPase in the presence of Mn2+ ions are studied. It has been concluded from the saturation curves of ESR spectra that Mn2+ is localized at the distance approximately 40 A from the SH-group of the enzyme active centre. Platinum compound (K2PtCl4) changes the spectrum of ESR 1 due to the displacement of the label from the enzyme SH-groups and disintegration of the sarcoplasmic reticulum structure. Palladium compound (Pd(NH3)4Cl2) produced no effect on the ESR 1 spectrum.
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PMID:[Study of sarcoplasmic reticulum Ca-ATPase by the paramagnetic label--paramagnetic probe method]. 22 72

The presence of an H+/K(+)-ATPase and its contribution to the regulation of intracellular pH (pHi) was investigated in Caco-2 cells. The H+/K(+)-ATPase was detected immunologically using the monoclonal antibody 5-B6, which was raised against hog gastric H+/K(+)-ATPase. Cell pH was determined using the pH-sensitive dye 2',7'-bis(carboxyethyl)-carboxyfluorescein. Control pHi, measured in HCO(3-)-free medium, was 7.62 +/- 0.03 (n = 27) when cells were cultured for 14 days and decreased to 7.40 +/- 0.03 (n = 18) after 35 days in culture. Recovery of pHi following a NH+4/NH3 pulse could be reduced by either 100 microM SCH 28080 or 1 mM amiloride, or by removing extracellular Na+. The inhibitory effects of SCH 28080 and amiloride were additive, demonstrating the involvement of a gastric-like H+/K(+)-ATPase and a Na+/H+ exchanger in regulating pHi. Recovery rates at pHi 6.8 were not significantly different in cells cultured for up to 21 days, but were significantly lower in cells cultured for 28 and 35 days. This decrease in recovery rate was due to a decrease in the SCH-28080-insensitive recovery, indicating a reduction of the relative importance of Na+/H+ exchange to the recovery. Recovery of pHi was also inhibited by 1 mM N-ethylmaleimide. However, it is unlikely that N-ethyl-maleimide inhibited a vacuolar type of H+-ATPase, since bafilomycin A1 had no effect on pHi recovery. In conclusion, Caco-2 cells contain a SCH-28080-sensitive mechanism for regulating pHi, which is most conveniently studied after 28 days in culture, when the relative contribution of a Na+/H+ exchanger to pHi regulation is decreased.
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PMID:The colon carcinoma cell line Caco-2 contains an H+/K(+)-ATPase that contributes to intracellular pH regulation. 133 76

The three-dimensional structure of Na,K-ATPase has been analyzed with electron microscopy and image processing. The enzyme, purified from pig kidney outer medulla, was arranged in a new form of tetragonal two-dimensional membrane crystals after incubation with cobalt-tetrammine-ATP, a stable MgATP complex analogue. Each continuous protein domain, as delineated by negative stain, consists of two alpha beta-protomers related by a dyad axis. The two rod-like regions are connected by a bridge displaced about 20 A away from the center of the structure toward the lipid bilayer. The domain connecting the two promoters is more constricted and closer to the center of the structure in the Co(NH3)4ATP-induced crystals than in the vanadate-induced p21 crystals. These observations suggest that the difference between previously analyzed dimers of two-dimensional p21 crystals induced with vanadate/magnesium and dimers of p4 crystals induced with Co(NH3)4ATP reflects two different conformational states of the enzyme.
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PMID:Three-dimensional structure of Na,K-ATPase determined from membrane crystals induced by cobalt-tetrammine-ATP. 133 71

The interaction of sarcoplasmic reticulum Ca(2+)-ATPase with the Mg.ATP analogues Rh(H2O)4ATP and Co(NH3)4ATP have been examined. Co(NH3)4ATP slowly inactivates Ca(2+)-ATPase in a first order process, with a rate constant of 1.13 x 10(-3) s-1 and an apparent inactivation constant, KI, of 32 mM. Rh(H2O)4ATP likewise inactivates sarcoplasmic reticulum Ca(2+)-ATPase, but the plot of reciprocal apparent inactivation rate constants versus 1/[Rh(H2O)4ATP] is biphasic. The chi-intercepts of this plot yield apparent inactivation constants for the inhibition of Ca(2+)-ATPase by Rh(H2O)4ATP of KI1 = 30 microM and KI2 = 221 microM. The corresponding values of k2, the maximal first-order rate constant for inhibition in these two phases, are 1.16 and 2.19 x 10(-4)s-1. Tridentate Rh(H2O)3ATP also inhibits Ca(2+)-ATPase, but only after much longer incubation times. Ca(2+)-ATPase inactivation is accompanied by incorporation of radioactivity from gamma-32P into an acid-precipitable enzyme. Both processes were dependent on the presence of Ca2+ ions and were quenched by excess ATP. The first-order rate constant for inactivation of Ca(2+)-dependent ATPase activity in this experiment was 2.19 x 10(-4)s-1, and the first-order rate constant for Ca(2+)-dependent E-P formation was 2.07 x 10(-4)s-1, in excellent agreement with the value for inactivation. A linear relationship is observed between ATPase inactivation and E-P formation. Moreover, atomic absorption analysis demonstrates that the phosphorylation of Ca(2+)-ATPase by Rh(H2O)4ATP is accompanied by incorporation and tight binding of rhodium, with a stoichiometry of one rhodium incorporated per ATPase molecule phosphorylated. The characteristics of ATPase inactivation and phosphorylation (i.e., Ca2+ dependence, ATP competition, agreement of rate constants, and stoichiometric rhodium incorporation) suggest that Rh(H2O)4ATP is binding to the catalytic nucleotide site on Ca(2+)-ATPase and producing a highly stable, phosphorylated intermediate.
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PMID:Inactivation and phosphorylation of sarcoplasmic reticulum Ca(2+)-ATPase by Mg.ATP analogues Rh(III)-ATP and Co(III)-ATP. 153

The interaction of ruthenium red, [(NH3)5Ru-O-Ru(NH3)4-O-Ru(NH3)5]Cl6.4H2O, with various Ca2(+)-binding proteins was studied. Ruthenium red inhibited Ca2+ binding to the sarcoplasmic reticulum protein, calsequestrin, immobilized on Sepharose 4B. Furthermore, ruthenium red bound to calsequestrin with high affinity (Kd = 0.7 microM; Bmax = 218 nmol/mg protein). The dye stained calsequestrin in sodium dodecyl sulfate-polyacrylamide gels or on nitrocellulose paper and was displaced by Ca2+ (Ki = 1.4 mM). The specificity of ruthenium red staining of several Ca2(+)-binding proteins was investigated by comparison with two other detection methods, 45Ca2+ autoradiography and the Stains-all reaction. Ruthenium red bound to the same proteins detected by the 45Ca2+ overlay technique. Ruthenium red stained both the erythrocyte Band 3 anion transporter and the Ca2(+)-ATPase of skeletal muscle sarcoplasmic reticulum. Ruthenium red also stained the EF hand conformation Ca2(+)-binding proteins, calmodulin, troponin C, and S-100. This inorganic dye provides a simple, rapid method for detecting various types of Ca2(+)-binding proteins following electrophoresis.
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PMID:Interaction of ruthenium red with Ca2(+)-binding proteins. 169 45

Inactivation of Na+/K(+)-ATPase activity by the MgPO4 complex analogue Co(NH3)4PO4 leads, in everted red blood cell vesicles, to the parallel inactivation of 22Na+/K+ flux and 86Rb/Rb+ exchange, but leaves the 22Na+/Na(+)-exchange activity and the uncoupled ATP-supported 22Na+ transport unaffected. Furthermore, inactivation of purified Na+/K(+)-ATPase by Co(NH3)4PO4 leads to a parallel decrease of the capacity of the [3H]ouabain receptor site, when binding was studied by the Mg2+/Pi-supported pathway (ouabain-enzyme complex II) but the capacity of the ouabain receptor site was unaltered, when the Na+/Mg2+/ATP-supported pathway (ouabain-enzyme complex I) was used. No change in the dissociation constants of either ouabain receptor complex was observed following inactivation of Na+/K(+)-ATPase. When eosin was used as a marker for the high-affinity ATP-binding site of the E1 conformation, formation of stable E'2.Co(NH3)4PO4 complex led to a shift in the high-affinity ATP-binding site towards the sodium form. This led to an increase in the dissociation constant of the enzyme complex with K+, from 1.4 mM with the unmodified enzyme to 280 mM with the Co(NH3)4PO4-inactivated enzyme. It was concluded, that the effects of Co(NH3)4PO4 on the partial activities of the sodium pump are difficult to reconcile with an alpha, beta-protomeric enzyme working according the Albers-Post scheme. The data are consistent with an alpha 2, beta 2 diprotomeric enzyme of interacting catalytic subunits working with a modified version of the Albers-Post model.
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PMID:Blocking of Na+/K+ transport by the MgPO4 complex analogue Co(NH3)4PO4 leaves the Na+/Na(+)-exchange reaction of the sodium pump unaltered and shifts its high-affinity ATP-binding site to a Na(+)-like form. 169 57

The synthetase subunit of Escherichia coli carbamyl phosphate synthetase has two catalytic nucleotide-binding domains, one involved in the activation of HCO3- and the second in phosphorylation of carbamate. Here we show that a Glu841----Lys841 substitution in a putative ATP-binding domain located in the carboxyl half of the synthetase abolishes overall synthesis of carbamyl phosphate with either glutamine or NH3 as the nitrogen source. Measurements of partial activities indicate that while HCO3(-)-dependent ATP hydrolysis at saturating concentrations of substrate proceeds at higher than normal rates, ATP synthesis from ADP and carbamyl phosphate is nearly completely suppressed by the mutation. These results indicate Glu841 to be an essential residue for the phosphorylation of carbamate in the terminal step of the catalytic mechanism. The Lys841 substitution also affects the kinetic properties of the HCO3- activation site. Both kcat and Km for ATP increase 10-fold, while Km for HCO3- is increased 100-fold. Significantly, NH3 decreases rather than stimulates Pi release from ATP in the HCO3(-)-dependent ATPase reaction. The increase in kcat of the HCO3(-)-dependent ATPase reaction, and an impaired ability of the Lys841 enzyme to catalyze the reaction of NH3 with carboxy phosphate, strongly argues for interactions between the two catalytic ATP sites that couple the formation of enzyme-bound carbamate with its phosphorylation.
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PMID:Mutational analysis of carbamyl phosphate synthetase. Substitution of Glu841 leads to loss of functional coupling between the two catalytic domains of the synthetase subunit. 173 23

The progress of research in the Central Nervous System (CNS) had led to the consideration of neurons and glia as indissociable functional complexes. Neuron-glia interactions are essential for the maturation of the CNS. Glial cells release trophic factors for neurons (NGF) and neurons release trophic factors for glia (GGF). Furthermore, the latter provide a substrate for the migration of neurons and guidance of axons by mean of adhesion molecules. In adults, the interactions between neurons and glial cells serve to maintain homeostasis. Thus, the glial cells perform the restoration of the metabolic equilibrium overthrown by the transmission of the nerve impulse and provide the glucose required for neuronal activity. The nerve impulse provokes increases in the cellular space of CO2, K+, NH3 and neurotransmitters which must be taken up to allow neuronal activity to continue (in normal conditions). Astrocytes perform the uptake of the extracellular K+ by means of passive ionic channels, ionic voltage-dependent channels and a sodium-potassium-ATPase-dependent pump. The oligodendrocytes are involved in the metabolism of CO2 by converting CO2 into carbonic acid by means of carbonic anhydrase. Oligodendrocytes and astrocytes play a role in terminating neural transmission by the uptake of the amino acid neurotransmitters, such as GABA, glutamate and aspartate. The catabolism of glutamate to glutamine by means of glutamine synthetase allows both the conversion of an excitatory amino acid into a neutral amino acid (which can diffuse in the extracellular space without causing neural transmission) and the reduction of cerebral NH3 content.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Neuron-glia interactions]. 178 93


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