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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The nucleotide binding capacity and affinity of the isolated beta subunit from Escherichia coli F1-ATPase have been studied with radiolabeled ADP and ATP by an equilibrium dialysis technique. Each mole of beta subunit in the presence of EDTA bound 1 mol of ADP or ATP with Kd values of 25 microM and 50-100 microM, respectively. At a saturating concentration, aurovertin enhanced the affinity of ADP or ATP for the isolated beta subunit by 3-6-fold. The Kd values for the binding of ADP or ATP were also assessed through the enhancing effect of ADP on [14C]aurovertin binding (Issartel, J.-P., Klein, G., Satre, M., & Vignais, P.V. (1983) Biochemistry 22, 3485-3492); the Kd values determined by this approach were several times lower than in the absence of aurovertin, in agreement with results obtained by direct titration with radiolabeled ADP or ATP.
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PMID:Evidence for a nucleotide binding site on the isolated beta subunit from Escherichia coli F1-ATPase. Interaction between nucleotide and aurovertin D binding sites. 624 80

We have investigated the suitability of 5'-p-fluorosulfonylbenzoyladenosine (FSBA) as an ATP site affinity probe for the canine kidney Na+, K+-ATPase. The purified enzyme is slowly inactivated by this compound in suitable buffers, losing about half of its activity over a two-hour period. The rate of inactivation is more rapid in 0.1 M KCl than in 0.1 M NaCl. Low concentrations of ATP protect the enzyme against inactivation, with half-maximal effects at 4 microM ATP in 0.1 M NaCl and 350 microM ATP in 0.1 M KCl. ADP also protects against FSBA inhibition, but AMP is ineffective when present at 100 microM levels. This pattern is consistent with the previously described nucleotide specificity of the Na+, K+-ATPase. Addition of protective amounts of ATP after inactivation has occurred does not restore enzyme activity, indicating that inhibition is irreversible. Measurement of the concentration-dependence of FSBA inactivation suggests an apparent Kd for binding of this compound well above 1 mM, the solubility limit of the analog. This finding is reinforced by the failure of 1 mM FSBA to compete effectively with ATP for the high-affinity ATP site of the enzyme. Nevertheless, attachment of the analog to this site is indicated by its ability to prevent [3H]-ADP binding in proportion to the number of sites it has inactivated. Studies with [3H]-FSBA show that about 1 mole of the analog attaches specifically to the alpha subunit per mole of enzyme inactivated. A similar amount of nonspecific labeling also occurs with negligible effect on enzyme activity. These findings suggest that FSBA may be useful in probing the topography of the high-affinity ATP binding site of the Na+, K+-ATPase and related enzymes.
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PMID:5'-p-fluorosulfonylbenzoyladenosine as an ATP site affinity probe for Na+, K+-ATPase. 626 29

The maximal content of mitochondrial isoenzyme of creatine kinase (CK) in rat heart mitochondria does not exceed 12.5 moles per mole of ATP-ADP translocase. This value was obtained by titration of mitochondrial CK activity in aged mitochondria by 5,5'-dithiobis-(2-nitrobenzoate) (DTNB) and 2,4-dinitrofluorobenzene (DNFB) and by a more complex and accurate method. The essential thiol groups of membrane-bound mitochondrial CK (and its enzymic activity) can be specifically protected by phosphocreatine (12 mM) + ADP (1-5 mM) against inactivation by DTNB. Mitochondria with protected SH-groups of CK and with groups inactivated by DTNB were repeatedly incubated with DTNB under identical conditions and the number of additionally reacted sulfhydryl groups and the changes in CK activity were measured. The differences in the number of additionally reacted SH-groups correlated with the changes in the CK activity, which made it possible to calculate the molar ratios of mitochondrial CK to cytochrome c oxidase and ATP-ADP translocase (2.16 +/- (0.4): 1:2, respectively).
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PMID:[Determination of molar content of creatine kinase in heart mitochondria by SH-reagents]. 627 Dec 62

The incorporation of N-ethylmaleimide into the 30,000-Mr component of beef-heart mitochondria has been studied as a function of various ligands to the ADP/ATP carrier and the isolation of the N-ethylmaleimide-labeled protein is reported. 1. The incorporation of N-ethylmaleimide into the 30,000-Mr component is specifically stimulated by ADP and ATP. Thus by differential incorporation of N-ethylmaleimide, the 30,000-Mr component is preferentially labeled. 2. Addition of carboxyatractylate inhibits, whereas bongkrekate tolerates, the incorporation of N-ethylmaleimide. 3. After solubilization by Triton the purification of N-ethylmaleimide-labeled protein is facilitated in the presence of bongkrekate but not of carboxyatractylate, in agreement with the postulated existence of only a bongkrekate-N-ethylmaleimide-protein complex. The labeled protein was purified to homogeneity on hydroxyapatite in Triton and subsequently, after denaturation in dodecylsulfate, on Sepharose 6B. 4. The identify of the isolated labeled protein with the formerly isolated bongkrekate-protein or carboxyatractylate-protein complexes is confirmed by the isoelectric point and amino acid composition. 5. Two moles of N-ethylmaleimide must be incorporated into the 30,000-Mr component in order to inhibit fully the binding of one mole carboxyatractylate. This corresponds to one -SH group per unit.
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PMID:Incorporation of N-ethylmaleimide into the membrane-bound ADP/ATP translocator. Isolation of the protein labeled with N-[3H]ethylmaleimide. 627 30

The substrate specificity of diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase from Physarum polycephalum for dinucleoside polyphosphates has been determined by high-performance liquid chromatography (HP-LC). Elution of a strong anion-exchange resin with a pH and ionic strength gradient of ammonium phosphate separates a series of monoadenosine and diadenosine polyphosphates. Most of the corresponding guanine nucleotides are also resolved on this HPLC system. One mole each of Ap4A and Gp4G is symmetrically hydrolyzed to 2 mol of ADP and GDP, respectively. Ap3A, Ap5A, Ap6A, and Ap4 are hydrolyzed, and in each case ADP is one of the products. Gp3G, Gp5G, Gp6G, and Gp4 are also substrates, and in each case GDP is one of the products. AMP, ADP, ATP, Ap2A, ADPR, GMP, GDP, GTP, NAD+, and NADP+ are not substrates. No hydrolysis of the cap dinucleotides m7Gp3Am and m7Gp3Cm was detected by HPLC. Diadenosine tetraphosphate pyrophosphohydrolase preparations were also assayed for adenylate kinase, nucleotide diphosphate kinase, NAD(P)+ pyrophosphohydrolase, phosphodiesterase, cyclic nucleotide phosphodiesterase, phosphatase, and ribonuclease activities. These enzymic activities were not detectable in diadenosine tetraphosphate pyrophosphohydrolase. The symmetrical hydrolysis of Ap4A and Gp4G is an unique catalytic property that distinguishes diadenosine tetraphosphate pyrophosphohydrolase from P. polycephalum from diadenosine tetraphosphate phosphohydrolases from other organisms.
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PMID:Diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase from Physarum polycephalum. Substrate specificity. 629 57

Pyruvate, Pi dikinase in extracts of chloroplasts from mesophyll cells of Zea mays is inactivated by incubation with ADP plus ATP. This inactivation was associated with phosphorylation of a threonine residue on a 100 kDa polypeptide, the major polypeptide of the mesophyll chloroplast stroma, which was identified as the subunit of pyruvate, Pi dikinase. The phosphate originated from the beta-position of ADP as indicated by the labelling of the enzyme during inactivation in the presence of [beta-32P]ADP. During inactivation of the enzyme up to 1 mole of phosphate was incorporated per mole of pyruvate, Pi dikinase subunit inactivated. 32P label was lost from the protein during the Pi-dependent reactivation of pyruvate, Pi dikinase.
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PMID:Regulation of C4 photosynthesis: regulation of pyruvate, Pi dikinase by ADP-dependent phosphorylation and dephosphorylation. 631 Dec 12

The calcium pump of sarcoplasmic reticulum possesses high-affinity calcium-binding and ATP-binding sites. At 0 degrees C pH 6.8 and in the absence of calcium, about 3.5 nmol/mg of high-affinity ATP-binding sites are titrated with a dissociation constant, Kd, of 5 microM. In the presence of Ca2+, ATP phosphorylates the enzyme at a much lower concentration: K 1/2 = 100 nM. In the absence of ATP the calcium ions reversibly bind to the high-affinity calcium sites (6.5 nmol/mg); however the following is shown in this paper. 1. Phosphorylation of the enzyme in the presence of calcium leads to the immediate occlusion of the calcium ions bound to the high-affinity sites. 2. Two moles of calcium are occluded per mole of phosphoenzyme formed. 3. Occlusion can be reversed by ADP. 4. Transport is a slower process which occurs in the presence of Mg2+ at the same rate as the spontaneous decay of the phosphoenzyme. Experiments performed in the absence of magnesium reveal another divalent cation binding site which is probably directly involved in ATP and Pi binding. The nature of the cation bound to this site determines the stability and ADP-sensitivity of the phosphoenzyme.
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PMID:Occlusion of divalent cations in the phosphorylated calcium pump of sarcoplasmic reticulum. 644 98

Cibacron Blue F3GA and its immobilized derivatives have been shown before to bind and inhibit nucleotide-dependent enzymes and, among them, myosin subfragment 1. Experiments have been carried out to examine the mechanism of the subfragment 1--dye interaction. Binding of subfragment 1 to immobilized dye (Affi-Gel Blue) does not involve the ATP binding site on myosin. Subfragment 1 hydrolyzes MgATP and CaATP while bound to the Affi-Gel Blue column. Inactivated subfragment 1, which contains [3H]ADP noncovalently trapped at the active site, binds and elutes from the Affi-Gel Blue column in the same manner as unmodified, active protein. Free Cibacron Blue inhibits the ATPase activity of subfragment 1. The inhibition is pH, salt, and time dependent. Complete inhibition correlates with the noncovalent binding of four to five dye molecules per mole of subfragment 1. Three to four of these dye molecules can be preferentially removed from subfragment 1 in the presence of 1 M KCl without relieving the inhibition. This inhibition, which can be traced to one dye molecule per subfragment 1, is reversible and is facilitated in the presence of MgADP and MgATP, suggesting that the dye does not bind at the active site of subfragment 1. Our observations are explained in terms of hydrophobic and electrostatic protein--dye interactions.
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PMID:Interaction of myosin subfragment 1 with Cibacron Blue F3GA. 645 18

A study of creatine kinase modification by ATP gamma-(N-(2-chloroethyl)-N-methyl)amide was performed. The attachment 1,7-1,8 moles of analogue per mole of functional dimer results in full inactivation of the enzyme. The substrates, ATP and ADP, protect the enzyme both against inactivation and covalent binding of analogue. The affinity modification rate depends on the reagent and magnesium ion concentrations and pH of the reaction mixture. The dissociation constants (1,0 and 1,5 mM) for the enzyme-analogue complexes and the affinity modification maximal rate constants (2,1 X 10(-3) and 1,2 X 10(-3) c-1) in the absence and presence of Mg2+ ions were estimated. Some differences in the affinity modification rates were observed for the nonidentical M and M'-subunits of creatine kinase. The data obtained are indicative of a histidine residue alkylation by the ATP analogue. This histidine (pK 7,7) may function as a general acid-base catalyst in deprotonation of the guanidinium group of creatine as the latter is phosphorylated by ATP.
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PMID:[Affinity modification of creatine kinase from the rabbit skeletal muscle by gamma-amide of ATP--a nitrogen mustard derivative]. 654 33

The time course of magnesium adenosine triphosphate (Mg ATP) cleavage in chemically skinned muscle fibres of the rabbit was measured by a method in which Mg ATP cleavage was initiated by photolytic release of ATP from P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate (caged ATP) and terminated by rapid freezing 50 ms to 8 s later. Up to 5 mM-ATP was released following a single 50 ns laser pulse at 347 nm. Mg ATP cleavage was measured at 19 degrees C in the presence and absence of calcium ions, for fibres near rest length and stretched beyond overlap of the myofilaments. At full overlap and in the absence of calcium (less than 10(-8) M) and nucleotide, the fibres developed rigor tension. Following the laser pulse the tension decreased to that of a relaxed fibre in two distinct phases. The first phase lasted about 40 ms and was followed by a second phase during which tension decreased to zero with an approximately exponential time course with a rate constant of 11 s-1. In the presence of 2 X 10(-5) M-free calcium ions, the initial phase following the laser flash lasted approximately 13 ms, and was followed by an exponential rise of tension with a rate constant of 28 s-1. The active tension reached by the muscle fibres was 54 kN/m2. For fibres stretched beyond overlap, no change in tension was observed following the release of Mg ATP. Under all conditions the time course of Mg ATP cleavage was biphasic, and consisted of a rapid initial burst of ADP formation, complete within 50 ms, followed by a slower steady-state rate of Mg ATP cleavage. The number of molecules of Mg ATP cleaved during the burst was approximately equal to the number of myosin subfragment 1 heads for fibres at full myofilament overlap, and equal to 0.7 molecules per myosin subfragment 1 head for fibres stretched beyond overlap. At full overlap in the presence of calcium ions, the steady-state rate equalled 1.8 mol Mg ATP cleaved per mole myosin subfragment 1 head per second. In all other cases the steady-state rate of Mg ATP cleavage was at least 10-fold less. When fibres at full overlap were pre-incubated with 2 mM-ADP, the initial phase of the tension response was somewhat prolonged, but the burst of ADP formation was also complete within 50 ms.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The kinetics of magnesium adenosine triphosphate cleavage in skinned muscle fibres of the rabbit. 661 12


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