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

8-Azido-ATP is a substrate for the ATP synthase in submitochondrial particles with a Vmax equal to 6% of the Vmax with ATP. The Km values for 8-azido-ATP are similar to those for ATP. ATP synthase in submitochondrial particles can bind maximally 2 mol 8-N-ATP or 8-N-ADP per mole and the inhibition of ATP hydrolysis by covalently bound N-ATP or N-ADP is proportional to the saturation of the enzyme with inhibitor, similar to the results obtained with isolated F1. Both 8-N-ATP and 8-N-ADP are bound mainly to the beta subunits and at all levels of saturation the distribution of the label is 77% to the beta and 23% to the alpha subunits. It is proposed that the binding of 8-azido-AXP itself is mainly to the beta subunit, but that part of the nitreno radicals formed during excitation with light reacts with an amino acid of the alpha subunit, due to the location of the binding site at an interface between a beta and an alpha subunit. Partial saturation with 8-N-ATP, under conditions that the concentration of 8-azido-ATP during the incubation is intermediate between the low and high Km values, does not abolish the apparent negative cooperativity of ATP hydrolysis. It is concluded that this apparent cooperativity is not due to the presence of two different catalytic sites, nor to a cooperativity between the two catalytic sites, but to interaction between the catalytic sites and regulatory sites.
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PMID:The use of 8-azido-ATP and 8-azido-ADP as photoaffinity labels of the ATP synthase in submitochondrial particles: evidence for a mechanism of ATP hydrolysis involving two independent catalytic sites? 286 13

2-Azidoadenosine 5'-diphosphate (2-azido-ADP) labeled with 32P in the alpha-position was prepared and used to photolabel the nucleotide binding sites of beef heart mitochondrial F1-ATPase. The native F1 prepared by the procedure of Knowles and Penefsky [Knowles, A. F., & Penefsky, H. S. (1972) J. Biol. Chem. 247, 6617-6623] contained an average of 2.9 mol of tightly bound ADP plus ATP per mole of enzyme. Short-term incubation of F1 with micromolar concentrations of [alpha-32P]-2-azido-ADP in the dark in a Mg2+-supplemented medium resulted in the rapid supplementary binding of 3 mol of label/mol of F1, consistent with the presence of six nucleotide binding sites per F1. The Kd relative to the reversible binding of [alpha-32P]-2-azido-ADP to mitochondrial F1 in the dark was 5 microM in the presence of MgCl2 and 30 microM in the presence of ethylenediaminetetraacetic acid. A linear relationship between the percentage of inactivation of F1 and the extent of covalent photolabeling by [alpha-32P]-2-azido-ADP was observed for percentages of inactivation up to 90%, extrapolating to 2 mol of covalently bound [alpha-32P]-2-azido-ADP/mol of F1. Under these conditions, only the beta subunit was photolabeled. Covalent binding of one photolabel per beta subunit was ascertained by electrophoretic separation of labeled and unlabeled beta subunits based on charge differences and by mapping studies showing one major radioactive peptide segment per photolabeled beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Photoaffinity labeling of mitochondrial adenosinetriphosphatase by 2-azidoadenosine 5'-[alpha-32P]diphosphate. 286 80

The photoreactive nucleotides [2-3H]8-azido-ATP and [2-3H]8-azido-ADP could be used to label the nucleotide binding sites on isolated mitochondrial F1-ATPase to a maximum of 4 mol of nucleotide per mol F1, also when the F1 was depleted of tightly bound nucleotides. At a photolabel concentration of 300-1000 microM, label was found on both alpha and beta subunits in a typically 1:3 ratio, independent of the total amount bound. Under these conditions the covalent binding of two nucleotides is needed for full inactivation (Wagenvoord, R.J., Van der Kraan, I. and Kemp, A. (1977) Biochim. Biophys. Acta 460, 17-24). At lower concentrations of [2-3H]8-azido-ATP (20 microM), it was found that covalent binding of only 1 mol of nucleotide per mole F1 was required for complete inactivation to take place indicating catalytic site cooperativity in the mechanism of ATP hydrolysis. Under those conditions, radioactivity was only found on the beta subunits, which would indicate that the catalytic site is located on a beta subunit and that a second site is located on the alpha/beta interface. It is found that four out of the six nucleotide binding sites are exchangeable and can be labelled with 8-azido-AT(D)P, i.e., two catalytic sites and two non-catalytic sites.
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PMID:Demonstration of two exchangeable non-catalytic and two cooperative catalytic sites in isolated bovine heart mitochondrial F1, using the photoaffinity labels [2-3H]8-azido-ATP and [2-3H]8-azido-ADP. 287 64

When the heat-activated chloroplast F1 ATPase hydrolyzes [3H, gamma-32P]ATP, followed by the removal of medium ATP, ADP, and Pi, the enzyme has labeled ATP, ADP, and Pi bound to it in about equal amounts. The total of the bound [3H]ADP and [3H]ATP approaches 1 mol/mol of enzyme. Over a 30-min period, most of the bound [32P]Pi falls off, and the bound [3H]ATP is converted to bound [3H]ADP. Enzyme with such remaining tightly bound ADP will form bound ATP from relatively high concentrations of medium Pi with either Mg2+ or Ca2+ present. The tightly bound ADP is thus at a site that retains a catalytic capacity for slow single-site ATP hydrolysis (or synthesis) and is likely the site that participates in cooperative rapid net ATP hydrolysis. During hydrolysis of 50 microM [3H]ATP in the presence of either Mg2+ or Ca2+, the enzyme has a steady-state level of about one bound [3H]ADP per mole of enzyme. Because bound [3H]ATP is also present, the [3H]ADP is regarded as being present on two cooperating catalytic sites. The formation and levels of bound ATP, ADP, and Pi show that reversal of bound ATP hydrolysis can occur with either Ca2+ or Mg2+ present. They do not reveal why no phosphate oxygen exchange accompanies cleavage of low ATP concentrations with Ca2+ in contrast to Mg2+ with the heat-activated enzyme. Phosphate oxygen exchange does occur with either Mg2+ or Ca2+ present when low ATP concentrations are hydrolyzed with the octyl glucoside activated ATPase. Ligand binding properties of Ca2+ at the catalytic site rather than lack of reversible cleavage of bound ATP may underlie lack of oxygen exchange under some conditions.
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PMID:Bound adenosine 5'-triphosphate formation, bound adenosine 5'-diphosphate and inorganic phosphate retention, and inorganic phosphate oxygen exchange by chloroplast adenosinetriphosphatase in the presence of Ca2+ or Mg2+. 287 34

F1-ATPase of rat liver was examined for its capacity to interact with both metal ions and nucleotides and for the effect of covalent ATPase inhibitors on these interactions. As isolated, rat liver F1 contains about 2 mol of Mg2+/mol of F1, 1 mol of which can be removed or exchanged. The remaining mole of Mg2+ per mole of F1 remains very tightly associated with F1 and is recovered in the alpha gamma fraction after cold denaturation. Rat liver F1 also contains as isolated a nearly equivalent amount of nucleotide (approximately 1.7 mol/mol of F1) which is readily removed by incubation at room temperature followed by column centrifugation. The "2 Mg2+ enzyme" binds almost 3 mol of 5'-adenylyl imidodiphosphate (AMP-PNP)/mol of F1 in the presence or absence of added divalent cation. When divalent cation is present as Co2+, an equivalent activator to Mg2+ in the ATPase reaction, 1 mol of F1 binds 3 mol of both AMP-PNP and Co2+. under these conditions, the very tight Mg2+ site remains loaded, the exchangeable Mg2+ site is replaced with AMP-PNPCo, and two additional AMP-PNPCo sites are filled. At this point, ADP can be loaded onto the enzyme as a fourth nucleotide at a site separate and distinct from the AMP-PNP sites. Significantly, rat liver F1 contains only a single readily detectable ADP binding site in the presence or absence of divalent cation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ligand binding studies of the F1 moiety of rat liver ATP synthase: implications about the enzyme's structure and mechanism. 288 76

The photolabeling of chloroplast F1 ATPase, following exposure to Mg2+ and 2-azido-ATP and separation from medium nucleotides, results in derivatization of two separate peptide regions of the beta subunit. Up to 3 mol of the analogue can be incorporated per mole of CF1, with covalent binding of one moiety or two moieties per beta subunit that can be either AMP, ADP, or ATP derivatives. These results, the demonstration of noncovalent tight binding of at least four [3H]adenine nucleotides to the enzyme and the presence of three beta subunits per enzyme, point to six potential adenine nucleotide binding sites per molecule. The tightly bound 2-azido nucleotides on CF1, found after exposure of the heat-activated and EDTA-treated enzyme to Mg2+ and 2-azido-ATP, differ in their ease of replacement during subsequent hydrolysis of ATP. Some of the bound nucleotides are not readily replaced during catalytic turnover and covalently label one peptide region of the beta subunit. They are on noncatalytic sites. Other tightly bound nucleotides are readily replaced during catalytic turnover and label another peptide region of the beta subunit. They are at catalytic sites. No alpha-subunit labeling is detected upon photolysis of the bound 2-azido nucleotides. However, one or both of the sites could be at an alpha-beta-subunit interface with the 2-azido region close to the beta subunit, or both binding sites may be largely or entirely on the beta subunit.
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PMID:Chloroplast F1 ATPase has more than three nucleotide binding sites, and 2-azido-ADP or 2-azido-ATP at both catalytic and noncatalytic sites labels the beta subunit. 288 81

In mitochondria and submitochondrial particles (SMP), the rate of ATP synthesis is restricted by the rate of energy production by the respiratory chain. Fractional inactivation of the ATP synthase complexes (F0F1) of bovine heart SMP by covalent modifiers increased the rate of ATP synthesis per mole of active F0F1. Thus, by use of SMP containing fractionally inactivated F0F1 complexes, a synthetic rate of 420 mol of ATP (mol of F0F1.s)-1 was measured, which extrapolated to a Vmax of 440 s-1. At this extrapolated point, the turnover rate of F0F1 complexes was independent of the rate of energy production by the respiratory chain. These results have been discussed in relation to the effect of fractional inactivation of the F0F1 complexes of SMP on the steady-state free energy of the system. The above rate of ATP synthesis is comparable to the rate of ATP hydrolysis by SMP (400-520 s-1) in the absence of energy coupling constraints and control by the ATPase inhibitor protein. More interestingly, this rate is also comparable to the rate of ATP synthesis by chloroplast F0F1 under high light intensity (approximately 420 s-1). Under the conditions specified, bovine heart SMP and chloroplasts show similar apparent Km values for ADP. Thus, it appears that the mammalian and chloroplast ATP synthase complexes are similar not only in structure but also in catalytic efficiency for ATP synthesis.
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PMID:Estimation of the turnover number of bovine heart F0F1 complexes for ATP synthesis. 289 47

This article reviews the current status of information regarding the role of energy in the process of oxidative phosphorylation by mitochondria. The available data suggest that in submitochondrial particles (SMP) energy is utilized for the binding of ADP and Pi and for the release of ATP bound at the catalytic sites of F1-ATPase. The process of ATP synthesis on the surface of F1 from F1-bound ADP and Pi appears to be associated with negligible free energy change. The rate of energy production by the respiratory chain modulates the kinetics of ATP synthesis between a low Km (for ADP and Pi)-low Vmax mode and a high Km-high Vmax mode. The Km extremes for ADP are 2-3 microM and 120-150 microM, and Vmax for ATP synthesis at high rates of energy production by bovine-heart SMP is about 440 S-1 (mole F1)-1 at 30 degrees C, which corresponds to 11 mumol ATP (min.mg of protein)-1. The interaction of dicyclohexylcarbodiimide (DCCD) or oligomycin at the proteolipid (subunit c) of the membrane sector (F0) of the ATP synthase complex alters the mode of ATP binding at the catalytic sites of F1, probably to one of lower affinity. It has been suggested that protonic energy might be conveyed to the catalytic sites of F1 in an analogous manner, i.e., via conformation changes in the ATP synthase complex initiated by proton-induced alterations in the structure of the DCCD-binding proteolipid. Finally, the relationship between the steady-state membrane potential (delta psi) and the rates of electron transfer and ATP synthesis has been discussed. It has been shown, in agreement with the delocalized chemiosmotic mechanism, that under appropriate conditions delta psi is exquisitely sensitive to changes in the rates of energy production and consumption.
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PMID:Role of energy in oxidative phosphorylation. 290 62

The protein ATPase inhibitor entraps about five nucleotides in pig heart mitochondrial F1, one at least being a triphosphate [Di Pietro, A., Penin, F., Julliard, J.H., Godinot, C., & Gautheron, D.C. (1988) Biochem. Biophys. Res. Commun. 152, 1319-1325]. The fate of these nucleotides was studied during ATP synthesis driven by NADH oxidation in reconstituted inverted submitochondrial particles. Iodinated F1, containing 0.7 mol of endogenous nucleotides/mol, was first loaded with tritiated adenine nucleotides in the presence or absence of the protein inhibitor and then reassociated with F1-depleted submitochondrial particles (ASU particles) to reconstitute an efficient NADH-driven ATP synthesis. In the absence of the protein inhibitor, 1.7 mol of labeled nucleotides remained bound per mole of reassociated F1, 0.8-0.9 mol being rapidly exchangeable against medium ADP or ATP, as measured after rapid filtration through nitrocellulose filters. In the presence of the protein inhibitor, as many as 3.25 mol of labeled nucleotides remained bound per mole of reassociated F1. Under hydrolysis conditions where ATPase activity was highly inhibited, no release of tritiated nucleotide occurred. In contrast, under ATP synthesis conditions where the protonmotive force was generated by NADH oxidation, the progressive reversal of inhibition by the protein inhibitor was correlated to a concomitant release of tritiated nucleotide. When ATP synthesis became fully active, about one nucleotide was completely exchanged whereas more than three nucleotides remained tightly bound and did not appear to be directly involved in ATP synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fate of nucleotides bound to reconstituted Fo-F1 during adenosine 5'-triphosphate synthesis activation or hydrolysis: role of protein inhibitor and hysteretic inhibition. 290 4

Four mechanisms for the allosteric regulation of the calcium and magnesium ion activated adenosinetriphosphatase (Ca,Mg-ATPase) of sarcoplasmic reticulum were examined. Negative cooperativity in substrate binding was not supported by 3H-labeled 5'-adenylyl methylenediphosphate (AMPPCP) binding, which was best fit by a single class of sites. Although calcium had no effect on the absence of cooperativity, it did increase the affinity of the enzyme for AMPPCP. Allosteric regulation via an effector site for AMPPCP or ATP on the same ATPase chain was eliminated by the stoichiometry of ATP and AMPPCP binding, 1 mol of site per mole of enzyme. The possibility that AMPPCP acts at an effector site was eliminated by showing that it competitively inhibits the rate of phosphoenzyme formation. Allosteric regulation of kinetics via site-site interaction in an oligomer was eliminated by showing that the inhibition of ATPase activity by fluorescein isothiocyanate is linearly dependent upon its incorporation into the sarcoplasmic reticulum. The fourth mechanism considered was stimulation of ATPase activity by the binding of ATP or AMPPCP at the active site after departure of ADP but before the departure of inorganic phosphate. This hypothesis was supported by site stoichiometry and by the observation that AMPPCP or ATP stimulates v/EP, the rate of ATP hydrolysis for a given level of phosphoenzyme. Computer simulation of this branched monomeric model could duplicate all experimental observations made with AMPPCP and ATP as allosteric regulators. The condition that the affinity of ATP binding to the enzyme be reduced when it is phosphorylated, which is required by the computer model, was confirmed experimentally.
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PMID:Mechanism of allosteric regulation of the Ca,Mg-ATPase of sarcoplasmic reticulum: studies with 5'-adenylyl methylenediphosphate. 293 90


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