UMLS:C0027960 - FACTA Search

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

During the inactivation of the nucleotide-free F1-ATPase at pH 7.0, by p-fluorosulfonyl[14C]benzoyl-5'-adenosine ([14C]FSBA) in the presence of 20% glycerol, about 4.5 g atoms of 14C are incorporated/350,000 g of enzyme. Isolation of the subunits has shown: (a) over 90% of the incorporated label is associated with the alpha and beta subunits; (b) the amount of label incorporated into the alpha subunit is about 0.5 g atoms/mol which is nonspecifically associated with a number of tyrosine and lysine residues; (c) the amount of radioactivity incorporated into the beta subunit is about 0.9 g atoms/mol which correlates with the degree of inactivation of the enzyme and resides on a single tyrosine residue; (d) up to 2.2 mol of alpha subunit have been isolated from each mole of inactivated enzyme; and (e) about 2 mol of beta subunit have been isolated from each mole of inactivated enzyme. These results account for the incorporation of 4.5 g atoms of 14C which are incorporated/mol of ATPase during inactivation if there are three copies each of the alpha and beta subunit present in the enzyme. It has also been shown that 4-chloro-7-nitrobenzofurazan (NBD-Cl) and FSBA react with different tyrosine residues when they inactivate the ATPase. In addition, it has been shown that the ATPase inactivated with FSBA retains the capacity to bind up to 2.2 mol of [14C]ADP/350,000 g of enzyme.
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PMID:On the subunit stoichiometry of the F1-ATPase and the sites in it that react specifically with p-fluorosulfonylbenzoyl-5'-adenosine. 15 96

1. Conventional electrophysiological techniques were used to record from isolated rat phrenic nerve-hemidiaphragm preparations. After periods of rest (20 min) or nerve stimulation (7/sec for 20 min) the bathing medium of the preparation was removed and assayed for adenosine triphosphate (ATP) and adenosine diphosphate (ADP) using a sensitive modification of the firefly luciferase method (Silinsky, 1974). 2. In the presence of tubocurarine and normal (2 mM) calcium, fourteen periods of nerve stimulation (eight preparations) caused the appearance of ATP and/or ADP in amounts ranging from 28 to 641 p-mole. Experiments using carbachol (30 muM or 1 mM) suggested that this nucleotide efflux was not produced by a secondary action of released acetylcholine (ACh). 3. Stimulation of isolate phrenic nerve trunks at 7/sec for 20 min did not cause the efflux of ATP or ADP. 4. In solutions of normal osmotic pressure and reduced calcium concentrations (0-1 mM or 'calcium-free'), stimulation failed to release adenine nucleotide from non-contracting preparations. 5. Diaphragms were bathed in normal calcium and indirectly stimulated at 11/sec for 80-90 min in the presence of 5 times 10-minus 5 M hemicholinium-3. After all detectable signs of ACh release were eliminated, nerve stimulation failed to release ATP or ADP. 6. These results in conjunction with experiments on the hydrolysis of exogenous ATP suggest that ATP is released from the motor nerve ending and is subsequently degraded by enzymatic activity. It is also suggested that the released nucleotide may be derived from the cholinergic vesicle.
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PMID:On the association between transmitter secretion and the release of adenine nucleotides from mammalian motor nerve terminals. 16 62

The time course of binding of N-ethylmaleimide (NEM) to the SR was measured at pH 7.5 in the presence or absence of ATP or ADP. The following results were obtained. 1. Both in the presence and absence of nucleotide, the ATPase [EC 3.6.1.3] activity decreased linearly with increase in the amount of NEM bound to the fragmented sarcoplasmic reticulum (SR), and was inhibited almost completely by the binding of 2 moles of NEM per 10(5) g of the SR protein. 2. The amount of NEM incorporated into the ATPase (M.W.=105,000) was measured by SDS disc-gel electrophoresis. It was shown that the ATPase activity was inhibited almost completely by the binding of 2 moles of NEM per mole of ATPase. 3. The rate of binding of NEM to SR decreased by 30-40% in the presence of either ATP or ADP. The concentrations of both ATP and ADP for half-saturation were 0.1-0.2mM. 4. The effect of nucleotide on the rate of binding of NEM was not changed by the presence of Ca2+ and Mg2+ ions. Similar effects were also observed even when the SR membranes were solubilized with Triton X-100. It is suggested from these results that one or two SH groups are located in the active site of the SR ATPase, and that conformational changes are induced by the addition of ATP and ADP.
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PMID:Chemical modification of the Ca2+-dependent ATPase of sarcoplasmic reticulum from skeletal muscle. I. Binding of N-ethylmaleimide to sarcoplasmic reticulum: evidence for sulfhydryl groups in the active site of ATPase and for conformational changes induced by adenosine tri- and diphosphate. 18 70

1. Two moles of 2-hydroxy-5-nitrobenzyl group bound selectively to one mole of heavy meromyosin when it was treated with 2-hydroxy-5-nitrobenzyl bromide, a specific reagent for tryptophanyl residues. The binding with ADP, the size of the initial burst of Pi liberation and the difference absorption spectrum with and without ADP of the bound 2-hydroxy-5-nitrobenzyl groups were measured with heavy meromyosin modified with various amounts of reagent. The properties of the modified heavy meromyosin did not change until the molar binding ratio of the reagent, rH, was about 1, but the properties changed remarkably when rH increased from 1 to 2. 2. Subfragment-1 was prepared from the modified heavy meromyosin by trypsin [EC 3.4.21.4] digestion. The molar binding ratio of the reagent in subfragment-1, rS, was found to be less than 0.1 when rH of the starting heavy meromyosin was less than 0.8. However, rS was about 0.5 in subfragment-1 prepared from heavy meromyosin of rH about 2. The results indicate that only one mole of 2-hydroxy-5-nitrobenzyl group, which was bound with lower reactivity than the other, was bound to a head part of heavy meromyosin. 3. Subfragment-1 fraction prepared from the modified heavy meromyosin could be separated into two fractions by DE-32 cellulose column chromatography; the subfragment-1 portion which eluted later showed a higher rS than that eluted in front. The binding with ADP, the size of the initial burst of Pi liberation and the difference absorption spectrum induced by ATP were measured with the modified subfragment-1 separated by DE-32 cellulose column chromatography. The ADP-binding ability and the size of the initial burst were not dependent on rS, and coincided with those of subfragment-1 prepared from unmodified heavy meromyosin. 4. The results of ADP binding studies suggest that heavy meromyosin is constituted from nonidentical subunits, and that there is an interaction between them which controls the ADP binding. Two tryptophanyl residues having specific reactivity toward 2-hydroxy-5-nitrobenzyl bromide are assumed to be involved in the interaction.
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PMID:The role of tryptophanyl residues in heavy meromyosin as studied by chemical modification with 2-hydroxy-5-nitrobenzyl bromide. 18 81

Platelets from most patients with type IIa hyperlipoproteinemia (IIa) aggregate in the presence of lower concentrations of epinephrine and adenosine diphosphate (ADP) than are necessary to aggregate normal platelets. We have observed a comparable functional alteration in human platelets made cholesterol-rich in vitro by incubation in a milieu artificially rich in free cholesterol relative to phospholipid. We therefore examined platelet aggregation and lipid composition of platelets and of plasma low-density lipoprotein (LDL) in 19 individuals with IIa (including three homozygotes), seven normolipidemic individuals with symptomatic, angiographically-documented coronary atherosclerosis (atherosclerosis group), and 23 asymptomatic, normolipidemic subjects (control group). More than 99 percent of platelet cholesterol was unesterified. There was a 7 percent increase in the cholesterol content of whole platelets per mole of platelet phospholipid (C/PL) in IIa as compared to normal controls. This resulted from a 22 percent increase in the C/PL of IIa platelet membranes with no change in the C/PL of the soluble or granule fraction. The C/PL of IIa platelets was 6 percent greater than that of platelets from patients with atherosclerosis. As compared to those of normal controls, IIa platelets aggregated in response to a ninefold lower concentration of epinephrine (p less than 0.001) and a twofold lower concentration of ADP (p less than 0.02). The response of atherosclerosis platelets to these agents was comparable to that of controls. In all groups, there was a negative correlation between the log concentration of epinephrine required to produce complete platelet aggregation and the platelet C/PL (r = -0.06; p less than 0.002). The composition of LDL isolated from the plasma of patients with IIa was characterized by a 39 percent increase in the amount of free cholesterol relative to protein and a 35 percent increase in C/PL, as compared with control LDL. These values were increased 23 and 19 percent, respecitvely, when IIa was compared with the atherosclerosis group. In all groups the C/PL of LDL correlated well with the C/PL of platelets (r = =0.61; p less than 0.001). However, a simple cause-and-effect relationship did not appear to exist since (1) erythrocyte membrane C/PL was not affected and (2) normal platelets or erythrocytes underwent no change in C/PL during 18 hours' incubation in IIa plasma. These studies demonstrate that LDL and platelets in IIa contain an increased amount of free cholesterol relative to its principal solubilizer, phospholipid. In platelets this correlates with an increased sensitivity to aggregating agents. Moreover, the similarity between the functional abnormality in IIa platelets and that previously observed in normal platelets made cholesterol-rich in vitro suggests that the lipid composition of platelet membranes may have a direct effect on the function of platelets in man.
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PMID:Abnormalities of cholesterol-phospholipid composition in platelets and low-density lipoproteins of human hyperbetalipoproteinemia. 18 56

1. Binding of MG-ADP to both heart and fast skeletal myosin was found with 3 methods to proceed in 2 steps. One mole of MG-ADP binds with high affinity (K approximately equal to 10(6) M-1) and subsequently a second with lower affinity (K approximately equal to 10(2)-10(4) M-1) per myosin. Only one mole of MG-ADP was found to bind with the high affinity to isolated myosin heads. This implies that binding of MG-ADP to intact myosin exhibits negative cooperativity. 2. When a nucleotide is bound, the 2 heads of a single myosin molecule adopt different conformations since on each head a different type of essential thiol group was found to be the most reactive towards N-ethylmaleimide. In the presence of MG-pyrophosphate a thiol-1 is the most reactive essential group in both heads. Therefore, the nucleoside moiety seems to be required for this latter type of head-head interaction.
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PMID:Evidence for head-head interactions in myosin from cardiac and skeletal muscles. 19 85

The kinetic properties of pH jump-induced phosphorylation in thylakoidal membranes of spinach chloroplasts were investigated, and the following results were obtained. 1. The pH jump-induced P incorporation proceeded linearly with time for at least 2 sec after the start of the reaction. Phosphate was incorporated mainly into ATP. The amounts of incorporation into ADP during 0.1 and 2.0 sec were 0.02 and 0.1 mole/400 moles chl, respectively. The amounts of P incorporation into ADP and the beta-position of ATP during a 2 sec reaction were less than 5 and 0.1% of the total amount of P incorporation, respectively. Even in the absence of added ADP, ATP was formed by the pH jump, but the amount was very small, i.e., less than 1% of that in the presence of a saturating amount of ADP. Formation of ATP was not enhanced by the addition of 0.1 mM AMP, instead of ADP. 2. The dependence of the rate of ATP formation, v, induced by a pH jump from 3.85 to 8.11 on the concentrations of ADP and Pi was given by v=Vopt/[1+psi1/[ADP]) (1 + psi2/[Pi)], where the values of the constants, Vopt, psi1, and psi2 were 14--20 moles/10-6 g chl/sec, 12.5-15 muM and 11-20 mM, respectively, at 0 degrees. 3. The dependence of v on the concentration of protons was given by v=Va/[1 + psi H-a/[H+,-a])-2], and v =Vb/[1 + ([H+,-b]/psiH-b)-2], in the acidic and basic phases, respectively. The values of the constants psi H-a and psi H-b were 10-5.7 and 10-7.9 M, respectively. 4. ATP formation was initiated by adding one of the substrates, ADP or Pi, at various times after after the pH jump in the presence of the other substrate. The rate decreased logarithmically with increase in the time between the pH jump and the start of the reaction. When phosphorylation was initiated by adding Pi after the pH jump in the presence of ADP, the decay constant of v was about 0.08 sec-1, which was one-third of that observed when the order of addition of ADP and Pi was reversed.
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PMID:pH jump-induced phosphorylation of adenosine diphosphate in thylakoidal membranes. 23 94

Rat and calf adrenal cortex homogenates were found to contain three different malic enzymes. Two were strictly NADP+-dependent and were localized, one each, in the cytosol and the mitochondrial fractions, respectively. These two enzymes appear to be identical to those described by Simpson and Estabrook (Simpson, E. R., and Estabrook, R. W. (1969) Arch. Biochem. Biophys. 129, 384-395). The third was NAD(P)+-linked and was present in the mitochondrial fraction only. All three malic enzymes separated as distinct bands during electrophoresis on 5 percent polyacrylamide slab gels at pH 9.0. Marker enzymes and the mitochondrial malic enzymes migrated together in intact mitochondria during sucrose density gradient centrifugations despite changes in the equilibrium position of the mitochondria promoted by energy-dependent calcium phosphate accumulation. In adrenal cortex mitochondria subfractionated by the method of Sottocasa et al. (SOTTOCASA, G.L., KUYLENSTIERNA, B., ERNSTER, L., and BERGSTAND, A. (1967) J. Cell Biol. 32, 415-438), both malic enzymes were associated with the inner membrane-matrix space. Sonication solubilized the two malic enzymes along with the matrix space marker enzymes. The NAD(P)+-dependent malic enzyme was purified 100-fold from calf adrenal cortex mitochondria. The final preparation was free of malic dehydrogenase, fumarase, the strictly NADP+-linked malic enzyme and adenylate kinase. Either Mn24 orMg2+ was required for activity and 1 mol of pyruvate was formed for each mole of NAD+ and NADP+ reduced. The pH optima with NAD+ and NADP+ were 6.5 tp 7.0 and 6.0 to 6.5, respectively. Michaelis-Menten kinetics were observed on the alkaline side. Fumarate, succinate, and isocitrate were positive and ATP and ADP were negative modulators of the regulatory enzyme. The modulators did not influence the stoichiometry and they were not metabolized during the reaction. Under Vmax conditions the ratios for the rate of NAD+:NADP+ reduction were 1.76 and 1.15 at pH 7.4 and 6.0, respectively. The apparent Michaelis constants also differed depending on the pH and the coenzyme. At pH 7.4 (in the presence of 5 mM fumarate) and at pH 6.0 (no fumarate) the Km values for (-)-malate, NAD+, and Mn2+ were 1.7, 0.16, and 0.15 mM, and 0.31, 0.06, and 0.09 mM, respectively. At pH 7.4 (5MM fumarate) and pH 6.0 (no fumarate), the Km values for (-)-malate, NADP+, and Mn2+ were 6.5, 0.62, and 0.59 mM, and 0.68. 0.12, and 0.31 mM, respectively. The apparent Ki values for ATP with NAD+ and NADP+ as coenzyme were 0.42 and 0.27 mM, respectively.
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PMID:The mitochondrial malic enzymes. I. Submitochondrial localization and purification and properties of the NAD(P)+-dependent enzyme from adrenal cortex. 23 89

A calorimetric procedure for determining deltaH, deltaG, deltaS and Keq of a bimolecular reaction with two or more products is described. By using this method the thermodynamic parameters of the phosphofructokinase reaction are determined. At pH 7.0 and 25 degrees C a reaction enthalpy of-6.96kcal/mole was found after correction for the neutralization enthalpy of the buffer and of the enthalpy difference of the magnesium complexes of ATP and ADP, respectively. The free energy of the phosphofructokinase reaction has been found under these conditions to be -3.96kcal/mole.
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PMID:[Microcalorimetric determination of thermochemical parameters of the phosphofructokinase reaction]. 24 Nov 84

Ribulose-diphosphate carboxylase from Thiobacillus novellus has been purified to hemogeneity as observed by polyacrylamide gel electrophoresis and U.V. light observation during sedimentation velocity analysis. The optimum pH for the enzyme with Tris-HCl buffers was about 8.2. Concentrations of this buffer in excess of 80 mM were inhibitory. The apparent Km for RuDP was about 14.8 muM with a Hill value of 1.5, for HCO3- the apparent Km was about 11.7 mM with an n value of 1.18 and for Mg2+ about 0.61 mM. The enzyme was specific for this cation. Relatively high concentrations of either Hg2+ or pCMB were required before significant inhibition was observed. Activity declined slowly during a 4-hr incubation period in either 3.0 M or 8.0 M urea. Incubation for 12 hrs resulted in complete loss of activity which was not prevented by 10 mM Mg2+ and was not reversed by dialysis and subsequent addition of 10 mM cysteine. Polyacrylamide gel electrophoresis revealed a loss of the major band and the appearance of 2 new bands. SDS polyacrylamide gel electrophoresis gave an average M.W. of 73500 +/- 2500 for the slower moving band and 12250 +/- 2500 for the faster moving. However, incubation in urea for up to 40 hrs revealed a decrease in the M.W. of the slower moving band to about 60000. The Ea for the enzyme was calculated to be about 18.85 kcal mole-1, with the possibility of a "break" between 40 and 50 degrees C. The Q10 was 3.07 between 20 and 30 degrees C whereas between 30 to 40 degrees C it was 3.31. Only phosphorylated compounds caused significant inhibition of enzyme activity. They included ADP, FDP, F6P, G6P, PEP, 6PG, 2-PGA, R1P, R5P, and Ru5p.
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PMID:Properties and regulation of ribulose diphosphate carboxylase from Thiobacillus novellus. 24 94

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