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)

To directly assess the possible role of ADP in muscle fatigue, we have studied the effect of physiological MgADP levels on maximum Ca(2+)-activated isometric force and unloaded shortening velocity (Vus) of single skinned fiber segments from rabbit fast-twitch (psoas) and slow-twitch (soleus) muscles. MgADP concentration was changed in a controlled and well-buffered manner by varying creatine (Cr) in solutions, which also contained MgATP, phosphocreatine (PCr), and creatine kinase (CK). To quantify ADP as a function of Cr added, we determined the apparent equilibrium constant (K') of CK for the conditions of our experiments (pH 7.1, 3 mM Mg2+, 12 degrees C): K' = (sigma [Cr]. sigma [ATP])/(sigma [PCr]. sigma [ADP]) = 260 +/- 3 (SE). In this manner, ADP was altered essentially as occurs during stimulation in vivo but without the concomitant changes in pH and P(i), which affect force and Vus. As ADP (and Cr) was increased, force and Vus decreased in both fiber types; at the highest ADP level used, 200 microM, normalized force was 96.6 +/- 1.7% for psoas (n = 6) and 93.7 +/- 2.8% for soleus (n = 6), and Vus was 80.4 +/- 2.4% for psoas and 91.3 +/- 7.7% for soleus. Diffusion-reaction calculations indicated that radial gradients of metabolite concentrations within fibers could not explain the small effects of ADP on fiber mechanics, and experiments verified that metabolite levels were well buffered within fibers by the CK reaction. Exogenous CK was added to bathing solutions at 290 U/ml, threefold above that necessary to maintain Vus independent of CK concentration; in the absence of PCr and exogenous CK, at least a fourfold increased MgATP was necessary to maintain Vus at the control level. Adenylate kinase activity was not detectable; thus myofibrillar adenosine-triphosphatase and exogenous CK activities were the major determinants of nucleotide levels within activated cells. Cr alone (in absence of PCr and exogenous CK) also decreased force and Vus, presumably by a nonspecific mechanism. Over the physiological range, altered ADP had little or no effect on force or Vus in well-buffered conditions. It is therefore likely that other factors decrease force and Vus during muscular fatigue.
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PMID:Effect of physiological ADP concentrations on contraction of single skinned fibers from rabbit fast and slow muscles. 786 87

In order to assess the relationship between cytosolic [ATP] or [ATP]/[ADP] and the intracellular Na+ concentration ([Na+]i), we have used the phosphate trap 2-deoxy-D-glucose (DG) to alter the high energy phosphate levels in rat cardiomyocytes. Pyruvate-perfused rat hearts were treated with 2 mM DG in the presence of 10IU/l of insulin for 28 min, followed by perfusion with DG without insulin for 60 min. The DG + insulin treatment resulted in dramatic changes in the 31P NMR spectra: phosphocreatine (PCr) and total ATP decreased (to 15 and 35%, respectively) and deoxyglucose-6-phosphate accumulated, with little change in either inorganic phosphate or intracellular pH. These changes corresponded to a decrease in cytoplasmic [ATP] (from 7.6 to 1.8 mM), [ATP]/[ADP] (from 494 to 24) and ATP affinity [A(ATP), by 8.9 kJ/mol] and an increase in [ADP] (five-fold) and free [Mg2+] (two-fold). Subsequent perfusion with DG--insulin resulted in slow recovery of PCr, [ATP]/[ADP] and A(ATP) such that the "low energy" state lasted an additional 16 min during which ATP remained low and constant. There were no detectable changes in the intracellular Na+ content as assessed by shift reagent-aided 23Na NMR at the end of DG + insulin treatment (98 +/- 18%, 28-36 min of the protocol). In addition, there was no change in the Rb+ influx rate as measured by 87Rb NMR at the beginning of insulin washout which was achieved by replacing 20% of the KCl with RbCl ([K+] = 3.76 mM, [Rb+] = 0.94 mM). During DG + insulin treatment the pressure-rate product (PRP) decreased by half and was restored upon insulin washout to 80% of its initial value both in the presence and in the absence of the shift reagent [5 mM Dy (triethylenetetraminehexaacetate)3-]. These data imply that unfavorable thermodynamic [low A(ATP)] and kinetic (low [ATP] and [ATP]/[ADP]) conditions induced by DG treatment do not inhibit Na+, K(+)-ATPase activity. We speculate that during anoxia when changes in [ATP]/[ADP] are comparable to those induced by DG treatment, the observed increase in [Na+]i is not due to inhibition of the Na+ pump by reduced [ATP] or [ATP]/[ADP].
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PMID:Relationships between cytosolic [ATP], [ATP]/[ADP] and ionic fluxes in the perfused rat heart: A 31P, 23Na and 87Rb NMR study. 786 98

Protective effect of alpha-tocopherol on the structure and function of brain cell membranes was investigated by measuring Na+,K(+)-ATPase activity and products of lipid peroxidation (fluorescent compounds) in brain cell membranes obtained from newborn piglets. Four groups of anesthetized, ventilated piglets were studied: five hypoxic piglets and five normoxic piglets were pretreated with free alpha-tocopherol (20 mg/kg/dose i.m.), five additional hypoxic piglets received i.m. placebo and five normoxic piglets served as control. Placebo and alpha-tocopherol were given 48 and 3 h prior to onset of hypoxia. Hypoxic hypoxia was induced and cerebral hypoxia was documented as a decrease in the ratio of phosphocreatine to inorganic phosphate (PCr/P(i)) using 31P NMR spectroscopy. PCr/P(i) decreased from baseline of 2.62 +/- 0.54 to 1.05 +/- 0.27 in alpha-tocopherol-pretreated and from 2.44 +/- 0.48 to 1.14 +/- 0.30 in the placebo-pretreated group during hypoxia. Na+,K(+)-ATPase activity was unchanged in both normoxic and hypoxic alpha-tocopherol-pretreated groups. However, in placebo-pretreated hypoxic group, Na+,K(+)-ATPase activity decreased as compared with control (44.9 +/- 9.7 vs. 61.8 +/- 5.7 mumol P(i)/mg protein/h, P < 0.005). The level of fluorescent compounds increased in placebo-pretreated but not in alpha-tocopherol-pretreated group as compared with control. During hypoxia, serum alpha-tocopherol levels were higher in alpha-tocopherol-pretreated groups as compared with placebo-pretreated hypoxic group. The present data indicates that alpha-tocopherol protects brain cell membranes in newborn piglets from lipid peroxidative damage during tissue hypoxia probably by being incorporated in cell membrane and also as circulating antioxidant.
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PMID:Protective effect of alpha-tocopherol on brain cell membrane function during cerebral cortical hypoxia in newborn piglets. 798 74

Rabbit portal veins were permeabilized using Staphylococcus aureus alpha-toxin, and adenosinetriphosphatase (ATPase) was measured as the formation of [3H]ADP, [3H]AMP, and [3H]adenosine from [3H]ATP in the solution bathing the muscle. The resting ATPase (1.96 +/- 0.15 mM/min, n = 13) is approximately 5-10 times higher than that measured in Triton X-100-permeabilized muscles (0.28 +/- 0.01 mM/min, n = 4), with nucleotide accumulating as ADP, AMP, and adenosine. The ATPase activity is also seen when the intact muscle is incubated in a Krebs solution containing 1 mM MgATP (2.76 +/- 0.10 mM/min, n = 73). This suggests that it is due primarily to an ecto-ATPase. The ectoenzyme is capable of hydrolyzing both ATP and ADP, and in both cases there is a higher rate at 3 than at 1 mM nucleotide. The high resting ATPase compromises the control of nucleotide concentrations within the permeabilized tissue even in the presence of an ATP-regenerating system consisting of phosphocreatine (PCr, 35mM) and creatine kinase (1 mg/ml). Treatment of the intact muscle with the ectonucleotidase inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) followed by alpha-toxin permeabilization and inclusion of sodium azide in subsequent solutions reduces the ecto-ATPase by approximately 70%. Addition of PCr and creatine kinase then results in the maintenance of high [ATP] and low [ADP] in the muscle, and importantly, there are no significant changes in [ATP], [ADP], [adenosine/AMP], or the ADP-to-ATP ratio upon activation of the muscle in pCa 4.5. In general, the force output in high Ca2+ increased as the metabolic profile of the muscle improved. When ATPase was measured as the appearance of [32P]Pi from [32P]PCr and [gamma-32P]ATP, the alpha-toxin-permeabilized muscle subjected to the above treatment showed only approximately 30% higher total ATPase under activated conditions compared with the freeze-glycerinated Triton-treated portal vein. The suprabasal ATPase is similar in both preparations. We conclude that the reduction of the basal ATPase by the DIDS-azide treatment permits both rigorous control of nucleotide contents and accurate measurement of ATPase activity in alpha-toxin-permeabilized smooth muscle.
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PMID:Metabolic characteristics of alpha-toxin-permeabilized smooth muscle. 802 97

This study tests the hypothesis that magnesium, a selective non-competitive antagonist of the NMDA receptor, will attenuate hypoxia-induced alteration in NMDA receptors and preserve MK-801 binding characteristics during cerebral hypoxia in vivo. Anesthetized, ventilated and instrumented newborn piglets were divided into three groups: normoxic controls were compared to untreated hypoxic and Mg(2+)-treated hypoxic piglets. Cerebral hypoxia was induced by lowering the FiO2 to 5-7% and confirmed biochemically by a decrease in the levels of phosphocreatine (82% lower than control). The Mg(2+)-treated group received MgSO4 600 mg/kg over 30 min followed by 300 mg/kg administered during 60 min of hypoxia. Plasma Mg2+ concentrations increased from 1.6 +/- 0.1 mg/dl to 17.7 +/- 3.3 mg/dl. 3H-MK-801 binding was used as an index of NMDA receptor modification. The Bmax in control, hypoxic and Mg(2+)-treated hypoxic piglets was 1.09 +/- 0.17, 0.70 +/- 0.25 and 0.96 +/- 0.14 pmoles/mg protein, respectively. The Kd for the same groups were 10.02 +/- 2.04, 4.88 +/- 1.43 and 8.71 +/- 2.23 nM, respectively. The Bmax and Kd in the hypoxic group were significantly lower compared to the control and Mg(2+)-treated hypoxic groups, indicating a preservation of NMDA receptor number and affinity for MK-801 during hypoxia with Mg2+. The activity of Na+, K+ ATPase, a marker of neuronal membrane function, was lower in the hypoxic group compared to the control and Mg(2+)-treated hypoxic groups. These findings show that MgSO4 prevents the hypoxia-induced modification of the NMDA receptor and attenuates neuronal membrane dysfunction.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Protective effect of MgSO4 infusion on nmda receptor binding characteristics during cerebral cortical hypoxia in the newborn piglet. 803 41

Single turnover experiments were performed on myosin-bound ADP by measuring the time course of incorporation of [3H]ADP following rapid formation of [3H]ATP by photolysis of caged [3H]ATP. Permeabilized rabbit portal veins were incubated in a solution at 20 degrees C with 1 mM MgATP, 20 mM phosphocreatine, 1 mg/ml creatine phosphokinase, and containing [14C]ATP and high specific activity caged [3H]ATP. At variable times following a UV flash, the muscle was frozen, nucleotides were extracted, and the ratio 3H:14C in ADP was compared to that in ATP. At rest, the exchange of bound ADP occurred with a rate constant of 0.004 s-1. When the myosin light chain was about 80% thiophosphorylated, and the muscle was generating maximum isometric force, there appeared a fast phase of ADP exchange (44% of the total) which had a rate constant of 0.2 s-1. The change in rate of ADP exchange on myosin is sufficient to explain the measured increase in ATPase activity upon thiophosphorylation of the myosin light chain. A simple analysis of the data suggests that there is a 50-fold increase in the cycling rate of cross-bridges in the muscle upon phosphorylation under isometric conditions. The fraction of ADP exchanged at 10 s following photolytic release of [3H]ATP was found to be approximately linearly related to the degree of thiophosphorylation of the myosin light chain. This supports the idea that phosphorylation of the light chain causes the transition of myosin from the resting (slow ATPase) cycle into the activated (fast ATPase) cycle, and that the fraction of myosin in the fast cycle is directly determined by the degree of light chain phosphorylation. The data are also consistent with the cooperativity model described previously by Vyas et al.
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PMID:Cross-bridge cycling at rest and during activation. Turnover of myosin-bound ADP in permeabilized smooth muscle. 812 47

The effect of hypoxia on the N-methyl-D-aspartate (NMDA) receptor/ion channel complex in the brain cell membrane of the newborn piglet was studied. Experiments were conducted on newborn piglets, 2-4 days of age, that were anesthetized and mechanically ventilated. Hypoxic hypoxia was induced in the experimental group by lowering the FiO2 to 5-7%. The control group was ventilated under normoxic conditions. Tissue hypoxia was documented biochemically by decreased levels of ATP and phosphocreatine (PCr) in the hypoxic group (52% and 81% lower than the normoxic group, respectively). [3H]MK-801 binding characteristics (Bmax = number of receptors, Kd = dissociation constant) were used as an index of NMDA receptor modification. In hypoxic brains, Bmax decreased from the control level of 1.13 +/- 0.15 pmol/mg protein to 0.68 +/- 0.23 pmol/mg protein (P < 0.01) and the Kd value decreased (reflecting increased affinity) from 9.46 +/- 1.68 nM in the control brains to 4.87 +/- 1.42 nM (P < 0.01) in the hypoxic brains. The Na+,K(+)-ATPase activity, an index of brain cell membrane function, decreased from a control value of 46.5 +/- 0.4 to 40.5 +/- 2.3 mumol inorganic phosphate (Pi) mg protein/h (P < 0.005) during hypoxia. The results of this study indicate that hypoxia in newborn piglets modifies the NMDA receptor in the cerebral cortex, resulting in an increased affinity of the receptor channel. Hypoxia-induced modification of the NMDA ion/receptor complex may be a potential mechanism of cerebral excitotoxicity.
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PMID:Hypoxia-induced modification of the N-methyl-D-aspartate receptor in the brain of the newborn piglet. 817 16

1. Rat small intestinal basolateral membrane vesicles (BLMVs) were prepared and found to be 31% non-vesiculated and 69% vesiculated, 4.9% right side out and 63.8% inside out. 2. Thiamine uptake by BLMVs followed a hyperbolic time course reaching equilibrium after 60-90 min incubation. Uptake was not affected by the transmembrane potential or by the presence or absence of Na+ or K+ in the incubation medium. 3. At concentrations below 1.25 microM, [3H]thiamine was taken up mainly by a saturable mechanism with an apparent Michaelis-Menten constant (Km) = 1.32 microM and maximal flux (Jmax) = 1.93 pmol (mg protein)-1 (4 s)-1. At higher concentrations, a non-saturable mechanism prevailed. 4. Only 29% of [3H]thiamine taken up by the vesicles was membrane bound, the remaining being translocated into the vesicular space. No thiamine phosphoesters could be detected inside the vesicles. 5. In the absence of ATP, the Na(+)-K(+)-ATPase inhibitors ouabain, frusemide and vanadate reduced thiamine uptake by 35, 30 and 15% respectively. 6. In experiments conducted with K+ inside the vesicles and Na+, Mg2+ and ATP outside, the time course of thiamine uptake by BLMVs displayed an overshoot (80-90% increment) at 30 s incubation as compared to controls. When ATP was replaced with phosphocreatine, or when NaCl was replaced with isosmotic amounts of KCl, the overshoot disappeared. 7. The thiamine analogues pyrithiamine, amprolium and 4'-oxythiamine decreased the ATPase-dependent transport of [3H]thiamine by 100, 86 and 31% respectively. 8. These results provide evidence that the transport of thiamine by BLMVs is coupled directly to the hydrolysis of ATP (primary active transport).
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PMID:Thiamine outflow from the enterocyte: a study using basolateral membrane vesicles from rat small intestine. 825 15

Cerebral capillaries represent the responsible structure for the establishment of the BBB. We have demonstrated the presence within both pericytes and endothelial cells of abundant cytoplasmic vesicles. We have noticed the presence of Na+/K(+)-ATPase-, alpha-actin-, phosphocreatine- and clathrin-like molecules within cerebellar capillaries and their microenvironment. These facts suggest the importance of the contractile and transport mechanisms in the blood-brain barrier. We have also demonstrated the close contact between different nervous components of the cerebellar cortex with the basement membrane that surrounds capillary wall. We suggest that these observations represent the morphological evidence of neurogenic control of brain circulation.
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PMID:Molecular and ultrastructural basis of the blood-brain barrier function. Immunohistochemical demonstration of Na+/K+ ATPase, alpha-actin, phosphocreatine and clathrin in the capillary wall and its microenvironment. 829 29

Myofibrillar creatine kinase (CK) serves as one microcompartment of the phosphorylcreatine shuttle by providing ATP as substrate for adenosinetriphosphatase (ATPase). During perinatal heart development, augmentations of myofibrillar ATPase and CK occur in concert with increased contractile performance. The maximal reaction velocity (Vmax) for CK doubles during development in both intact native myofibril and enzyme extracted from myofibril. The absence of alterations in ADP and creatine phosphate substrate Michaelis constants (Km), isoenzyme composition, or total number of -SH groups suggests active site function (Vmax) is influenced indirectly via a subunit domain effect on enzyme conformation.
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PMID:Perinatal enhancement of cardiac myofibrillar creatine kinase activity without change in enzyme Km. 836 67


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