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 glucose transporter of Trypanosoma brucei procyclic forms was characterized and compared with its bloodstream form counterpart. Measuring the glucose consumption enzymatically, we determined a saturable uptake process of relatively high affinity (Km = 80 microM, Vmax = 4 nmol min-1 10(-8) cells), which showed substrate inhibition at glucose concentrations above 1.5 mM (Ki = 21 mM). Control experiments measuring deoxy-D-[3H]Glc uptake under zero-trans conditions indicated that substrate inhibition occurred on the level of glycolysis. Temperature-dependent kinetics revealed a temperature quotient of Q10 = 2.33 and an activation energy of Ea = 64 kJ mol-1. As shown by trans-stimulation experiments, glucose uptake was stereospecific for the D isomer, whereas L-glucose was not recognized. Inhibitor studies using either the uncoupler carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone (5 microM), the H+/ATPase inhibitor N,N'-dicyclohexylcarbodiimide (20 microM), the ionophor monensin (1 microM), or the Na+/K+-ATPase inhibitor ouabain (1 mM) showed insignificant effects on transport efficiency. The procyclic glucose transporter was subsequently enriched in a plasma-membrane fraction and functionally reconstituted into proteoliposomes. Using Na+-free conditions in the absence of a proton gradient, the specific activity of D-[14C]glucose transport was determined as 2.9 nmol min-1 (mg protein)-1 at 0.2 mM glucose. From these cumulative results, we conclude that glucose uptake by the procyclic insect form of the parasite occurs by facilitated diffusion, similar to the hexose-transport system expressed in bloodstream forms. However, the markedly higher substrate affinity indicates a differential expression of different transporter isoforms throughout the lifecycle.
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PMID:Glucose uptake occurs by facilitated diffusion in procyclic forms of Trypanosoma brucei. 861 69

1. Myofibrillar ATP consumption and isometric tension (P0) were determined in chemically skinned skeletal muscle fibres from human rectus abdominis and vastus lateralis muscle. Fibres were classified in four groups (I, IIA, IIB, IIA/B or mixed) based on myosin heavy chain composition. 2. ATP consumption (+/- S.E.M.) at 20 degrees C varied from 0.41 +/- 0.06 mmol l-1 s-1 in type IIB fibres (n = 5) to 0.10 +/- 0.01 mmol l-1 s-1 in type I fibres (n = 13). 3. The ratio between ATPase activity and P0 (tension cost) differed significantly between fast type II and slow type I fibres. At 12 degrees C tension cost was lower than the values found previously in corresponding fibre types in the rat. 4. The relative increase in ATPase activity for a 10 degrees C temperature change (Q10), determined in the range from 12 to 30 degrees C, was temperature independent and amounted to 2.60 +/- 0.06. The increase in P0 with temperature was smaller and declined when the temperature increased. 5. From these measurements, estimates were obtained for the maximum rate of isometric ATP consumption and force development at muscle temperature in vivo (35 degrees C).
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PMID:Myofibrillar ATPase activity in skinned human skeletal muscle fibres: fibre type and temperature dependence. 878 97

Cardiac myocyte Ca transport systems, such as sarcoplasmic reticulum Ca-ATPase and sarcolemmal Na/Ca exchange (Na/Ca), are critically dependent on temperature. The purpose of this work was to study the effect of temperature on cellular Ca compartmentation and its exchange characteristics in intact functional neonatal cultured myocytes. The Na/Ca mediated Ca exchange (CaNa/Ca)--including its sarcoplasmic reticulum (SR) and sarcolemmal (SL) contributions, a slow exchange component related to mitochondrial Ca and the La displaceable Ca pool were studied combining isotopic and gas-dissection techniques for membrane isolation. The major findings of this study are: (i) The amount of Ca exchanged through CaNa/Ca is clearly dependent on temperature (Q10 approximately 1.6) in the range studied (17-37 degrees C); (ii) the addition of 1 microM nifedipine does not modify the temperature dependence of CaNa/Ca; (iii) the sarcolemmal bound fraction contributing to CaNa/Ca is not changed by temperature; (iv) the increase in CaNa/Ca with temperature is explained by an increment in the contribution of SR-Ca to CaNa/Ca; (v) a fraction of SR which does not exchange via CaNa/Ca at low temperatures can be released and mobilized by caffeine-this caffeine sensitive fraction is reduced as temperature is increased and is no longer measurable as a separate entity at 37 degrees C; (vi) if we consider (iv) and (v) together, SR content would be temperature dependent with a Q10 of approximately 1.5; (vii) a La displaceable pool, which represents over 66% of the total exchangeable Ca, increases in the range of 22-33 degrees C with a Q10 of 1.25 which is consistent with a pool distribution of 70% SL-bound and 30% SR-derived [Post J.A., Langer G.A. Cellular origin of the rapidly exchangeable calcium pool in the cultured neonatal rat heart cell. Cell Calcium 1992; 13: 627-634]; and (viii) the rate constant for the mitochondrial Ca component increases by 60% from 22 degrees C to 37 degrees C, but Ca content in this organelle is not modified over this temperature range.
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PMID:The effects of temperature upon calcium exchange in intact cultured cardiac myocytes. 916 Jan 62

1. The rate of appearance of inorganic phosphate (Pi) and hence the ATPase activity of rabbit psoas muscle in single permeabilized muscle fibres initially in rigor was measured following laser flash photolysis of the P3-1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP) in the presence and absence of Ca2+. Pi appearance was monitored from the fluorescence signal of a Pi-sensitive probe, MDCC-PBP, a coumarin-labelled A197C mutant of the phosphate-binding protein from Escherichia coli. Fibres were immersed in oil to optimize the fluorescence signal and to obviate diffusion problems. The ATPase activity was also measured under similar conditions from the rate of NADH disappearance using an NADH-linked coupled enzyme assay. 2. On photolysis of NPE-caged ATP in the presence of Ca2+ at 20 degrees C, the fluorescence increase of MDCC-PBP was non-linear with time. ATPase activity was 41 s-1 in the first turnover based on a myosin subfragment 1 concentration of 150 microM. This was calculated from a linear regression of the fluorescence signal reporting 20-150 microM of Pi release. Tension was at 67% of its isometric level by the time 150 microM Pi was released. ATPase activities were 36 and 31 s-1 for Pi released in the ranges of 150-300 microM and 300-450 microM, respectively. The ATPase activity had a Q10 value of 2.9 based on measurements at 5, 12 and 20 degrees C. 3. An NADH-linked assay showed the ATPase activity had a lower limit of 12.7 s-1 at 20 degrees C. The response to photolytic release of ADP showed that the rate of NADH disappearance was partially limited by the flux through the coupled reactions. Simulations indicated that the linked assay data were consistent with an initial ATPase activity of 40 s-1. 4. On photolysis of NPE-caged ATP in the absence of Ca2+ the ATPase activity was 0.11 s-1 at 20 degrees C with no discernible rapid transient phase of Pi release during the first turnover of the ATPase. 5. To avoid the rigor state, the ATPase rate in the presence of Ca2+ was also measured on activation from the relaxed state by photolytic release of Ca2+ from a caged Ca2+ compound, nitrophenyl-EGTA. At 5 degrees C the ATPase rate was 5.8 and 4.0 s-1 in the first and second turnovers, respectively. These rates are comparable to those when NPE-caged ATP was used. 6. The influence of ADP and Pi on the ATPase activities was measured using the MDCC-PBP and NADH-linked assays, respectively. ADP (0.5 mM) decreased the initial ATPase rate by 23%. Pi (10 mM) had no significant effect. Inhibition by ADP, formed during ATP hydrolysis, contributed to the decrease of ATPase activity with time. 7. The MDCC-PBP assay and NPE-caged ATP were used to measure the ATPase rate in single permeabilized muscle fibres of the semitendinosus muscle of the frog. At 5 degrees C in the presence of Ca2+ the ATPase activity was biphasic being 15.0 s-1 during the first turnover (based on 180 microM myosin subfragment 1). Tension was 74% of its isometric level by the time 180 microM Pi was released. During the third turnover the ATPase rate decreased to about 20% of that during the first turnover. 8. ATPase activity in isometric rabbit muscle fibres during the first few turnovers is about an order of magnitude greater than that when a steady state is reached. Possible reasons and the consequences for understanding the mechanism of muscular contraction are discussed.
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PMID:ATPase kinetics on activation of rabbit and frog permeabilized isometric muscle fibres: a real time phosphate assay. 917 99

Proton (H+) conductive pathways are suggested to play roles in the regulation of intracellular pH. We characterized temperature-sensitive whole cell currents in mouse bone marrow-derived mast cells (BMMC), immature proliferating mast cells generated by in vitro culture. Heating from 24 to 36 degrees C reversibly and repeatedly activated a voltage-dependent outward conductance with Q10 of 9.9 +/- 3.1 (mean +/- SD) (n = 6). Either a decrease in intracellular pH or an increase in extracellular pH enhanced the amplitude and shifted the activation voltage to more negative potentials. With acidic intracellular solutions (pH 5.5), the outward current was detected in some cells at 24 degrees C and Q10 was 6.0 +/- 2.6 (n = 9). The reversal potential was unaffected by changes in concentrations of major ionic constituents (K+, Cl-, and Na+), but depended on the pH gradient, suggesting that H+ (equivalents) is a major ion species carrying the current. The H+ current was featured by slow activation kinetics upon membrane depolarization, and the activation time course was accelerated by increases in depolarization, elevating temperature and extracellular alkalization. The current was recorded even when ATP was removed from the intracellular solution, but the mean amplitude was smaller than that in the presence of ATP. The H+ current was reversibly inhibited by Zn2+ but not by bafilomycin A1, an inhibitor for a vacuolar type H(+)-ATPase. Macroscopic measurements of pH using a fluorescent dye (BCECF) revealed that a rapid recovery of intracellular pH from acid-load was attenuated by lowering temperature, addition of Zn2+, and depletion of extracellular K+, but not by bafilomycin A1. These results suggest that the H+ conductive pathway contributes to intracellular pH homeostasis of BMMC and that the high activation energy may be involved in enhancement of the H+ conductance.
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PMID:A highly temperature-sensitive proton current in mouse bone marrow-derived mast cells. 922 99

1. The Na+-K+ pump current was studied in smooth muscle cells from mesenteric resistance arteries of guinea-pigs by the use of the perforated patch-clamp technique in the presence of blockers for various ion channels and exchangers. 2. When the Na+ concentration in the pipette solution ([Na+]i) was 50 mM, an increase in the extracellular K+ concentration ([K+]o) from 0 to 10 mM caused an outward current. Both the removal of K+ from the bath solution and the application of 10 microM ouabain abolished this current. Thus, this K+-induced and ouabain-sensitive current was considered to be the Na+-K+ pump current. 3. The amplitude of the Na+-K+ pump current increased as the membrane potential was made more positive until around 0 mV, while the amplitude saturated at more positive potentials than 0 mV. 4. An increase in [K+]o or [Na+]i amplified the Na+-K+ pump current. For [K+]o, the binding constant (Kd) was 1.6+/-0.3 mM and the Hill coefficient (nH) was 1.1+/-0.2 (n = 6). For [Na+]i, Kd was 22+/-5 mM and nH was 1.7+/-0.5 (n = 4-19). 5. The presence of various monovalent cations other than Na+ in the bath solution also evoked the Na+-K+ pump current. The order of potency was K+ >= Rb+ > Cs+ >> Li+. 6. Ouabain inhibited the Na+-K+ pump current in a dose-dependent manner with a Kd of 0.35+/-0.03 microM and an nH of 1.2+/-0.1 (n = 6-8). 7. The Na+-K+ pump current increased as temperature increased. The temperature coefficient (Q10; 26-36 C) was 1.87 (n = 9). 8. In summary the present study characterized for the first time the Na+-K+ pump current in vascular smooth muscle cells by the use of the voltage-clamp method. The use of this method should provide essential information for Na+,K+-ATPase-mediated changes in the cell functions of vascular smooth muscle cells.
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PMID:Sodium-potassium pump current in smooth muscle cells from mesenteric resistance arteries of the guinea-pig. 1043 51

It is well known that ouabain, a specific inhibitor of Na-K ATPase-dependent transport, interferes with renal tubular salt reabsorption. In this study, we employed radiochemical methods to measure the kinetics of [3H]ouabain binding to slices of rabbit renal medulla and high resolution quantitative autoradiography to determine the location and number of cellular binding sites. The kinetics obeyed a simple bimolecular reaction with an association constant of 2.86 +/- 0.63 SD x 10(3) M-1 min-1 and a dissociation constant of 1.46 x 10(-3) min-1, yielding an equilibrium binding constant of 0.51 x 10(-6) M. Binding was highly dependent upon temperature. At a concentration of 10(-6) M, the rate of accumulation between 25 degrees C and 35 degrees C exhibited a Q10 of 1.8. At 0 degree C the rate of ouabain dissociation was negligible. The specificity of binding was demonstrated with increasing potassium concentrations. At a concentration of 1 microM, 6 mM, and 50 mM K+ produced a 2.5- and 7-fold decrease, respectively, in the rate of ouabain accumulation observed at zero K+. Binding was completely inhibited by 1 mM strophanthin K. The major site of ouabain binding was the thick ascending limb; little or no binding was observed in thin limbs and collecting ducts. Moreover, binding was confined to the basolateral membranes. From autoradiographic grain density measurements, it was estimated that each cell contains over 4 x 10(6) ouabain binding sites or Na-K ATPase molecules. These results taken together with physiological and biochemical observations suggest that Na-K ATPase plays a key role in salt reabsorption by this segment.
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PMID:Ouabain binding to renal tubules of the rabbit. 1060 38

1. Transport-P is an uptake process for amines in peptidergic neurones of the hypothalamus. It differs from other uptake processes by its anatomical location in post-synaptic neurones, its functional properties and by the structure of its ligands. Transport-P accumulates amines in intracellular vesicles, derives its energy from the electrochemical proton gradient and is linked to vacuolar-type ATPase (V-ATPase). Transport-P is blocked by antidepressants. We have now studied the release of amines following uptake by Transport-P in a cell line of hypothalamic peptidergic neurones. 2. Release of prazosin was not inhibited by the antidepressant desipramine; as Transport-P is blocked by desipramine, this indicated that amines are released by a mechanism which is independent of Transport-P. 3. Release of prazosin was sensitive to temperature and conformed to the Arrhenius equation. Release was minimal in the range 0-25 degrees C but accelerated exponentially at higher temperatures up to 33 degrees C. The activation energy for the release of prazosin is 83.1 kJ x mol(-1), corresponding to a temperature quotient (Q10) value of 3. 4. Release was accelerated by the organic base chloroquine, the ionophore monensin, bafilomycinA1 which inhibits V-ATPase and by increasing extracellular pH. Thus, retention of prazosin requires an intracellular proton gradient which is generated by V-ATPase. 5. Fluorescence microscopy demonstrated that release of BODIPY FL prazosin was temperature dependent and was accelerated by chloroquine and monensin. 6. Thus, following uptake by Transport-P, amines are accumulated in acidified intracellular stores. Their retention in peptidergic neurones requires intracellular acidity. The amines are released by a temperature-dependent process which is resistant to antidepressants.
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PMID:Release of amines from acidified stores following accumulation by Transport-P. 1118 26

Different tissues display distinct sensitivities to defective mitochondrial oxidative phosphorylation (OXPHOS). Tissues highly dependent on oxygen such as the cardiac muscle, skeletal and smooth muscle, the central and peripheral nervous system, the kidney, and the insulin-producing pancreatic beta-cell are especially susceptible to defective OXPHOS. There is evidence that defective OXPHOS plays an important role in atherogenesis, in the pathogenesis of Alzheimer's disease, Parkinson's disease, diabetes, and aging. Defective OXPHOS may be caused by abnormal mitochondrial biosynthesis due to inherited or acquired mutations in the nuclear (n) or mitochondrial (mt) deoxyribonucleic acid (DNA). For instance, the presence of a mutation of the mtDNA in the pancreatic beta-cell impairs adenosine triphosphate (ATP) generation and insulin synthesis. The nuclear genome controls mitochondrial biosynthesis, but mtDNA has a much higher mutation rate than nDNA because it lacks histones and is exposed to the radical oxygen species (ROS) generated by the electron transport chain, and the mtDNA repair system is limited. Defective OXPHOS may be caused by insufficient fuel supply, by defective electron transport chain enzymes (Complexes I - IV), lack of the electron carrier coenzyme Q10, lack of oxygen due to ischemia or anemia, or excessive membrane leakage, resulting in insufficient mitochondrial inner membrane potential for ATP synthesis by the F0F1-ATPase. Human tissues can counteract OXPHOS defects by stimulating mitochondrial biosynthesis; however, above a certain threshold the lack of ATP causes cell death. Many agents affect OXPHOS. Several nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit or uncouple OXPHOS and induce the 'topical' phase of gastrointestinal ulcer formation. Uncoupled mitochondria reduce cell viability. The Helicobacter pylori induces uncoupling. The uncoupling that opens the membrane pores can activate apoptosis. Cholic acid in experimental atherogenic diets inhibits Complex IV, cocaine inhibits Complex I, the poliovirus inhibits Complex II, ceramide inhibits Complex III, azide, cyanide, chloroform, and methamphetamine inhibit Complex IV. Ethanol abuse and antiviral nucleoside analogue therapy inhibit mtDNA replication. By contrast, melatonin stimulates Complexes I and IV and Gingko biloba stimulates Complexes I and III. Oral Q10 supplementation is effective in treating cardiomyopathies and in restoring plasma levels reduced by the statin type of cholesterol-lowering drugs.
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PMID:Mitochondrial medicine--molecular pathology of defective oxidative phosphorylation. 1131 62

Effects of Cu2+ on a non-specific conductance and H+-ATPase activity in the plasma membrane of the freshwater alga Nitella flexilis L. Agardh was studied using a conventional microelectrode voltage-clamp technique. We show that a Cu2+-induced increase in the non-specific conductance is related to the formation of pores in the plasma membrane. Pore formation is the result of unidentified chemical reactions, since the Q10 for the rate of increase of conductance over time was about 3. Various oxidants and antioxidants (10 mmol/l H2O2, 10 mmol/l ascorbate, 100 microg/ml superoxide dismutase, and 100 microg/ml catalase) did not alter Cu2+-induced changes in the plasma membrane conductance, suggesting that the effect of Cu2+ was unrelated to peroxidation of plasma-membrane lipids. In contrast, organic and inorganic Ca2+-channel antagonists (nifedipine, Zn2+, Cd2+, Fe2+, Ni2+) inhibited the Cu2+-induced non-specific conductance increase. This suggests that changes in Ca2+ influx underlie this effect of Cu2+. Decreasing the pH or the ionic strength of external solutions also inhibited the Cu2+-induced plasma-membrane conductance increase. Copper was also found to inhibit plasma-membrane H+-ATPase activity with half-maximal inhibition occurring at about 5-20 micromol/l and full inhibition at about 100-300 micromol/l. The Hill coefficient of Cu2+ inhibition of the H+-ATPase was close to two.
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PMID:Characteristics of non-specific permeability and H+-ATPase inhibition induced in the plasma membrane of Nitella flexilis by excessive Cu2+. 1152 15


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