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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)

A new method for determination of 32Pi-ATP and [14C]ADP-ATP exchange rates is described. It is based upon separation of nucleotides and Pi by thin-layer chromatography on commercial aluminum or plastic sheets precoated with silica gel. The method permits avoiding special procedures for stopping of the reaction and for preparation of aliquots for thin-layer chromatography separation. It also allows to separate all adenine nucleotides and Pi in one chromatography procedure, and to work with a double label. The volume of the reaction mixture was 50-100 microliters. Aliquots (2-6 microliters) of the reaction mixture were taken at various moments without stopping the reaction and were layered immediately on a heated silica gel sheets. The nucleotides and Pi were separated in a solvent system which consisted of dioxane, isopropanol, 25% ammonia, and water (4:2:3:4, v/v). The nucleotide spots were detected in ultraviolet light, cut out, and their radioactivity was measured with a liquid scintillation counter. The method for measurement of kinetics of exchange reactions catalyzed by reconstituted into liposomes H+-ATPase complexes from beef heart mitochondria and high plant chloroplasts, is described.
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PMID:Microquantitative determination of Pi-ATP and ADP-ATP exchange kinetics using thin-layer chromatography on silica gel. 631 Oct 50

Acute and sustained hyperammonemia in mice resulted in a decrease of the transition temperature of Arrhenium plots of synaptosomal (Na+-K+)ATPase. The activation energies in both phases of the plots were increased. "In vitro" addition of ammonia produced similar changes. This seems to indicate that ammonia alters the physical properties of synaptosomal membranes. The "in vitro" interaction of ammonia and ethanol at the membrane level was also investigated. Both agents together produced a further shift in the transition temperature and affected the activation energies. The relevance of these findings regarding the mechanism of ammonia toxicity and the protective effect of ethanol thereon is discussed.
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PMID:Effects of ammonia on synaptosomal membranes. 632 65

The human large intestine absorbs Na+, Cl- and water from its lumen and secretes HCO-3 and some K+. The primary event in absorption is thought to be the active transport of Na+ ions out of the cell and across the baso-lateral cell membrane, by the energy requiring Na+-K+ ATPase. This leads in turn to Na+ entry into the cell via its luminal border and the creation of a potential across the mucosa which drives the transport of other ions. Cl- is coupled to HCO-3 secretion through a common carrier and K+ enters the intestinal lumen partly through an active secretory pathway. Most ions probably cross the epithelium by both transcellular and paracellular (shunt) pathways, water moving in response to solute transport. However the colon is not normally perfused by a saline-bicarbonate solution. It contains an active microflora which ferment 30 g or more of carbohydrate daily, derived from diet and intestinal secretions, with the production of at least 300 mmol of short chain fatty acids (acetic, propionic and butyric acids). About 6 g of urea is also degraded to NH3. These metabolic processes result in the generation of solutes which are then transported across the mucosa and which alter the pattern of water and electrolyte transport significantly. Short chain fatty acids are rapidly absorbed by passive diffusion as the undissociated acids, although anion transport, possibly through a paracellular route, is also feasible. Their absorption leads to the accumulation in the lumen of HCO3, a rise in pH, fall in pCO2 and stimulation of Na+ and water transport. The effect on Na+ transport is thought to indicate the presence of a Na+/H+ exchange in the cell membrane. The amounts of these organic solutes produced in the colon each day are probably greater than the total numbers of inorganic ions such as Na+, K+, Cl- and HCO-3 and as such must be taken into account in any understanding of overall transport processes in the large intestinal epithelium.
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PMID:Colonic absorption: the importance of short chain fatty acids in man. 637 78

The H+-translocating ATPase from rat liver mitochondria can be disaggregated selectively to yield two distinct, stable complexes of the rutamycin-insensitive ATPase. The two ATPase complexes can be purified to homogeneity by zone sedimentation in a glycerol gradient. Based on their electrophoretic mobility in 5% polyacrylamide gels, the aggregates have been designated as type I (Rf = 0.49) ATPase and type II (Rf = 0.56) ATPase. These two complexes of the ATPase differ in ATP hydrolytic activity, in stability, in mobility on 5% polyacrylamide gel electrophoresis, in subunit composition, and in ability to reassociate with submitochondrial particles which are highly depleted in ATPase activity. The type II ATPase is similar to the F1-ATPase, but the type I ATPase contains a 26.5-kilodalton subunit not present in the type II enzyme. This 26.5-kilodalton subunit is equimolar with the gamma subunit of the ATPase (based on Coomassie blue dye binding); its presence seems to be correlated to the altered properties of the type I ATPase. Type I ATPase reconstitutes rutamycin-sensitive ATPase activity in submitochondrial particles treated with trypsin, urea, ammonia, and 1.5% silicotungstic acid. The type II ATPase does not reconstitute rutamycin-sensitive ATPase activity in these ATPase-depleted submitochondrial particles unless it is supplemented with the 26.5-kilodalton subunit isolated from the type I ATPase. The 26.5-kilodalton protein has thus been functionally identified as important for the binding of the ATPase to the membrane by providing a direct link to the membrane or by binding to the ATPase putting it in an appropriate conformation for binding.
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PMID:Selective disaggregation of the H+-translocating ATPase. Isolation of two discrete complexes of the rutamycin-insensitive ATPase differing in mitochondrial membrane-binding properties. 645 Feb 7

Three apparently different modifications of submitochondrial particles (SMP) or ATP synthase preparations (complex V) inhibit oxidative phosphorylation and ATP-32Pi exchange activities, all of which are reversible by addition of mono- or dithiols. (a) Triphenyltin chloride inhibits ATP synthesis and hydrolysis without uncoupling. The inhibition by triphenyltin chloride is reversible by addition of beta-mercaptoethanol, dithiothreitol, or dihydrolipoamide. (b) Factor B is a water-soluble protein of Mr (11-12) X 10(3), contains a vicinal dithiol, and is required for energy transfer to and from F1-ATPase when tested with SMP-rendered factor B deficient by extraction with ammonia-ethylenediaminetetraacetic acid (EDTA) (AE-SMP). Treatment of factor B with mono- and dithiol modifiers, such as p-(chloromercuri)benzenesulfonate (PCMPS), Cd2+, or diazenedicarboxylic acid bis(dimethylamide) (diamide), inhibits factor B. This inhibition is reversed by addition to modified factor B of appropriate mono- and dithiol compounds. Preparations of AE-SMP are partially F1 deficient and partially uncoupled. The uncoupling can be repaired completely by addition of factor B or low levels of oligomycin, or to a large extent by addition of F1-ATPase + oligomycin sensitivity conferring protein. (c) SMP, AE-SMP, and complex V can be completely uncoupled by treatment at 30 degrees C with phenylarsine oxide, Cd2+, diamide, PCMPS, monobromobimane, and mono- and bifunctional maleimides. The uncoupling by these reagents is potentiated by membrane energization. Uncoupling by diamide is greater than or equal to 80% reversed by dihydrolipoamide or beta-mercaptoethanol, the former being much more potent. Dithiothreitol and dithioerythritol are poorly effective.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Thiols in oxidative phosphorylation: inhibition and energy-potentiated uncoupling by monothiol and dithiol modifiers. 647 76

The water content and amounts of sodium, potassium and chloride were measured in the brains of normal rats, rats with PCA, normal rats fed ammoniated cationic exchange resin, and rats with PCA fed the resin. Plasma electrolytes and ammonia levels were also measured, and sodium and chloride spaces were calculated. Rats with PCA showed increased water content, sodium space and chloride space in the brainstem compared to controls. Rats with PCA fed ammoniated resin showed increased chloride content and Na+:K+ ratio in the brainstem, and an increased chloride space in the brainstem. In these rats the chloride spaces in the cerebrum and cerebellum exceeded the sodium spaces. It is concluded that high circulating ammonia levels can in vivo produce ionic shifts which may interfere with nervous function. It is also concluded that increased cytoplasmic osmolarity produced by ammonium ion-induced stimulation of (Na+ + Ka+) ATPase may result in the appearance of swollen astrocytes in conventional electron micrographs.
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PMID:The brain in experimental portal-systemic encephalopathy. II. Water and electrolyte changes. 687 7

The binding of N-acetyl-L-glutamate, the physiological allosteric activator, to rat liver carbamoyl-phosphate synthetase (ammonia) was studied by techniques of rate of dialysis and of ultracentrifugation in the Airfuge. There is one binding site for acetylglutamate per enzyme monomer (Mr 165 000). K+, Mg2+ (free) and ATP were required to demonstrate binding. The concentrations of ATP required indicate that binding of ATPA (the ATP molecule that yields Pi) is needed. HCO-3 was not essential, but it enhanced binding of acetylglutamate. Glycerol also favored binding. Plots of Kd values versus the reciprocal of free Mg2+ and ATP concentrations are linear and indicate that ATPA, K+ and Mg2+ bind before acetylglutamate. In the presence of these ligands and HCO-3, ammonia increased drastically the Kd value for acetylglutamate, whereas in absence of HCO-3 ammonia had little effect. This suggests that acetylglutamate dissociates with the products and explains the higher Km for acetylglutamate in the synthetase (overall) reaction than in the ATPase (partial) reaction. In the absence of ATP acetylglutamate was bound with high affinity if ADP and carbamoyl phosphate were present. ADP or carbamoyl phosphate alone did not promote substantial binding. Binding of acetylglutamate at low concentration was slow; it was accelerated at higher concentrations of the activator. Exchange of bound acetylglutamate with acetylglutamate in solution was fast. A scheme proposed earlier for allosteric activation of the enzyme [Rubio, V., Britton, H. G. and Grisolia, S. (1983) Eur. J. Biochem. (in preparation)] is refined to incorporate the new information. Binding of ATPA, K+ and Mg2+ and formation of 'active CO2' (the central complex) are greatly favored by acetylglutamate.
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PMID:Binding of N-acetyl-L-glutamate to rat liver carbamoyl phosphate synthetase (ammonia). 688 68

Sodium vanadate is a potent inhibitor of Na-K-adenosine triphosphatase. p-Aminohippurate (PAH) and tetraethylammonium accumulation in rat renal cortical slices was inhibited by vanadate in a dose-dependent manner at medium vanadate concentrations from 10(-6) to 10(-3) M. Inhibition was reversible at vanadate concentrations less than 1.5 x 10(-5) M. The slice content of vanadium (7.5-325 micrometer V/g wt. of tissue) was linearly related to medium vanadate concentrations ranging from 10(-5) to 10(-3) M. The ability of slices to generate glucose and ammonia was not impaired by medium vanadate concentrations up to 5 x 10(-4) M, a concentration that maximally inhibited organic ion accumulation. Increasing medium K+ concentrations potentiated vanadate inhibition of PAH accumulation which correlated with inhibition of sodium pump activity, as determined by 42K+ uptake. Intraperitoneal administration of vanadate (1 or 5 mg V/kg) to rats produced a profound diuresis and natriuresis during the 1st hr. Inhibition of PAH accumulation of renal slices from these rats was related to tissue vanadium concentrations. These data suggest that vanadate exerts its action on proximal tubule transport of PAH via inhibition of Na-K-adenosine triphosphatase.
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PMID:Effects of vanadate on organic ion accumulation in rat renal cortical slices. 691 88

Biochemical changes in the air-breathing catfish, Clarias batrachus (Linn.) exposed to a sublethal level of carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) at 0.5 ppm concentration in ambient water for a period of 30 days were assessed. A small reduction in growth rate was observed in the fish treated with 0.5 ppm carbofuran for 60 days although no mortality or any apparent symptom of toxicity could be noted. Studies were carried out on the activities of certain enzymes of intermediary metabolism viz., glucose 6-phosphatase, alkaline phosphatase, acid phosphatase, Na+, K+-ATPase, GOT and GPT in certain vital tissues of the fish exposed to carbofuran (0.5 ppm) for 30 days. Exposure to carbofuran resulted in sharp inhibition of acetylcholinesterase activity in brain of the fish which recovered rather rapidly after terminating pesticide treatment and maintaining the fish in clean freshwater. Ratio of the levels of calcium/phosphorus in serum showed significant diminution in experimental groups of fish compared to controls. Level of ammonia in serum of experimental fish was markedly increased while excretion of ammonia by fish showed concomitant decrease. The bioaccumulation level of the pesticide and its degraded product, 3-hydroxy-carbofuran in liver tissue was measured by gas chromatography. A rationale of the effect of carbofuran on metabolism vis-a-vis toxicity in the fish has been suggested.
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PMID:Certain biochemical responses in the air-breathing catfish Clarias batrachus exposed to sublethal carbofuran. 712 66

1. Some factors determining the distribution of ammonia between hepatocytes and the suspension medium and between mitochondria and cell cytosol were examined. 2. Intracellular [ammonia] of isolated hepatocytes was similar to that in freeze-clamped rat liver. The intra- to extracellular [ammonia] ratio ('ammonia ratio') of cells incubated without added substrates was as great as 70. 3. High ammonia ratios were found only within the physiological range of extracellular [ammonia] i.e. 0.03 mM. At higher external [NH4Cl] the ammonia ration decreased until at 20 mM it approached 1.0. 4. On addition of NH4Cl (10.0 mM) ammonia entered hepatocytes rapidly until at about 2.5 min internal and external ammonia concentration were similar. The final steady state distribution of ammonia was not reached until 60 min, when the internal concentration was slightly higher than that externally. 5. Intracellular [K+] decreased when intracellular [ammonia] increased. The sum of intracellular [K+] plus [NH4(+)] remained approximately constant. 6. In anaerobic cells the high endogenous ammonia ratio was not maintained. 7. The high physiological ammonia ratio was not abolished by ouabain. Thus the (Na+ + K+)-ATPase does nt appear to be responsible for the maintenance of the ammonia ratio. 8. A high ammonia ratio also existed between hepatocyte cytosol and mitochondria. 9. On addition of fructose, which depletes the cells of Pi and adenine nucleotides, the ammonia content of the cells decreased parallel with Pi and ATP. In the presence of fructose much ammonia was removed by the formation of alanine. However, when pyruvate was added to stimulate alanine formation there was no effect on the ammonia distribution. This showed that alanine formation alone was not responsible for decreased intracellular [ammonia] with fructose. 10. Incubation with adenosine led to a large increase in intracellular ATP and ammonia content as well as in the ammonia ratio. Incubation with alanine also led to increased intracellular ammonia production but in this case ammonia was released into the medium and high ammonia ratios did not occur. 11. Some mechanisms for maintenance of a high ammonia ratio between cells and medium are discussed.
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PMID:The distribution of ammonia between hepatocytes and extracellular fluid. 740 49


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