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 ATPase activity of rabbit isolated renal tubule segments was measured using a microtechnique in which the hydrolysis of ATP was enzymatically coupled to the appearance of an alkali-converted, highly fluorescent form of nicotinamide adenine dinucleotide. The methods are simple, reproducible, and have a high sensitivity in which picomole quantities of hydrolyzed ATP can readily be measured. Several methods for permeabilizing the cell membranes for measurement of Na+-K+-ATPase activity were evaluated, including osmotic (distilled water or 300 mM imidazole) and temperature (freezing) shock and addition of the nonionic detergent octylglucoside. An octylglucoside concentration of 0.5% was found to cause a maximum activation of the Na+-K+-ATPase and was comparable with that observed when tubules were permeabilized by exposure to distilled water and freezing. Incubation of tubules in 300 mM imidazole was less effective in permeabilizing the cell membranes. In all subsequent studies, the cells were permeabilized by exposure to distilled water and freezing as done by others. The methods were used to assay for the basal levels of Na+-K+-ATPase in the superficial proximal convoluted tubule, the superficial proximal straight tubule, and the cortical collecting tubule and were found to average 44.9 +/- 6.3, 26.4 +/- 2.4, and 11.8 +/- 2.2 pmol ADP X mm-1 X min-1, respectively. Furthermore, elevation of plasma mineralocorticoids by daily injections of deoxycorticosterone acetate (2 mg X kg-1 X day-1) for 4-15 days caused a doubling in the Na+-K+-ATPase activity of the cortical collecting duct, confirming the results of others. The methods presented can easily be adapted for microanalysis of other ATPases.
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PMID:Micromethodology for measuring ATPase activity in renal tubules: mineralocorticoid influence. 609 64

Maintenance of cell calcium homeostasis and transepithelial transport of this cation require its extrusion from the cell against a steep electrochemical gradient. Because it has been proposed that a membrane Ca-ATPase activated by micromolar concentrations of Ca2+ prevailing in the cell participates in these processes, we attempted in this study to determine whether such an enzyme is present in the rabbit nephron. A magnesium-dependent ATPase, maximally activated by Ca2+ (Ca-Mg-ATPase) concentrations between 1.1 and 2.3 microM (apparent Km = 0.3-0.4 microM), was found in all segments of the nephron. Ca-Mg-ATPase (pmol.mm-1.h-1) was highest in the distal convoluted tubule (243) and cortical collecting tubule (208), intermediate in the proximal convoluted tubule (140) and medullary thick ascending limb of Henle's loop (135), and lower in the pars recta (97), cortical thick ascending limb (50), and medullary collecting tubule (51). The enzyme was insensitive to ouabain and vanadate, but was inhibited by ruthenium red in a dose-dependent manner (Ki congruent to 2.10(-6) M). Sodium azide, an inhibitor of mitochondrial ATPase, did not affect Ca-Mg-ATPase, suggesting that the enzyme was located in the plasma membrane. The Ca-Mg-ATPase activity measured in most segments of the rabbit nephron in this study appears sufficient to account in theory for the active component of the unidirectional (lumen-to-bath) calcium flux found in the corresponding region of the nephron with in vitro single tubule microperfusion techniques.
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PMID:High-affinity Ca-Mg-ATPase along the rabbit nephron. 617 29

Na-K-ATPase activity was determined in 10 segments of the rat nephron using a fluorometric microassay method [4]. The enzyme activity showed three peaks (greater than 200 pmol ADP min-1 mm-1) along the nephron of normal rats. These peaks were in the S1 portion of the proximal tubule, the medullary thick ascending limb from the inner stripe and the distal convoluted tubule. Feeding the rats a low potassium diet for 8 weeks produced a significant decrease in Na-K-ATPase activity in the cortical collecting duct, but no significant change in this enzyme in any other segment. The low potassium diet did not produce a significant change in Mg-ATPase in any nephron segments. We conclude that Na-K-ATPase activity along the rat nephron shows a pattern that is qualitatively similar to that seen in the rabbit nephron [4]. However, quantitatively the Na-K-ATPase activity in the rat nephron is greater than in the corresponding segments of the rabbit nephron. The results are consistent with the greater rate of glomerular filtration and Na+ reabsorption per rat nephron. Furthermore, our results suggest that the decrease in potassium excretion during potassium deficiency is modulated, at least in part, by the level of Na-K-ATPase activity in the cortical collecting duct.
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PMID:Effect of low potassium-diet on Na-K-ATPase in rat nephron segments. 628 58

The direct effects of acute changes in K+ concentration on HCO-3 (JnettCO2) and volume reabsorption (Jv) were examined in isolated perfused rabbit proximal convoluted tubules (PCT). Increasing ambient K+ concentration from 5 to 8 mM did not change JnettCO2 (94.5 +/- 16.1 vs. 98.8 +/- 17.7 pmol X mm-1 X min-1) or Jv (1.27 +/- 0.15 vs. 1.24 +/- 0.16 nl X mm-1 X min-1). In contrast, reducing ambient K+ concentration from 5 to 2 mM inhibited JnettCO2 by 22% and Jv by 29%. Reducing luminal K+ concentration from 5 to 0 mM with constant bath K+ concentration at 5 mM did not affect JnettCO2 or Jv. Further reductions in bath K+ concentration to 0.5 and 0 mM showed a similar dependence of both fluxes on K+ concentration. Half-maximum inhibition of JnettCO2 was obtained at 1.1 mM ambient K+ concentration and of Jv at 0.85 mM. At zero bath K+ concentration JnettCO2 was 6.6 +/- 2.5 pmol X mm-1 X min-1 and Jv was 0.03 +/- 0.04 nl X mm-1 X min-1. To determine whether this rate of acidification was significantly different from zero, we examined the ability of the PCT to generate tCO2 concentration gradients with zero bath K+ concentration at slow perfusion rates. The tCO2 concentration gradient generated (0.94 mM) was not different from that found when the perfusate was inserted directly into the collection pipette in the absence of a tubule (0.71 mM). These data are consistent with the view that HCO-3 reabsorption is totally dependent on the Na+-K+-ATPase pump system.
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PMID:Effect of potassium concentration on bicarbonate reabsorption in the rabbit proximal convoluted tubule. 629 11

HCO-3 transport (JHCO-3) in early juxtamedullary proximal convoluted tubules isolated from infant rabbits during the 1st 3 wk of life is about one-third that in tubules obtained from adults. A rapid increase in transport ensues during wk 4 through 6, so that near-mature levels are attained by the end of this time. Because the pattern for development of glucose absorption was similar and because both HCO-3 and glucose absorption are driven by the lumen-to-cell Na+ flux, the activity of Na-K-ATPase (the Na+-extruding pump) was considered to be a critical mediator. A kinetic microassay (which couples ATP hydrolysis to NADH oxidation) allowed the measurement of Na-K-ATPase and ouabain-insensitive ATPase on the same tubular segment. Three to nine early juxtamedullary proximal convoluted tubules were obtained after collagenase treatment of the kidney and four to six rabbits were studied at each week of life. The mean activity of Na-K-ATPase during the 1st wk of life was 44.5 +/- 3.5 pmol X min-1 X mm-1, one-third of the adult level. During an interim period of development (2-6 wk), enzyme activity gradually reached 60% of adult levels (76.3 +/- 3.0 at 6 wk), while transport of HCO-3 and glucose, studied previously in other animals, attained mature rates. Only in the 7th wk did the enzyme activity reach that of the adult (106.8 +/- 6.8 in wk 7 vs. 128.4 +/- 14.0 in adult rabbits).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Development of solute transport in rabbit proximal tubule. III. Na-K-ATPase activity. 633 Nov 74

To determine the number of Na-K-ATPase units and the enzyme's turnover rate along the rabbit nephron, the specific binding of [3H]ouabain and the Na-K-ATPase activity were measured in single nephron segments microdissected from collagenase-treated kidneys. The highest density of Na-K-ATPase (20-30 fmol X mm-1) was found in the distal convoluted tubule and the medullary thick ascending limb. Binding was intermediate (10 fmol X mm-1) in the proximal convoluted tubule and connecting tubule, and it was lowest (2-7 fmol X mm-1) in the pars recta, the cortical thick ascending limb, and the collecting tubule. In the medullary thick ascending limb, Scatchard analysis of the specific [3H]ouabain binding indicated a dissociation constant of 1.8 microM. The pump activity was proportional to the number of catalytic units, indicating that the maximal turnover rate of Na-K-ATPase (2,000 ATP molecules per minute per ouabain binding site) was similar in the various segments of the nephron. The method developed for quantitating [3H]ouabain binding is technically simple enough to permit simultaneous measurement of the enzyme in large numbers of tubules and sufficiently sensitive to determine the number of Na-K-ATPase units in each region of the nephron.
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PMID:Quantitation of [3H]ouabain binding and turnover of Na-K-ATPase along the rabbit nephron. 633 Dec

Previous studies have shown that Na-K-ATPase activity in the cortical collecting tubule of the rabbit increases significantly with mineralocorticoid stimulation and decreases significantly with adrenalectomy. The present study examined the effects of 10(-4) M ouabain on K secretion in the isolated perfused cortical collecting tubules from normal, adrenalectomized, and mineralocorticoid-stimulated animals. Potassium secretion was similar in the tubules from adrenalectomized (3.42 +/- 0.53 pmol X mm-1 X min-1) and from normal rabbits (3.38 +/- 0.36 pmol X mm-1 X min-1). K secretion was greater (15.1 +/- 3.0 pmol X mm-1 X min-1) in tubules from animals receiving 11-deoxycorticosterone acetate (DOCA). Ouabain inhibition of K secretion was 74% for the adrenalectomized group, 86% for the normal group, and 98% for the DOCA-treated group. The degree of inhibition was statistically equivalent among the three groups and not statistically different from complete inhibition of K secretion. Ouabain had no effect on the lumen-positive transepithelial voltage of the cortical collecting tubules from adrenalectomized rabbits but reduced the lumen-negative voltage in tubules from normal rabbits and reversed the polarity of the transepithelial voltage in cortical collecting tubules from DOCA-treated animals. Thus adrenal mineralocorticoids may be necessary for maximal K secretion by the cortical collecting tubule, but they are not essential to maintain K secretion during "normal" K intake. In all groups K secretion is totally dependent on Na-K-ATPase. The lumen-positive transepithelial voltage from DOCA-treated animals after addition of ouabain suggests an additional effect of mineralocorticoid to stimulate secretion of cations (protons) or reabsorption of anions.
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PMID:Effect of ouabain on K secretion in cortical collecting tubules from adrenalectomized rabbits. 649 87

We have previously demonstrated that significant luminal acidification in the K-replete rabbit inner stripe of the outer medullary collecting duct (OMCDi) occurs via a renal H-K-adenosinetriphosphatase (H-K-ATPase) sensitive to several of the gastric H-K-ATPase inhibitors. To investigate further the mechanism of K-dependent luminal acidification in K-replete OMCDi, we examined the effects of luminal K removal, luminal addition of Ba in the presence and absence of luminal 5.0 nM bafilomycin A1 (BAF), and basolateral addition of Ba on net bicarbonate flux (JtCO2, pmol.mm-1.min-1) and transepithelial voltage (VT, mV). Removal of K from the perfusate inhibited JtCO2 by 74% (13.4 +/- 4.0 for control, 3.5 +/- 1.4 pmol.mm-1.min-1 for experimental, P < 0.05) and was statistically equivalent to the degree of inhibition previously observed under identical experimental conditions by either 10 microM Sch-28080 or 10 microM A-80915A. Approximately 50% inhibition of JtCO2 was observed following luminal application of 2.0 mM Ba2+, and the degree of inhibition was statistically equivalent regardless of whether BAF was present (12.2 +/- 2.7 for control, 6.0 +/- 1.4 pmol.mm-1.min-1 for 2.0 mM Ba2+, P < 0.05; 9.6 +/- 1.2 for control with 5 nM BAF, 5.7 +/- 1.3 pmol.mm-1.min-1 for 2.0 mM Ba2+ with 5 nM BAF, P < 0.05). Additionally, increasing the luminal concentration of Ba2+ from 2.0 to 4.0 mM resulted in no further inhibition of JtCO2 (8.5 +/- 1.7 for control, 3.9 +/- 1.3 pmol.mm-1.min-1 for 4.0 mM Ba2+, P = 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Luminal acidification in K-replete OMCDi: inhibition of bicarbonate absorption by K removal and luminal Ba. 763 25

1. Myofibrillar ATPase activity, isometric tension (Po) and unloaded shortening velocity (Vo) were determined in single skinned fibres isolated from rat hindlimb muscles during maximal calcium activation at 12 degrees C. In each fibre, myosin heavy chain (MHC) isoforms were identified using electrophoresis and immunocytochemistry. ATPase activity was determined spectrophotometrically from NADH oxidation in a coupled enzyme assay. 2. On the basis of their MHC isoform composition, the fibres (n = 102) were divided into five groups containing the slow isoform, I MHC, or one of the fast isoforms, IIB MHC, IIA MHC, IIX MHC, or a mixture of the latter three. ATPase activity was significantly higher in IIB than in 2X and IIA fibres (0.230 +/- 0.010, 0.178 +/- 0.023 and 0.168 +/- 0.026 nmol mm-3 s-1, respectively). Mixed fibres had intermediate values. ATPase activity in slow fibres was considerably less (0.045 +/- 0.006 nmol mm-3 s-1). 3. The ratio between ATPase activity and Po, i.e. tension cost, was found to be 2.90 +/- 0.09, 2.56 +/- 0.14, 1.89 +/- 0.22, 1.52 +/- 0.13 and 0.66 +/- 0.004 pmol ATP nM-1 mm-1 s-1 in IIB, mixed, IIX, IIA and slow fibres, respectively. All the differences were statistically significant except that between IIA and IIX fibres. 4. Within each group of fibres with the same MHC composition, ATPase activity was found to correlate with Po, but not Vo. However, ATPase activity was found to correlate with Vo when all the fibre types were pooled together. 5. In thirty-seven fast fibres the MLC ratio, i.e. the proportion of the fast alkali light chain isoform, MLC3f, to the amount of the regulatory light chain, MLC2f, was determined. IIB fibres had the highest proportion of MLC3f and IIA fibres, the lowest. 6. A multiple regression analysis, used to distinguish between the effects of MHC and MLC composition, showed that ATPase activity was insensitive to the MLC ratio, whereas it had a significant impact on Vo. 7. The results obtained in this study indicate that in rat skeletal muscle fibres: (a) ATPase activity during isometric contractions and tension cost are strongly dependent on MHC isoform composition, and (b) there is no evidence that the alkali MLC ratio is a determinant of ATPase activity.
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PMID:Myofibrillar ATPase activity during isometric contraction and isomyosin composition in rat single skinned muscle fibres. 770 34

The mesonephros, the precursor of the metanephros, the definitive kidney, is the functional excretory organ in the 12- to 20-day-old rabbit fetus. It is believed to acidify allantoic fluid. To determine whether H+ excretion occurs in the distal nephron, we examined isolated perfused mesonephric collecting tubules by microcalorimetry and pH-sensitive fluorescent dyes. Collecting tubules secreted H+ (absorbed HCO3-) at rates twice those observed in the mature outer medullary collecting duct (OMCD) of the metanephric kidney. H+ secretion was not inhibited by ouabain (18.5 +/- 2.2 vs. 16.7 +/- 4.0 pmol.min-1.mm-1; n = 7, P = NS) but was reversibly inhibited by removing Cl- from the bathing solution (15.1 +/- 2.3 to -0.6 +/- 3.7 to 15.5 +/- 1.1 pmol.min-1.mm-1; n = 5, P < 0.05); luminal application of N-ethylmaleimide (NEM), an inhibitor of the H(+)-ATPase, also inhibited H+ secretion (n = 2). These results suggested that H+ secretion in the mesonephric collecting tubule is mediated by transporters similar to those of the OMCD. To test this hypothesis, we stained collecting tubules from 15-20 day embryos with 6-carboxyfluorescein (6-CF) diacetate to identify intercalated-like cells or perfused them with 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) to measure intracellular pH (pHi). We found that 139 +/- 15 cells/mm concentrated 6-CF or BCECF, consistent with the phenotype of metanephric intercalated cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:H+ secretion in rabbit mesonephric collecting tubule. 781 Jul 6


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