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)

A kinetic assay system which provides reliable measurements of Na-K-ATPase activity on 0.2 to 0.5-mm segments of renal proximal convoluted tubules isolated from collagenase-digested renal cortical slices is described. The use of collagenase digestion provides higher values for Na-K-ATPase, possibly by making the enzyme more accessible to the reaction system. The advantages of a kinetic vs an endpoint assay include the ability to use the same tubule as its own reference for the determination of total, ouabain-sensitive, and ouabain-insensitive ATPase activity. In addition, it allows dose-response studies on the effect of inhibitors on ATPase activity in the same tubule segment.
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PMID:A kinetic assay for Na-K-ATPase activity in isolated renal proximal tubules. 630 32

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

We have previously shown that inositol-1,4,5-trisphosphate (IP3) releases Ca2+ from an intracellular calcium store in permeabilized acinar cells of rat pancreas (H. Streb et al., 1983, Nature (London) 306:67-69). This observation suggests that IP3 might provide the missing link between activation of the muscarinic receptor and Ca2+ release from intracellular stores during stimulation. In order to localize the intracellular IP3-sensitive calcium pool, IP3-induced Ca2+ release was measured in isolated subcellular fractions. A total homogenate was prepared from acinar cells which had been isolated by a collagenase digestion method. Endoplasmic reticulum was separated from mitochondria, zymogen granules and nuclei by differential centrifugation. Plasma membranes and endoplasmic reticulum were separated by centrifugation on a sucrose step gradient or by precipitation with high concentrations of MgCl2. IP3-induced Ca2+ release per mg protein in the total homogenate was the same as in leaky cells and was sufficiently stable to make short separation procedures possible. In fractions obtained by either differential centrifugation at 7000 X g, sucrose-density centrifugation, or MgCl2 precipitation there was a close correlation of Ip3-induced Ca2+ release with the endoplasmic reticulum markers ribonucleic acid (r = 0.96, 1.00, 0.91, respectively) and NADPH cytochrome c reductase (r = 0.63, 0.98, 0.90, respectively). In contrast, there was a clear negative correlation with the mitochondrial markers cytochrome c oxidase (r = -0.64) and glutamate dehydrogenase (r = -0.75) and with the plasma membrane markers (Na+ + K+)-ATPase (r = -0.81) and alkaline phosphatase (r = -0.77) in all fractions analyzed. IP3-induced Ca2+ release was distributed independently of zymogen granule or nuclei content of the fractions as assessed by electron microscopy. The data suggest that inositol-1,4,5-trisphosphate releases Ca2+ from endoplasmic reticulum in pancreatic acinar cells.
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PMID:Effect of inositol-1,4,5-trisphosphate on isolated subcellular fractions of rat pancreas. 633 62

Arterial basement-membrane-like material was isolated from rabbit aortic myomedial cell cultures by sonication and differential centrifugation. Isolated basement-membrane-like material was shown to be free of both cellular and matrix contaminants, on the basis of determinations of DNA, RNA, cholesterol, phosphorus and (Na+ + K+)-activated ATPase, combined with electron microscopy. Amino acid analyses showed that arterial basement-membrane-like material was composed of predominantly non-collagenous amino acids. Evaluated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, reduced basement-membrane-like material comprised six major and about 30 minor components in the Mr range 10 000-600 000. One of the major peptides (Mr 225 000) was disulphide-linked. Periodic acid-Schiff staining of gels indicated that most high-molecular-weight components were glycoproteins. Two-dimensional gel electrophoresis resolved reduced basement-membrane-like material into more than 100 components, with pI from 5 to 7. The disulphide-linked Mr-225 000 peptide appeared heterogeneous, with pI of 5.6-6.0, and was considered to represent fibronectin. All major peptides were of non-collagenous nature, on the basis of their susceptibility to pepsin and resistance to collagenase. Purified myomedial basement-membrane-like material contained collagenous peptides, as indicated by the presence of hydroxyproline and hydroxylysine. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of pepsin-treated and reduced basement-membrane-like material revealed five high-molecular-weight collagenous components appearing in the Mr range 105 000-375 000 relative to type I collagen standards.
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PMID:Arterial basement-membrane-like material isolated and characterized from rabbit aortic myomedial cells in culture. 687 Aug 38

Various procedures to decontaminate and purify M leprae free of host tissue material resulted in total retention of their intracellular ATP and also infectiousness. The ATP content of one million M. leprae cells, isolated from either livers, spleens, or lymph nodes of infected armadillos, or a nude mouse foot pad or a human biopsy specimen, was in the range of 1.17 to 1.40 picograms. Suspensions could be decontaminated with 4% NaOH and all non-bacterial ATP could be eliminated by the combined action of trypsin, chymotrypsin, and collagenase initially, followed by Triton X-100 plus ATPase. These findings further assure that M. leprae are different from M. lepraemurium in that they can withstand even the severest purification procedures that are necessary in order for them to be used for sophisticated biochemical and metabolic studies.
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PMID:Adenosine triphosphate content of Mycobacterium leprae: effect of purification procedures. 704 15

Calcium-tolerant myocytes were isolated from adult rat ventricles by successive perfusion and incubation with buffer containing collagenase and hyaluronidase. Greater than 70% of the cells excluded trypan blue, maintained normal morphology, and contracted in response to an externally applied electric field. We have characterized metabolic defects present in isolated calcium-tolerance myocytes when exposed to low concentrations of extracellular calcium under aerobic and anaerobic conditions. In control cells exposed to 1.25 mM Ca2+, the following metabolic parameters were measured (in mumol/g protein): adenosine triphosphate (ATP) 28.8 +/- 3.3, creatine phosphate (CrP) 49.1 +/- 7.5, intracellular Na+ 37.7 +/- 8.1, intracellular K+ 352.9 +/- 49.3, cellular Ca2+ 12.3 +/- 1.8, as well as rate of protein synthesis 0.34 +/- 0.03 mumol . g protein-1 . h-1. In aerobic cells incubated in medium without added Ca2+, the corresponding values (in mumol/g protein) were ATP 27.9 +/- 4.4, CrP 25.3 +/- 4.3, intracellular Na+ 130.9 +/- 23.1, intracellular K+ 217.2 +/- 32.0, cellular Ca2+ 3.9 +/- 1.0, and rate of protein synthesis 0.09 +/- 0.02 mumol . g protein-1 . h-1. These data indicated major metabolic aberrations in myocytes exposed to medium low in Ca2+ (less than 10 microM). Metabolic depression was most severe in cells incubated in the absence of both Ca2+ and O2. It is postulated that Ca2+ removal resulted in an increase in Na+ and K+ permeability, causing a net gain of intracellular Na+ and loss of intracellular K+. These ionic shifts might stimulate the activity of membrane-associated Na+-K+-ATPase, accounting for lower levels of CrP.
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PMID:Effects of extracellular calcium removal and anoxia on isolated rat myocytes. 711 49

Extracellular matrix vesicles from rat alveolar bone were isolated by collagenase digestion and differential centrifugation. Further purification was performed by discontinuous sucrose density gradient centrifugation. Control tissues, kidney and liver, were processed according to the same procedures. Sucrose density gradient centrifugation of bone matrix vesicles revealed two peaks of enzymatic activity: "light" and "heavy" vesicle-enriched fractions. Electron micrographs revealed a higher degree of purification of the "light" rather than the "heavy" vesicle-enriched fraction. This coincided with the high levels of enzymatic activity detected in this fraction. Preparations obtained from kidney and liver had significantly lower levels of activity of alkaline phosphatase and ATPase as compared to the bone matrix vesicle fractions. There were also differences in the positions of enzyme activity peaks in the sucrose gradient fractions from the three tissues studied. Electron microscopic examination of kidney and liver fractions revealed structures larger than the purified bone matrix vesicles. In addition no electron-dense material was found within organelles from kidney and liver and they were studded with numerous ribosomes. Our observations indicate that the present method of isolation and purification yields fractions of matrix vesicles which are specific to bone and are significantly different from those obtained from kidney and liver.
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PMID:Purification and further characterization of isolated matrix vesicles from rat alveolar bone. 734 98

In preparation for pulse-chase autoradiography experiments and studies of cell surface changes of relevance to plasma membrane biogenesis, we have prepared a cell suspension from the salt gland of ducklings. The method used was a modification of previous methods used for pancreas and salivary gland and included digestions with collagenase and hyaluronidase, divalent cation chelation, and dispersion by gentle pipetting. Yields were 1.13 X 10(7) cells/g gland, and cell recovery was 45% by DNA assay. Recovery of Na,K-ATPase, a marker for salt gland secretory cells was 40--47%. Cell viability was strongly indicated by trypan blue exclusion and 3H-leucine incorporation. Transmission and scanning electron microscopy revealed that most cells retained ultrastructural features characteristic of the intact gland. Smaller cells (3--8 micrometers in diameter), exhibiting few surface microvilli and relatively few cytoplasmic organelles, likely represented the undifferentiated, peripheral cells from the tips of secretory tubules. Larger cells (5--10 micrometers in diameter), exhibiting prominent surface membrane folds enclosing numerous mitochondria, likely represented the functional, secretory cells of the salt gland tubules in various stages of differentiation. The surface folds presented as microvilli and microplicae in scanning electron micrographs.
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PMID:Preparation and characterization of single cells from the avian salt gland. 742 16

Nitric oxide (NO) is a messenger molecule that is produced from L-arginine by NO synthase (NOS). Some NOS isoforms are present in cells constitutively, whereas others can be induced by cytokines. Recent evidence suggests that NO inhibits intracellular pH regulation by the vacuolar H(+)-adenosinetriphosphatase (ATPase) in macrophages, which contain an inducible form of NOS. The vacuolar H(+)-ATPase is involved in proton secretion in intercalated cells in the collecting duct. We have therefore examined the effect of NO on bafilomycin-sensitive H(+)-ATPase activity in individual cortical collecting ducts (CCD) microdissected from collagenase-treated kidneys of normal rats using a fluorometric microassay. Incubation of CCD with the NO donors, sodium nitroprusside (0.1 and 1 mM) or 3-morpholino-sydnonimine hydrochloride (SIN-1, 30 microM), caused a dose-dependent decrease in H(+)-ATPase activity. Incubation of CCD with lipopolysaccharide (LPS) and interferon-gamma, which induces NOS in macrophages, decreased H(+)-ATPase activity by 85%. This effect was prevented by simultaneous incubation with N omega-nitro-L-arginine, a competitive inhibitor of NOS, indicating that the decrease in H(+)-ATPase activity was caused by NO production. Incubation with 8-bromo-guanosine 3',5'-cyclic monophosphate (cGMP) also inhibited H(+)-ATPase activity, suggesting that NO may exert its effect in the CCD via activation of guanylyl cyclase and production of cGMP. Immunohistochemistry using antibodies to the macrophage-type NOS revealed strong labeling of intercalated cells in the CCD, confirming the presence of NOS in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Nitric oxide inhibits bafilomycin-sensitive H(+)-ATPase activity in rat cortical collecting duct. 752 55


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