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)

We have fused full length and the carboxyl-half of human MDR1 cDNA with the E. coli lacZ gene via a collagen linker and allowed their expression in yeast Saccharomyces cerevisiae. Using antibodies against beta-galactosidase we partially purified the fusion proteins by immunoprecipitation and show here that the full length fusion protein has ATPase activity. By contrast, the fusion protein containing the carboxyl-half of P-glycoprotein did not show ATPase activity, indicating that both domains of P-glycoprotein are necessary. By treatment of the immunoprecipitated fusion protein with collagenase, P-glycoprotein was released from the beta-galactosidase moiety. The results shown here open the possibility for a large scale purification of P-glycoprotein using this site specifically cleavable fusion protein.
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PMID:Production of a site specifically cleavable P-glycoprotein-beta-galactosidase fusion protein. 136 54

This study was aimed at identifying the renal sites of the antinatriuretic action of insulin by evaluating whether this hormone may alter the function of Na-K-ATPase in specific nephron segments. For this purpose, possible actions of insulin on the rate of 86Rb uptake were evaluated in vitro on single segments of proximal convoluted tubule (PCT), thick ascending limb, and collecting tubule microdissected from collagenase-treated kidneys of normal rats. Results indicate that physiological concentrations of insulin inhibited by 44% the initial rate of ouabain-sensitive 86Rb uptake in the medullary and cortical thick ascending limb, whereas it increased it by 40% in proximal tubules and by 60% in both cortical and medullary collecting tubules. The kinetics and dose dependence of insulin actions were different in the thick ascending limb, the PCT, and the collecting tubule, with the latter less sensitive but displaying an earlier response to insulin than the PCT and the thick ascending limb.
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PMID:Sites of antinatriuretic action of insulin along rat nephron. 163 41

The sequence of Escherichia coli UvrA protein suggests that it may fold into two functional domains each possessing DNA binding and ATPase activities. We have taken two approaches to physically isolate polypeptides corresponding to the two putative domains. First, a 180 base pair DNA segment encoding multiple collagenase recognition sequences was inserted into UvrA's putative interdomain hinge region. This UvrA derivative was purified and digested with collagenase, and the resulting 70-kDa N-terminal and 35-kDa C-terminal fragments were purified. Both fragments possessed nonspecific DNA binding activity, but only the N-terminal domain retained its nucleotide binding capacity as evidence by measurements of ATP hydrolysis and by ATP photo-cross-linking. Together, the two fragments failed to substitute for UvrA in reconstituting (A)BC excinuclease and, therefore, were presumed to be unable to load UvrB onto damaged DNA. Second, the DNA segments encoding the two domains were fused to the beta-galactosidase gene. The UvrA N-terminal domain-beta-galactosidase fusion protein was overproduced and purified. This fusion protein had ATPase activity, thus confirming that the amino-terminal domain does possess an intrinsic ATPase activity independent of any interaction with the carboxy terminus. Our results show that UvrA has two functional domains and that the specificity for binding to damaged DNA is provided by the proper three-dimensional orientation of one zinc finger motif relative to the other and is not an intrinsic property of an individual zinc finger domain.
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PMID:Isolation and characterization of functional domains of UvrA. 182 51

PGE2 production by glomeruli is increased in a variety of glomerular diseases. Potentially, this process may affect mesangial cell protein synthesis and mesangial cell growth. Thus studies have been undertaken, using cultured human mesangial cells, to assess the effects of PGE2 on proline uptake, protein synthesis and cell proliferation. In the presence of 140 mM NaCl, incubation of mesangial cells with 0.01 to 1 microM PGE2 for 72 hours resulted in a marked decrease of 14C proline uptake, but did not modify 14C leucine uptake. Substitution of choline to sodium inhibited 14C proline uptake by 85% which became independent of PGE2, indicating that this PG specifically altered sodium-dependent proline uptake. Inhibition of this component reached 35 to 50% with 1 microM PGE2. The inhibitory effect of PGE2 on sodium-dependent proline uptake required a lag time of 48 hours, and was suppressed by ouabain, an inhibitor of Na+, K+ ATPase activity. PGE2 did not modify the Vmax of the transport system (1.007 vs. 1.023 nmol/mg/min) but increased (P less than 0.01) its Km (1.179 vs. 0.823 mM). 8-bromo-cyclic AMP also inhibited sodium-independent proline uptake, and PGE2 markedly increased cyclic AMP production. Taken together, these results suggested that PGE2 acted via cyclic AMP stimulation. PGE2 under identical conditions (1 microM, 72 hr incubation) produced a decrease in collagen synthesis estimated by the relative rate of collagen production after incubation of mesangial cells with 14C proline (percentage of 14C radioactivity in collagenase-sensitive proteins over total proteins). PGE2 also diminished the intracellular free proline pool. More generally, PGE2 inhibited cell proliferation and cell total proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of prostaglandin E2 on proline uptake and protein synthesis by cultured human mesangial cells. 196 49

Suspensions of renal proximal tubules (RPT) are the in vitro model for many biochemical and physiologic investigations. Inasmuch as there are numerous procedures for tubule isolation and the more commonly used enzymatic procedures may disrupt the basement membrane, there is a need for information comparing the influence of various isolation methods on RPT viability and function in long-term suspension. Rabbit RPT isolated a) enzymatically (ENZ) by in vitro collagenase digestion and Percoll size and density purification, and b) mechanically (MECH) by in vitro iron oxide perfusion and purification by sieving and magnetic removal of glomeruli were compared for viability, morphology, and functional stability during long-term suspension. RPT isolated by ENZ and MECH methods had excellent viability (less than 15% lactate dehydrogenase release), limited lipid peroxidation (less than 0.2 nmol MDA.mg protein-1), and stable nystatin-stimulated oxygen consumption (QO2) (38 and 36 nmol O2.mg protein-1.min-1) throughout 24 h of incubation. Basal QO2 was higher in ENZ than MECH tubules (27 and 19 nmol O2.mg protein-1.min-1, respectively), and was unchanged over 24 h in each preparation. The higher basal QO2 in ENZ tubules was ouabain-sensitive, suggesting an increased rate of Na+,K(+)-ATPase activity in these tubules. Total glutathione content (oxidized + reduced) in ENZ and MECH tubules increased over the 24-h incubation from 8 to 18 nmol.mg protein-1. gamma-Glutamyltranspeptidase (GGT) activity of the RPT homogenates was equivalent in both preparations and stable over time. The ratio of suspension GGT activity to homogenate GGT activity doubled (0.4 to 0.8) during the incubation period. MECH tubules retained their tubule structure during 24 h of incubation whereas the ENZ tubules had a striking loss of tubular morphology over time. These results show that ENZ- and MECH-isolated renal proximal tubule suspensions exhibit similar biochemical properties in long-term incubations but differ in ouabain-sensitive QO2 and the retention of tubular morphology. The loss of tubular morphology and the increase in the rate of Na+,K(+)-ATPase activity in ENZ tubules may be secondary to the disruption of the tubular basement membrane.
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PMID:Differences in enzymatic and mechanical isolated rabbit renal proximal tubules: comparison in long-term incubation. 197 32

We tested the hypothesis that the adenylate cyclase system and Na+, K(+)-ATPase are reciprocally related in rat pancreatic islets. We studied the effect of theophylline, caffeine, and dibutyryl cyclic AMP on Na+, K(+)-ATPase activity in a membrane preparation from collagenase-isolated rat islets. Theophylline, caffeine, or dibutyryl cyclic AMP, in concentrations of 1 mM, all inhibited Na+, K(+)-ATPase activity (44,62, and 43%, respectively). Kinetic analysis indicated that theophylline and dibutyryl cAMP inhibit Na+, K(+)-ATPase by different mechanisms; theophylline decreased Vmax and decreased apparent Km (ATP), whereas dibutyryl cAMP decreased Vmax and increased apparent Km (ATP). Similar inhibition of Na+, K(+)-ATPase by theophylline or dibutyryl cAMP was noted in a particulate fraction from rat kidney and in a purified porcine brain Na+, K(+)-ATPase preparation. The adenylate cyclase system and Na+, K(+)-ATPase may act reciprocally in pancreatic islets and in other tissues. In the beta cell this relationship may be essential in coordinating consumption of ATP in the stimulated, as opposed to the rest, state.
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PMID:Relationships between adenylate cyclase and Na+, K(+)-ATPase in rat pancreatic islets. 215 93

To determine the functioning rate of Na-K-ATPase in the rat nephron, a micromethod was developed to measure the rate of rubidium uptake in single nephron segments microdissected from collagenase-treated kidneys. Because the hydrolytic activity of Na-K-ATPase displayed the same apparent affinity for K and Rb ions, whereas the Vmax elicited by K was higher than that in the presence of Rb, experiments were performed in the presence of cold Rb plus 86Rb. Before the assay, tubules were preincubated for 10 min at 37 degrees C to restore the normal transmembrane cation gradients. 86Rb uptake was measured after washing out extracellular cations by rinsing the tubules in ice-cold choline chloride solution containing Ba2+. Rb uptake increased quasi-linearly as a function of incubation time up to 30 s in the thick ascending limb, 1 min in the proximal convoluted tubule, and 5 min in the collecting tubule, and reached an equilibrium after 5-30 min. The initial rates of Rb uptake increased in a saturable fashion as Rb concentration in the medium rose from 0.25 to 5 mM. In medullary thick ascending limb, the initial rate of Rb uptake was inhibited by greater than 90% by 2.5 mM ouabain and by 10(-5) M of the metabolic inhibitor carbonyl cyanide trifluoromethoxyphenylhydrazone. Correlation of Na-K-ATPase hydrolytic activity at Vmax and initial rates of ouabain-sensitive Rb uptake in the successive segments of nephron indicates that in intact cells the pump works at approximately 20-30% of its Vmax. Increasing intracellular Na concentration by tubule preincubation in a Rb- and K-free medium increased the initial rates of Rb intake up to the Vmax of the hydrolytic activity of the pump.
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PMID:Measurement of Na-K-ATPase-mediated rubidium influx in single segments of rat nephron. 216 56

The present work was undertaken to evaluate the distribution of the Na-ATPase activity in the different components of the rat kidney cortex. Suspensions of glomeruli, proximal and distal tubules were prepared following a collagenase digestion of outermost kidney cortex slices and a separation on a Percoll gradient. It was found that the Na-ATPase activity is higher in the fraction enriched in proximal tubules. The fraction enriched in glomeruli and in distal tubules show also a Na-ATPase activity, but it is lower.
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PMID:Ouabain-insensitive, Na-ATPase activity in pure suspensions of rat kidney proximal tubules. 216 45

Using hepatocytes isolated by collagenase perfusion, we studied the accumulation of 3H-noradrenaline. Cells incubated during 15 min in the presence of 0.4 mumol/l 3H-noradrenaline (without inhibition of noradrenaline metabolism) accumulated 8.32 +/- 1.77 pmol/10(6) cells (n = 3). The accumulation of 3H-noradrenaline in isolated parenchymal liver cells was sensitive to 10 mumol/l cocaine (inhibition 36.6 +/- 7.9%, n = 3) and 1 mumol/l desipramine (inhibition 27.2 +/- 6.9, n = 3). Accumulation of 3H-noradrenaline was temperature and sodium dependent (inhibition 33.2 +/- 9.4%, n = 9, when Na+ was replaced by Tris+) and was influenced by the inhibition of the membrane Na(+)-K(+)-adenosine triphosphatase (Na(+)-K(+)-ATPase) by 150 mumol/l ouabain (34.7 +/- 6.9% inhibition, n = 3). Accumulation of 3H-noradrenaline in the hepatocytes was not affected by the presence of uptake2 inhibitors, normetanephrine (30 mumol/l) and corticosterone (30 mumol/l), but was reduced by 30 mumol/l isoprenaline (76.3 +/- 5.0% inhibition, n = 6). Thus, the system that takes up and accumulates noradrenaline in the isolated rat liver cells possesses some characteristics of both, uptake1 and uptake2 systems and appears to be different from other extraneuronal cocaine-sensitive systems, such as the one reported for pulmonary endothelial cells.
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PMID:Accumulation of 3H-(+/-)-noradrenaline by isolated rat liver cells. 216 96

A recent hypothesis for the cellular mechanism of fluid secretion by lacrimal acini has been based, in part, on the results of subcellular fractionation analyses of lacrimal gland fragments which had been incubated for a brief period in vitro. An important assumption in those studies was that the ion transporters and neurotransmitter receptors measured in isolated subcellular fractions were associated with membranes derived from the acinar cells, since these comprise the bulk of the lacrimal gland mass. This study was undertaken to validate this assumption. Acinar complexes were isolated from rat exorbital lacrimal glands by digestion with collagenase, hyaluronidase, and DNase. Although terminal intralobular duct segments and myoepithelial cells were occasionally noted, the preparations appeared to be free of larger ducts, blood cells, blood vessels, and interstitial cells. Acinar cells were then disrupted, and the homogenates underwent the fractionation procedure used previously for lacrimal gland fragment preparations. This procedure involved a sequence of analyses by differential sedimentation, isopycnic centrifugation on sorbitol gradients, and partitioning in dextran-polyethyleneglycol two-phase systems. Calculated initial specific activities for sodium/potassium adenosinetriphosphatase (Na+/K(+)-ATPase), alkaline phosphatase, acid phosphatase, and succinate dehydrogenase were identical to those obtained from fragment preparations. Major membrane populations resolved by the sequential analyses, including one believed to represent endoplasmic reticulum membranes, two believed to be derived from the acinar cell basal-lateral membrane, and two believed to be derived from the Golgi complex, corresponded closely to populations resolved from lacrimal fragment preparations. In addition to validating the previous use of lacrimal gland fragment preparations in studies of acinar cell function, these results suggest that preparations of isolated lacrimal acini will be useful for future work on neurotransmitter-receptor regulation and basal-lateral plasma membrane dynamics in the lacrimal gland.
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PMID:Analytic subcellular fractionation of acini from rat lacrimal gland. 217 90


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