Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To better understand the adaptive strategies that led to freshwater invasion by hyper-regulating Crustacea, we prepared a microsomal (Na+, K+)-ATPase by differential centrifugation of a gill homogenate from the freshwater shrimp Macrobrachium olfersii. Sucrose gradient centrifugation revealed a light fraction containing most of the (Na+, K+)-ATPase activity, contaminated with other ATPases, and a heavy fraction containing negligible (Na+, K+)-ATPase activity. Western blotting showed that M. olfersii gill contains a single alpha-subunit isoform of about 110 kDa. The (Na+, K+)-ATPase hydrolyzed ATP with Michaelis Menten kinetics with K5, = 165+/-5 microM and Vmax = 686.1+/-24.7 U mg(-1). Stimulation by potassium (K0.5 = 2.4+/-0.1 mM) and magnesium ions (K0.5 = 0.76+/-0.03 mM) also obeyed Michaelis-Menten kinetics, while that by sodium ions (K0.5 = 6.0+/-0.2 mM) exhibited site site interactions (n = 1.6). Ouabain (K0.5 = 61.6+/-2.8 microM) and vanadate (K0.5 = 3.2+/-0.1 microM) inhibited up to 70% of the total ATPase activity, while thapsigargin and ethacrynic acid did not affect activity. The remaining 30% activity was inhibited by oligomycin, sodium azide and bafilomycin A. These data suggest that the (Na+, K+)-ATPase corresponds to about 70% of the total ATPase activity; the remaining 30%, i.e. the ouabain-insensitive ATPase activity, apparently correspond to F0F1- and V-ATPases, but not Ca-stimulated and Na- or K-stimulated ATPases. The data confirm the recent invasion of the freshwater biotope by M. olfersii and suggest that (Na+, K+)-ATPase activity may be regulated by the Na+ concentration of the external medium.
Comp Biochem Physiol B Biochem Mol Biol 2000 Jul
PMID:Characterization of (Na+, K+)-ATPase in gill microsomes of the freshwater shrimp Macrobrachium olfersii. 1100 72

Partial inhibition of Na/K-ATPase by ouabain causes hypertrophic growth and regulates several early and late response genes, including that of Na/K-ATPase alpha3 subunit, in cultured neonatal rat cardiac myocytes. The aim of this work was to determine whether ouabain and other hypertrophic stimuli affect Na/K-ATPase beta1 subunit gene expression. When myocytes were exposed to non-toxic concentrations of ouabain, ouabain increased beta1 subunit mRNA in a dose- and time-dependent manner. Like the alpha3 gene, beta1 mRNA was also regulated by several other well-known hypertrophic stimuli including phenylephrine, a phorbol ester, endothelin-1, and insulin-like growth factor, suggesting involvement of growth signals in regulation of beta1 expression. Ouabain failed to increase beta1 subunit mRNA in the presence of actinomycin D. Using a luciferase reporter gene that is directed by the 5'-flanking region of the beta1 subunit gene, transient transfection assay showed that ouabain augmented the expression of luciferase. These data support the proposition that ouabain regulates the beta1 subunit through a transcriptional mechanism. The effect of ouabain on beta1 subunit induction, like that on alpha3 repression, was dependent on extracellular Ca2+ and on calmodulin. Inhibitions of PKC, Ras, and MEK, however, had different quantitive effects on ouabain-induced regulations of beta1 and alpha3 subunits. The findings show that partial inhibition of Na/K-ATPase activates multiple signaling pathways that regulate growth-related genes, including those of two subunit isoforms of Na/K-ATPase, in a gene-specific manner.
Mol Cell Biochem 2000 Dec
PMID:Regulation of Na/K-ATPase beta1-subunit gene expression by ouabain and other hypertrophic stimuli in neonatal rat cardiac myocytes. 1120 57

In an attempt to understand the mechanism underlying the tissue-dependent function, the expression of NHE-1 protein and its sub cellular localization was examined in the rat GI-tract and other tissues. Rat NHE-1 polyclonal antibodies were raised in rabbits using a NHE-1 fusion protein antigen. The antibodies recognized a 110 kD protein in rats and mice, but not in human or rabbit RBCs. Colon, stomach, brain, spleen and kidney expressed NHE-1 protein abundantly, whereas the skeletal muscle the least abundant. Ouabain-sensitive-K+-stimulated p-nitrophenylphosphatase (PNPPase), the partial activity of the sodium pump and alkaline phosphatase (Apase) were used as the markers of the basolateral and apical membranes. NHE-1 was detected predominantly in the PNPPase enriched membrane fractions, but was also detected in the apical membrane enriched fractions in the kidney cortex, jejunum and colon at a lower level. NHE-1 was detected in the plasma membrane enriched fractions from the skeletal muscle and ventricle. Immunofluorescence data showed a similar localization pattern of NHE-1 in the colon and kidney sections. These findings suggest that NHE-1 is localized both on the apical and basolateral membrane. In view of its similar sub cellular localization in the GI-tract and kidney, but a different level of expression, might suggest that the level of protein, but not the sub cellular distribution is important to regulate its tissue-dependent function.
Mol Cell Biochem 2001 Mar
PMID:Expression and sub cellular localization of the sodium hydrogen exchanger isoform-1 in rat tissues: a possible functional relevance. 1135 47

The search for endogenous digitalis led to the isolation of ouabain from blood adrenals and hypothalamus. Additional cardiotonic steroids of the cardenolid and bufadienolide type seem to circulate in blood. Adrenal cortical cells in tissue culture release ouabain upon addition of angiotensin 11. Ouabain in blood is increased in 50% of Caucasians with low renin hypertension. Analogous to other steroid hormones, cardiotonic steroid hormones in blood are bound to a specific cardiac glycoside binding globulin. Since ouabain induced growth of myocytes in tissue culture, this effect probably mediates by partial inhibition of the sodium pump and consecutive rise of intracellular Ca2+ the thickening of the wall of arteries and myocardium. PST 2238, an antagonist of cardiac glycoside function at the sodium pump, leads in rats under prolonged therapy to a decrease of hypertension. The finding of ouabain as a new adrenal hormone of the Na+ metabolism and of ouabain antagonists opens new possibilities of therapy of hypertension and congestive heart failure.
Cell Mol Biol (Noisy-le-grand) 2001 Mar
PMID:Endogenous cardiotonic steroids. 1135 1

Ouabain is a well known inhibitor of the Na+ pump in all mammalian cells. We have demonstrated that ouabain at concentrations below those which inhibit the pump, i.e. 0.1 nM and 1.0 nM, induce proliferation of saphenous vein smooth muscle cells as measured by bromodeoxyuridine (BrdU) uptake. Ouabain at these low concentrations also activated MAPK. Proliferating concentrations of the drug did not increase levels of Ca(i)2+, suggesting no effect of this ion in the process. In addition, incubation of the cells in low levels of K+, which has been shown to inhibit the pump, had no effect on proliferation. These data show that low concentrations of ouabain that do not inhibit the Na+ pump can activate proliferation of vascular smooth muscle cells, suggesting that the pump complex may act as a transducing receptor.
Cell Mol Biol (Noisy-le-grand) 2001 Mar
PMID:Low concentrations of ouabain induce vascular smooth muscle cell proliferation. 1135 10

The kinetic properties of a gill Na(+), K(+)-ATPase from the freshwater shrimp Macrobrachium olfersii were studied using p-nitrophenylphosphate (PNPP) as a substrate. Sucrose gradient centrifugation of the microsomal fraction revealed a single protein fraction that hydrolyzed PNPP. The Na(+), K(+)-ATPase hydrolyzed PNPP (K(+)-phosphatase activity) obeying Michaelis-Menten kinetics with K(M)=1.72+/-0.06 mmol l(-1) and V(max)=259.1+/-11.6 U mg(-1). ATP was a competitive inhibitor of K(+)-phosphatase activity with a K(i)=50.1+/-2.5 micromol l(-1). A cooperative effect for the stimulation of the enzyme by potassium (K(0.5)=3.62+/-0.18 mmol l(-1); n(H)=1.5) and magnesium ions (K(0.5)=0.61+/-0.02 mmol l(-1), n(H)=1.3) was found. Sodium ions had no effect on K(+)-phosphatase activity up to 1.0 mmol l(-1), but above 80 mmol l(-1) inhibited the original activity by approximately 75%. In the range of 0-10 mmol l(-1), sodium ions did not affect stimulation of the K(+)-phosphatase activity by potassium ions. Ouabain (K(i)=762.4+/-26.7 micromol l(-1)) and orthovanadate (K(i)=0.25+/-0.01 micromol l(-1)) completely inhibited the K(+)-phosphatase activity, while thapsigargin, oligomycin, sodium azide and bafilomycin were without effect. These data demonstrate that the activity measured corresponds to that of the K(+)-phosphatase activity of the Na(+), K(+)-ATPase alone and suggest that the use of PNPP as a substrate to characterize K(+)-phosphatase activity may be a useful technique in comparative osmoregulatory studies of Na(+), K(+)-ATPase activities in crustacean gill tissues, and for consistent comparisons with well known mechanistic properties of the vertebrate enzyme.
Comp Biochem Physiol A Mol Integr Physiol 2001 Nov
PMID:Nitrophenylphosphate as a tool to characterize gill Na(+), K(+)-ATPase activity in hyperregulating Crustacea. 1169 3

We have constructed and characterized transgenic Drosophila lines with modified Na(+),K(+)-ATPase activity. Using a temperature dependent promoter from the hsp70 gene to drive expression of wild-type alpha subunit cDNA, we can conditionally rescue bang-sensitive paralysis and ouabain sensitivity of a Drosophila Na(+),K(+)-ATPase alpha subunit hypomorphic mutant, 2206. In contrast, a mutant alpha subunit (alpha(D369N)) leads to increased bang-sensitive paralysis and ouabain sensitivity. We can also generate temperature dependent phenotypes in wild-type Drosophila using the same hsp70 controlled alpha transgenes. Ouabain sensitivity was as expected, however, both bang sensitive paralysis or locomotor phenotypes became more severe regardless of the type of alpha subunit transgene. Using the Gal4-UAS system we have limited expression of alpha transgenes to cell types that normally express a particular Drosophila Na(+),K(+)-ATPase beta (Nervana) subunit isoform (Nrv1 or 2). The Nrv1-Gal4 driver results in lethality while the Nrv2-Gal4 driver shows reduced viability, locomotor function and uncontrolled wing beating. These transgenic lines will be useful for disrupting function in a broad range of cell types.
Comp Biochem Physiol B Biochem Mol Biol 2001 Dec
PMID:In vivo modification of Na(+),K(+)-ATPase activity in Drosophila. 1169 29

The transport characteristics of L- and D-histidine through the blood-lung barrier were studied in cultured rat lung microvascular endothelial cells (LMECs). L-Histidine uptake was a saturable process. The addition of metabolic inhibitors [2,4-dinitrophenol (DNP) and rotenone] reduced the uptake rate of L-histidine. Ouabain, an inhibitor of Na(+)-K(+)-ATPase, also reduced uptake of L-histidine. Moreover, the initial L-histidine uptake rate was reduced by the substitution of Na(+) with choline chloride and choline bicarbonate in the incubation buffer. The system N substrate, L-glutamic acid gamma-monohydroxamate, also inhibited uptake of L-histidine. However, system N-mediated transport was not pH sensitive. These results demonstrated that L-histidine is actively taken up by a system N transport mechanism into rat LMECs, with energy supplied by Na(+). Moreover, the Na(+)-independent system L substrate, 2-amino-2-norbornanecarboxylic acid (BCH), had an inhibitory effect on L-histidine uptake in Na(+) removal, indicating facilitated diffusion by a Na(+)-independent system L transport into the rat LMECs. These results provide evidence for there being at least two pathways for L-histidine uptake into rat LMECs, a Na(+)-dependent system N and Na(+)-independent system L process. On the other hand, the uptake of D-histidine into rat LMECs was not reduced by the addition of DNP, rotenone, or ouabain, or by Na(+) replacement. Although the uptake of D-histidine was reduced in the presence of BCH, the addition of L-glutamic acid gamma-monohydroxamate did not significantly decrease uptake of D-histidine. These results suggest that the uptake of D-histidine by rat LMECs has different characteristics compared with its isomer, L-histidine, indicating that system N transport did not involve D-histidine uptake.
Am J Physiol Lung Cell Mol Physiol 2002 Jun
PMID:Stereoselective transport of histidine in rat lung microvascular endothelial cells. 1200 73

The erythrocytes are widely used as model cells for studies of sodium-potassium pump (Na(+)-K(+) pump) in health and disease. Hence, to explore the possible role of the Na(+) transport across the cell membrane in the pathogenesis of pregnancy-induced hypertension (PIH), the present study was conducted to assess the Na(+)-K(+) pump functions in relation to its intrinsic kinetic properties using erythrocytes (RBC). Erythrocyte sodium concentration in pregnancy-induced hypertensive women was significantly (p<0.01) lower in comparison to normotensive pregnant women. On the contrary erythrocyte potassium was significantly higher (p<0.01) in PIH women as compared to normotensive pregnant women. Observed alterations in Na(+) and K(+) concentrations in erythrocytes were associated with significantly (p<001) increased Ouabain-sensitive sodium efflux rate and rate constants in erythrocytes from PIH women. Further, kinetic studies revealed that increased Ouabain-sensitive efflux rate constant in RBC from PIH women was accompanied by increased maximal velocity (V(max)) of Na(+)-K(+) pump. However, the affinity constant (K(m)) was unaltered in both the groups. Therefore, these findings suggest that increased Na(+)-K(+) pump activity in RBC of PIH women could be due to either increased numbers of Na(+)-K(+) pump units of increased numbers of active subunits of Na(+)-K(+) pump possibly due to specific plasma factors in PIH women.
J Biochem Mol Biol Biophys 2002 Jun
PMID:Alteration in ouabain-sensitive sodium potassium pump of erythrocytes during pregnancy induced hypertension: a kinetic study. 1218 49

We describe and compare the main kinetic characteristics of the (alpha beta)(2) form of rabbit kidney Na,K-ATPase. The dependence of ATPase activity on ATP concentration revealed high (K(0.5)=4 microM) and low (K(0.5)=1.4 mM) affinity sites for ATP, exhibiting negative cooperativity and a specific activity of approximately 700 U/mg. For p-nitrophenylphosphate (PNPP) as substrate, a single saturation curve was found, with a smaller apparent affinity of the enzyme for this substrate (K(0.5)=0.5 mM) and a lower hydrolysis rate (V(M)=42 U/mg). Stimulation of ATPase activity by K(+) (K(0.5)=0.63 mM), Na(+) (K(0.5)=11 mM) and Mg(2+) (K(0.5)=0.60 mM) all showed V(M)'s of approximately 600 U/mg and negative cooperativity. K(+) (K(0.5)=0.69 mM) and Mg(2+) (K(0.5)=0.57 mM) also stimulated PNPPase activity of the (alpha beta)(2) form. Ouabain (K(0.5)=0.01 microM and K(0.5)=0.1 mM) and orthovanadate (K(0.5)=0.06 microM) completely inhibited the ATPase activity of the (alpha beta)(2) form. The kinetic characteristics obtained constitute reference values for diprotomeric (alpha beta)(2)-units of Na,K-ATPase, thus contributing to a better understanding of the biochemical mechanisms of the enzyme.
Comp Biochem Physiol B Biochem Mol Biol 2003 Jul
PMID:Kinetic characterization of Na,K-ATPase from rabbit outer renal medulla: properties of the (alpha beta)(2) dimer. 1283 74


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