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 genetic system for directly synthesizing eukaryotic membrane proteins in Escherichia coli and assessing their ability to insert into the bacterial cytoplasmic membrane is described. The components of this system are the direct expression vector, pYZ4, and the mature beta-lactamase (BlaM) cassette plasmid, pYZ5, that can be used to generate translational fusions of BlaM to any synthesized membrane protein. The beta-subunit of sheep-kidney Na,K-ATPase (beta NKA), a class-II plasma membrane protein, was synthesized in E. coli using pYZ4, and BlaM was fused to a normally extracellular portion of it. The fusion protein conferred ampicillin resistance on individual host cells, indicating that the BlaM portion had been translocated to the bacterial periplasm, and that, by inference, the eukaryotic plasma-membrane protein can insert into the bacterial cytoplasmic membrane. A series of 31 beta NKA::BlaM fusion proteins was isolated and characterised to map the topology of the eukaryotic plasma membrane protein with respect to the bacterial cytoplasmic membrane. This analysis revealed that the organisation of the beta NKA in the E. coli cytoplasmic membrane was indistinguishable from that in its native plasma membrane.
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PMID:Correct insertion of a simple eukaryotic plasma-membrane protein into the cytoplasmic membrane of Escherichia coli. 226 59

cDNA fragments coding for the alpha and beta subunits of the Na,K-ATPase were separately ligated into the yeast expression vector YEp1PT in both the sense (YEpNKA(+)) and anti-sense (YEpNKA(-)) orientations with respect to the promoter. The recombinant plasmids were introduced into Saccharomyces cerevisiae strain UT4 by transformation. Total RNA from the transformed strains was isolated and analyzed by Northern hybridization. The resulting autoradiogram revealed strong signals indicative of a high level of transcriptional expression of both subunits in both orientations of the cDNA. 35S-Methionine labeled extracts were immunoprecipitated with antibodies specific for the beta subunit. A beta subunit translation product was produced from YEp beta NKA(+) but not from YEp beta NKA(-). Experiments to detect an alpha specific translation product are in progress.
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PMID:Development of a heterologous gene expression system for the Na,K-ATPase subunits in the yeast Saccharomyces cerevisiae. 285 34

A ligand for the digitalis receptor located on the membrane-embedded Na,K-ATPase (NKA; EC 3.6.1.37) has been isolated from bovine hypothalamus (hypothalamic inhibitory factor; HIF) and identified as isomeric ouabain (Tymiak et al., 1993, Proc. Natl. Acad. Sci. 90: 8189-8193). In analogy to cardioactive steroids (CS) derived from plants or from toad, HIF inhibits the Na/K-exchange process and the ATPase activity of isolated Na,K-ATPase although by a different molecular action mechanism. In the present work we show that, as plant-derived ouabain, HIF inhibits 86Rb-uptake by isolated human lymphocytes with an IC50 of about 20 nM; above this concentration HIF reduces cell viability in contrast to ouabain. The decrease in cell viability by excess HIF is accompanied by discrete morphological alterations (mitochondrial swelling) visible by transmission electron microscopy of ultra-thin sectioned peripheral blood mononuclear cells. Taken together the results show that the hypothalamic NKA inhibitor blocks NKA of isolated human lymphocytes with high potency at nanomolar concentrations without toxicity; concentrations exceeding the ones required to block 86Rb-uptake reduce cell viability, probably due to leak formation across the NKA molecule. Thus, lymphocytes constitute a potential target for HIF action and by their altered NKA status a possible messenger between the nervous and the immune system.
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PMID:Interaction of hypothalamic Na,K-ATPase inhibitor with isolated human peripheral blood mononuclear cells. 777 16

Lymphocytes are primordial immune cells with variable life times. Besides genetic programming, extracellular factors interacting with cell surface receptors might alter cell survival. We investigated whether the activity of the membrane-embedded Na,K-ATPase (EC3.6.1.37) or sodium pump (NKA) plays a role for cell survival since this ubiquitous system establishes the vital transmembrane Na and K gradients as well as the resulting high intracellular K/Na ratio required for macromolecule synthesis; furthermore, the system exposes an extracellular inhibitory receptors for cardioactive steroids and palytoxin. Isolated human lymphocytes were incubated in vitro and their viability assessed by exclusion of trypan blue. Various incubation conditions were compared; in RPMI-1640 medium cell viability was preserved for 30 h at 37 degrees C. Externally added ouabain, a hydrophilic cardioactive steroid, blocked the [86Rb]potassium uptake at nanomolar concentrations. Despite pump inhibition ouabain did not alter lymphocyte survival, even at 10 mM for 30 h. By contrast, the hydrophilic toxin palytoxin, the most potent animal poison described so far, killed all cells within 2 h at 10 nM; this toxin is known to act via the sodium pump and to provoke deadly cation-leaks by unmasking a channel component. Intracellular Na increased and K decreased as measured by atomic absorption spectrometry in presence of palytoxin; cell swelling was seen by electron microscopy. Ouabain protected the cells from the toxic effect of palytoxin. The results reveal a pivotal role of NKA integrity for lymphocyte survival.
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PMID:Role of cell membrane Na,K-ATPase for survival of human lymphocytes in vitro. 784 41

The coupling between the Na+/glucose cotransporter and Na(+)-K(+)-ATPase (NKA) described for epithelial cells (1) prompted us to study in rats with streptozocin-induced diabetes the effect of increased tubular glucose load on tubular Na+ reabsorption, NKA-dependent O2 consumption (QO2), and NKA activity. Filtered glucose is mainly reabsorbed in the proximal tubuli via the phlorizin-sensitive Na+/glucose cotransporter. In this study, the diabetic rats had a significantly higher renal blood flow (RBF), glomerular filtration rate (GFR), and Na+ reabsorption than the control rats. Total renal QO2 as well as QO2 in cortical tissue, which consists mainly of proximal tubular cells, was significantly higher in diabetic than in control rats. The increase in tissue QO2 was entirely caused by increased NKA-dependent QO2. NKA activity, measured as rate of ATP hydrolysis, was increased in cortical tubular but not glomerular tissue from diabetic rats. Phlorizin treatment abolished the increase in NKA activity, Na+ reabsorption, and QO2, as well as the increase in RBF and GFR in diabetic rats. We conclude that diabetes is associated with increased renal O2 metabolism secondary to the increase in coupled Na+ reabsorption via the Na+/glucose cotransporter and NKA. The increased oxygen consumption might contribute to the hyperperfusion and hyperfiltration in the diabetic kidney.
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PMID:Increased renal metabolism in diabetes. Mechanism and functional implications. 816 37

Sodium, potassium-ATPase (NKA) is an essential energy transduction mechanism in which the free energy released by hydrolysis of ATP is transferred to an electrochemical gradient across the cell membrane. The asymmetry of sodium in this gradient is coupled to membrane transport mechanisms which affect transmembrane movement of a range of solutes and electrolytes. Recent advances in the molecular biology of NKA have revealed important new aspects of structure-function relationships as well as illuminating the basis for variations in cardiac glycoside sensitivity of the enzyme. The search for endogenous mammalian counterparts of the cardiac glycosides, which regulate the activity of the enzyme by interacting from the extracellular surface at this receptor site, has moved ahead dramatically with evidence that ouabain is an endogenous product of the mammalian adrenal cortex. These advances, and problems raised by them, are explored in this review.
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PMID:Regulation of Na,K-ATPase by endogenous ouabain-like materials. 817 Oct 41

Forty-six commercially packaged teas and 78 teas prepared from purchased herbs were assayed for digoxin-like factors (DLF) by their crossreactivity with digoxin antibody (immuno-crossreactive DLF) and by their inhibition of ouabain binding to membrane Na,K-ATPase (NKA inhibitory DLF). Three packaged teas and 3 herbs gave NKA inhibitory DLF values > 30 micrograms digoxin equivalents/cup. Two packaged teas and 3 herbs gave immuno-crossreactive DLF values > .050 micrograms digoxin equivalent/cup. One herb, pleurisy root, had a crossreactive DLF value of 187 micrograms/cup and NKA inhibitory DLF equivalent to 3658 micrograms/cup. Plasma digoxin-like factors were measured after ingestion of the 3 commercially packaged herbal teas with highest values for NKA inhibitory DLF. After ingestion of each of the 3 teas, plasma NKA inhibitory DLF increase, in one case more than 100-fold. Two teas produced a measurable increase in plasma immuno-crossreactive DLF after ingestion. Some digoxin-like factors in human plasma may have a dietary source.
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PMID:Digoxin-like factors in herbal teas. 839 86

Gentamicin nephrotoxicity may arise in part from alterations in the expression of genes critical for renal proximal tubule metabolism. We tested the hypothesis that gentamicin suppressed the gene expression of the Na+/Ca2+ exchanger (NaCaX), glucose transporter 1 (GLUT1) and alpha 1-subunit of Na(+)-K(+)-ATPase (alpha 1-NKA) in renal tubules. The products of these genes mediate Na(+)-dependent Ca2+ efflux, glucose efflux and influx, and ATP-dependent Na+ efflux across tubular basolateral membranes, respectively. After 10 days of gentamicin intoxication (40 mg/kg ip, twice daily), levels of mRNAs encoding NaCaX and the cognate protein declined. GLUT1 mRNA levels increased, although GLUT1 protein levels were also reduced. Moreover, whereas alpha 1-NKA mRNA levels remained unchanged, alpha 1-NKA protein levels were also reduced. We suggest that the higher GLUT1 mRNA level is part of the stress response to tubular injury. However, regardless of the mRNA level, the most consistent effect of gentamicin was reduction of specific protein levels. We propose that failure to translate high levels of mRNA into proportionally high levels of protein, as in the case of GLUT1, may attenuate the expression of stress response gene products, and thus diminish the possibility of recovery in gentamicin intoxication.
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PMID:Studies of renal injury. I. Gentamicin toxicity and expression of basolateral transporters. 877 84

We have characterized the nucleotide sequence of Na+/K(+)-ATPase alpha subunit (NKA) cDNA in embryos of the sea urchin, Hemicentrotus pulcherrimus. The primer extension experiments showed that the sea urchin NKA gene generated multiple lengths of transcript. To obtain the 5'-ends of the transcripts, we isolated cDNA clones by the rapid amplification of cDNA ends (RACE). These clones were classified into 2 types on the basis of their 5' leader sequences. The sequences of the clones were identical except their 5' leaders. By Northern blot analysis, 1 of the 2 types of transcripts was always detectable in sea urchin embryos during early development, and another was not detected before the morula stage. Genomic PCR demonstrated that the two 5' leaders were coded by different exons separated by an intron in a single gene. These results show that the transcripts coding 2 isoforms were expressed from a single gene.
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PMID:Two types of Na+/K(+)-ATPase alpha subunit gene transcript in embryos of the sea urchin, Hemicentrotus pulcherrimus. 931 41

The mechanism by which increased dietary intake of calcium reduces blood pressure in the spontaneously hypertensive rat is unknown. The present studies were designed to determine if there were alterations in the activity of the major membrane ion translocating pump, sodium, potassium-ATPase (NKA), in the kidneys of hypertensive rats and whether increased dietary calcium intake affected the activity of this enzyme. Fifteen-week old SHR's were found to have lower total ATPase activity in microsomal preparations from the kidney than age matched Wistar-Kyoto animals. Both the ouabain-sensitive component (NKA) and the ouabain-insensitive component were lower in SHR. Increasing dietary calcium intake from 1% to 3% elevated both components of the ATPase activity in SHR, but was without effect in WKY. Measurement of membrane phospholipid composition suggested that altered phospholipid composition did not account for the reduced ATPase activity observed, but indicated a reduced density of ATPase in SHR. A technique has been devised for qualitative and quantitative analysis of Na, K-ATPase alpha isoforms using RT-PCR. This technique reveals that the alpha 1 isoform is the sole catalytic isoform present in the nephron. Accurate and precise quantification of the amount of gene expression in individual nephron segments is reported and will be applied to determine whether dietary calcium influences blood pressure by a mechanism which alters nephron NKA gene expression.
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PMID:Renal Na+, K+-ATPase in SHR: studies of activity and gene expression. 968 20


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