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)

Na,K-ATPases from various species differ greatly in their sensitivity to cardiac glycosides such as ouabain. The sheep and human enzymes are a thousand times more sensitive than the corresponding ones from rat and mouse. To define the region of the alpha 1 subunit responsible for this differential sensitivity, chimeric cDNAs of sheep and rat were constructed and expressed in ouabain-sensitive HeLa cells. The construct containing the amino-terminal half of the rat alpha 1 subunit coding region and carboxyl-terminal half of the sheep conferred the ouabain-resistant phenotype to HeLa cells while the reverse construct did not. This indicates that the determinants involved in ouabain sensitivity are located in the amino-terminal half of the Na,K-ATPase alpha subunit. By use of site-directed mutagenesis, the amino acid sequence of the first extracellular domain (H1-H2) of the sheep alpha 1 subunit, Gln-Ala-Ala-Thr-Glu-Glu-Glu-Pro-Gln-Asn-Asp-Asn, was changed to that of the rat, Arg-Ser-Ala-Thr-Glu-Glu-Glu-Pro-Pro-Asn-Asp-Asp. When expressed in HeLa cells, this mutated sheep alpha 1 construct, like the rat/sheep chimera, was able to confer ouabain resistance to these cells. Furthermore, similar results were observed when HeLa cells were transfected with a sheep alpha 1 cDNA containing only two amino acid substitutions. This double mutation was a Gln-111----Arg and Asn-122----Asp change at the amino terminus and carboxyl terminus, respectively, of the H1-H2 extracellular region. The resistant cells, whether transfected with the rat alpha 1 cDNA, the rat/sheep chimera, or the mutant sheep alpha 1 cDNAs, exhibited identical biochemical characteristics including ouabain-inhibitable cell growth, 86Rb+ uptake, and Na,K-ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Structure-function relationships in the Na,K-ATPase alpha subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme. 285 65

We have made use of a panel of mouse-hamster somatic cell hybrids and restriction fragment length polymorphisms between two mouse species (Mus musculus and Mus spretus) to determine the chromosomal localization of genes encoding the alpha and beta subunits of the Na,K-ATPase (Na+,K+-activated ATP phosphohydrolase, EC 3.6.1.3). DNA probes for three distinct isoforms of the Na,K-ATPase alpha subunit mapped to three different mouse chromosomes: the alpha 1 gene (Atpa-1) cosegregated with the Egf gene on chromosome 3; alpha 2 (Atpa-2) with the cytochrome P-450PB gene family/coumarin hydroxylase locus on chromosome 7; alpha 3 (Atpa-3) with the alpha-spectrin gene on chromosome 1. The Na,K-ATPase beta-subunit gene (Atpb) mapped to the same region of chromosome 1, but it was not tightly linked to the Atpa-3 gene. These results indicate that three isoforms of the Na,K-ATPase alpha subunit are encoded by three distinct genes. The dispersion of Na,K-ATPase genes suggests that their expression is not likely to be controlled by a common cis-acting regulatory element.
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PMID:Genes encoding alpha and beta subunits of Na,K-ATPase are located on three different chromosomes in the mouse. 288 48

We have isolated a cDNA clone for the rat brain Na,K-ATPase alpha subunit. A lambda gt11 cDNA expression library constructed from mRNA of 1- and 2-week-old rat brains was screened with an antibody reactive with rat brain Na,K-ATPase. A positive phage clone, lambda rb5, containing a 1200-base-pair cDNA insert expressed a beta-galactosidase-cDNA fusion protein that was reactive by immunoblotting with the Na,K-ATPase antibody. This fusion protein was also reactive in ELISA with a monoclonal antibody directed against the alpha subunit of the Na,K-ATPase. A 27S mRNA species exhibiting sequence hybridization to the cDNA insert of lambda rb5 was identified in rat brain, kidney, and liver, as well as in dog kidney. This 27S mRNA exhibited a tissue-specific pattern of abundance consistent with the relative abundance of Na,K-ATPase polypeptides in vivo: kidney greater than brain greater than liver. In a ouabain-resistant HeLa cell line, C+, which contains minute chromosomes and at least a 10-fold greater number of sodium pumps than parental HeLa cells, DNA sequences complementary to lambda rb5 cDNA were amplified approximately 40-fold. Analysis of the lambda rb5 cDNA sequence demonstrated a perfect nucleotide sequence match between a portion of the cDNA and the amino acid sequence of the Na,K-ATPase alpha-subunit fluorescein isothiocyanate binding site. Taken together, the data presented here demonstrate that the lambda rb5 cDNA clone is a portion of the gene coding for the rat brain Na,K-ATPase alpha subunit. The ATPase gene appears to be present in one or very few copies in the rat and human genomes and to be transcriptionally regulated in different rat tissues. In a ouabain-resistant human cell line, on the other hand, ouabain resistance appears to involve an increase in the number of gene copies coding for the Na,K-ATPase.
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PMID:Molecular cloning of rat brain Na,K-ATPase alpha-subunit cDNA. 299 74

The Cys127-Cys150 disulfide-bonded loop (L1) of the Torpedo californica Na,K-ATPase beta 1 subunit was substituted with the corresponding loop of the rat beta 1, mouse beta 2, or pig H,K-ATPase beta subunit. All the substituted mutant beta subunits assembled with the Na,K-ATPase alpha subunit in a trypsin-resistant manner. The mutants with L1 from the Na,K-ATPase beta subunit isoforms (rat beta 1 and mouse beta 2) each formed a functional complex with the Na,K-ATPase alpha subunit. On the other hand, the complex of the alpha subunit with the mutant beta subunit that was substituted with the pig H,K-ATPase beta subunit L1 was inactive as to ATP hydrolysis. Ser131 and Phe148 located within L1 of the pig H,K-ATPase beta subunit-substituted mutant were back-mutated to Pro131 and Arg148, respectively. The Phe148 to Arg mutation restored the ability of the mutant beta subunit substituted with the H,K-ATPase beta subunit L1 to form a functional complex with the alpha subunit. These results suggested that the Cys127-Cys150 loop of the Na,K-ATPase beta 1 subunit, especially Arg148, plays a critical role in the functional expression of Na,K-ATPase.
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PMID:Functional consequences of substitution of the disulfide-bonded segment, Cys127-Cys150, located in the extracellular domain of the Na,K-ATPase beta subunit: Arg148 is essential for the functional expression of Na,K-ATPase. 762 27

The topological organization of the Na,K-ATPase alpha subunit is controversial. Detection of extracellular proteolytic cleavage sites would help define the topology, and so attempts were made to find conditions and proteases that would permit digestion of Na,K-ATPase in sealed right-side-out vesicles from renal medulla. The beta subunit is predominantly extracellular and could mask the surface of the alpha subunit. Most of the tested proteases cleaved beta, and some digested it extensively. However, without further disruption of structure, there was still no digestion of the alpha subunit. Reduction (at 50 degrees C) of disulfide bonds that might stabilize the beta subunit fragments, or heating alone at 55 degrees C, permitted tryptic digestion of alpha at a site close to the C terminus, while simultaneously increasing digestion of beta. A 90-kDa N-terminal fragment of alpha was recovered, but the C-terminal fragment was further digested. Heating and reduction resulted in the extracellular exposure of a protein kinase A phosphorylation site, Ser-938, and the C terminus, both of which have been proposed to be located on the intracellular surface. At the same time, access to a distant protein kinase C phosphorylation site was not increased. The data suggest that the harsh treatment simultaneously resulted in alteration of the beta subunit and the extrusion of a segment of alpha that normally spans the membrane, without causing complete denaturation or opening the sealed vesicles. Preincubation with Rb+ was protective, consistent with prior evidence that it stabilizes the protein segments in the C-terminal third of alpha. We conclude that this portion of the alpha subunit contains a transmembrane structure with unique lability to heating.
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PMID:Topology of the Na,K-ATPase. Evidence for externalization of a labile transmembrane structure during heating. 772 85

There is considerable evidence that protein kinases play a role in regulation of the activity of the Na,K-ATPase, but the characteristics of direct kinase phosphorylation of Na,K-ATPase subunits are still not well understood. There are 36 sites that could qualify as protein kinase C motifs in rat alpha 1. Here we have used protein fragmentation with trypsin to localize the site of phosphorylation of the rat Na,K-ATPase alpha 1 subunit to within the first 32 amino acids of the N terminus and then used direct sequencing of the phosphorylated protein to determine which of two candidate serine residues was modified. The result was that at most 25% of the 32P was found on Ser-11, a site that is well conserved in Na,K-ATPase alpha 1 subunits. The remaining 75% or more of the 32P was found on Ser-18, a site that is absent in many Na,K-ATPase alpha subunit sequences. This accounts for the observation that dog and pig alpha 1 subunits can be phosphorylated by protein kinase C only to much lower levels than can rat alpha 1. It is also likely to be relevant to other known species-specific effects of protein kinase C on Na,K-ATPase.
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PMID:Structural basis for species-specific differences in the phosphorylation of Na,K-ATPase by protein kinase C. 777 68

Synthesis and assembly of most oligomeric plasma membrane proteins occurs in the ER. However, the role the ER plays in oligomerization is unknown. We have previously demonstrated that unassociated alpha and beta subunits of the Na,K-ATPase are targeted to the plasma membrane when individually expressed in baculovirus-infected Sf-9 cells. This unique property allows us to determine if assembly of these two polypeptides is restricted to the ER, or if it can also occur at the plasma membrane. To investigate the assembly of the Na,K-ATPase we have taken advantage of the ability of baculovirus-infected cells to fuse. Lowering the extracellular pH of the infected cells triggers an endogenously expressed viral protein to initiate plasma membrane fusion. When individual Sf-9 cells expressing either the Na,K-ATPase alpha or beta subunits are plated together and subjected to a mild acidic shock, they form large syncytia. In the newly continuous plasma membrane the separate alpha and beta polypeptides associate and assemble into functional Na,K-ATPase molecules. However, a hybrid ATPase molecule consisting of a Na,K-ATPase alpha subunit and a H,K-ATPase beta subunit, which efficiently assembles in the ER of coinfected cells, does not assemble at the plasma membrane of fused cells. When cells expressing the Na,K-ATPase alpha subunit are fused to cells coexpressing the Na,K-ATPase beta subunit and the H,K-ATPase beta subunit, the Na,K-ATPase alpha subunit selectively assembles with the Na,K-ATPase beta subunit. However, when cells are coinfected and expressing all three polypeptides, the Na,K-ATPase alpha subunit assembles with both beta subunits in the ER, in what appears to be a random fashion. These experiments demonstrate that assembly between some polypeptides is restricted to the ER, and suggests that the ability of the Na,K-ATPase alpha and beta subunits to leave the ER and assemble at the plasma membrane may represent a novel mechanism of regulation of activity.
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PMID:The alpha and beta subunits of the Na,K-ATPase can assemble at the plasma membrane into functional enzyme. 792 71

The phosphorylation state of the Na,K-ATPase alpha subunit has been examined in 32P-labeled sciatic nerves of control and streptozotocin-treated diabetic rats. Intact nerves were challenged with protein kinase (PK) modulators and alpha-subunit 32P labeling was analyzed after immunoprecipitation. In control nerves, the PKC activator phorbol 12-myristate 13-acetate (PMA) had little effect on alpha-subunit 32P labeling. In contrast, staurosporine, a PKC inhibitor, and extracellular calcium omission decreased it. In Ca(2+)-free conditions, PMA restored the labeling to basal levels. The cAMP-raising agent forskolin reduced the 32P labeling of the alpha subunit. The results suggest that nerve Na,K-ATPase is tonically phosphorylated by PKC in a Ca(2+)-dependent manner and that PKA modulates the phosphorylation process. In nerves of diabetic rats, PMA increased 32P labeling of the alpha subunit. In contrast to staurosporine or extracellular calcium omission, the decreased state of phosphorylation seen with forskolin was no longer significant in diabetic nerves. No change in the level of alpha-subunit isoforms (alpha 1 or alpha 2) was detected by Western blot analysis in such nerves. In conclusion, the altered effect of PK activators on Na,K-ATPase phosphorylation state is consistent with the view that a defect in PKC activation exists in diabetic nerves.
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PMID:In vivo phosphorylation of the Na,K-ATPase alpha subunit in sciatic nerves of control and diabetic rats: effects of protein kinase modulators. 801 40

The Na,K-ATPase is a heterodimer consisting of an alpha and a beta subunit. Both Na,K-ATPase subunits are encoded by multigene families. Several isoforms for the alpha (alpha 1, alpha 2, and alpha 3) and beta (beta 1, beta 2, and beta 3) subunits have been identified. All these isoforms are capable of forming functionally active enzyme. Although there is general agreement that the Na,K-ATPase consists of alpha and beta subunits in equimolar amounts, the quaternary structure of the Na,K-ATPase and its functional significance is unknown. Several studies have demonstrated that the enzyme exists within the plasma membrane as an oligomer of alpha beta dimers. However, because the alpha beta protomer seems to be catalytically competent, the possibility exists that higher oligomers are irrelevant to function. The ability to express different alpha isoforms in insect cells and the availability of isoform-specific antibodies has provided the opportunity to test for the existence of stable and specific associations among alpha subunits. By coexpressing different alpha-subunit isoforms in cultured cells, we demonstrate that the Na,K-ATPase alpha subunits specifically and stably associate into oligomeric complexes. This same association among alpha-subunit isoforms was demonstrated in the native enzyme from rat brain. The interaction between Na,K-ATPase alpha subunits is highly specific. When the Na,K-ATPase alpha subunit is coexpressed with the alpha subunit from the H,K-ATPase, the H,K subunit does not associate with the Na,K subunit. Moreover, expression of the truncated alpha 1T isoform with the full-length alpha subunit demonstrates that the C-terminal portion of the polypeptide is important in the alpha-subunit association. Although these results do not clarify the functional role of alpha alpha associations, they do establish their highly specific nature and suggest that oligomerization of alpha beta protomers may be important to the stability and physiological regulation of the enzyme.
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PMID:The alpha subunit of the Na,K-ATPase specifically and stably associates into oligomers. 807 19

2-Azido-ATP (2-N3-ATP) was investigated as a reagent for the identification of amino acids located within the catalytic ATP binding site of Na,K-ATPase. The enzymatic activity of Na,K-ATPase was inhibited up to 50% by 2-N3-ATP (K0.5 = 5-10 microM) after irradiation with ultra-violet light, and inhibition was prevented by 0.2 mM ATP. The binding of ATP to Na,K-ATPase (KD = 0.1 microM) was inhibited competitively by 2-N3-ATP. [alpha-32P]2-N3-ATP labels the alpha subunit of Na,K-ATPase, and the stoichiometry of covalent ATP-protectable incorporation of the probe into the protein is approximately equal to the stoichiometry of high-affinity binding of ATP to the Na,K-ATPase. 2-N3-ATP is also hydrolyzed by Na,K-ATPase as a substrate. From these data, it is concluded that 2-N3-ATP photochemically labels the Na,K-ATPase from within the catalytic ATP site on the protein. Trypsin digestion of Na,K-ATPase after photochemical labeling with [alpha-32P]2-N3-ATP generated a large 30-kDa fragment containing the radiolabeled nucleotide. This fragment was resistant to further cleavage by trypsin, but it could be digested further after denaturation in urea. High pressure liquid chromatography separation of tryptic peptides from the 30-kDa fragment and subsequent amino acid sequence analysis of the radiolabeled peptides identified the region between His496 and Arg510 of the Na,K-ATPase alpha subunit as the region labeled by [alpha-32P]2-N3-ATP. Gly502 was absent from all sequences of the radiolabeled peptides from this region, consistent with the derivatization of this amino acid by 2-N3-ATP and localization of Gly502 within the ATP binding site.
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PMID:Photochemical labeling and inhibition of Na,K-ATPase by 2-Azido-ATP. Identification of an amino acid located within the ATP binding site. 812 8


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