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)

The ouabain-resistant cell line H1C1 displays a 30-fold differential of reduced sensitivity to the structurally related cardiac glycosides digoxin and digitoxin (Baker, R. M. (1976) in Biogenesis and Turnover of Membrane Macromolecules (Cook, J.S., ed) pp. 93-103, Raven Press, New York). Since these ligand congeners differ only by the presence of a hydroxyl group at C-12 of digoxin we predicted that the H1C1 phenotype must reflect a mutation which alters the binding site of the cardiac glycoside receptor (Na,K-ATPase). Complementary DNA encoding the alpha 1 Na,K-ATPase was prepared from H1C1 cell total RNA by reverse transcription-coupled polymerase chain reaction and these cDNAs were cloned. Sequence analysis of the reverse transcriptase-polymerase chain reaction clones revealed several independent isolates containing a G > A transition at nucleotide 332 of the propeptide coding sequence, generating the amino acid substitution C108Y. The ability of this substitution to confer differential sensitivity for digoxin and digitoxin was tested and confirmed by expressing a human alpha 1 C108Y-Na,K-ATPase in wild type HeLa cells and assaying for inhibition of cell growth and inhibition of Na,K-ATPase activity. Phenylalanine or alanine substitutions of this cysteine also confer this pattern of ligand discrimination. Ouabain-resistant Na,K-ATPase substitutions, at positions other than Cys-108 failed to exhibit differential sensitivity indicating that this ligand discrimination is unique to Cys-108 substitutions rather than a general property of cardiac glycoside-resistant mutants. It is proposed that differential resistance of the C108Y receptor for these ligands is a consequence of altering two features of the ligand-receptor interaction; one, a disruption of a common hydrogen bond resulting in general loss of affinity for cardiac glycosides and the other, formation of a new H-bond between the C-12 hydroxyl of digoxin and the receptor, specifically augmenting the stability of this ligand-receptor complex.
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PMID:Identification of an amino acid substitution in human alpha 1 Na,K-ATPase which confers differentially reduced affinity for two related cardiac glycosides. 792 66

As the sole renal Na,K-ATPase isozyme, the alpha 1 Na,K-ATPase accounts for all active transport of Na+ throughout the nephron. This role in renal Na+ reabsorption and the primacy of the kidney in hypertension pathogenesis make it a logical candidate gene for salt-sensitive genetic hypertension. An adenine (A)1079-->thymine (T) transversion, resulting in the substitution of glutamine276 with leucine and associated with decreased net 86Rb+ (K+) influx, was identified in Dahl salt-sensitive/JR rat kidney alpha 1 Na,K-ATPase cDNA. However, because a Taq polymerase chain reaction amplification-based reanalysis did not detect the mutant T1079 but rather only the wild-type A1079 alpha 1 Na,K-ATPase allele in Dahl salt-sensitive rat genomic DNA, we reexamined alpha 1 Na,K-ATPase sequences using Taq polymerase error-independent amplification-based analyses of genomic DNA (by polymerase allele-specific amplification and ligase chain reaction analysis) and kidney RNA (by mRNA-specific thermostable reverse transcriptase-polymerase chain reaction analysis). We also performed modified 3' mismatched correction analysis of genomic DNA using an exonuclease-positive thermostable DNA polymerase. All the confirmatory test results were concordant, confirming the A1079-->T transversion in the Dahl salt-sensitive alpha 1 Na,K-ATPase allele and its transcript, as well as the wild-type A1079 sequence in the Dahl salt-resistant alpha 1 Na,K-ATPase allele and its transcript. Documentation of a consistent Taq polymerase error that selectively substituted A at T1079 (sense strand) was obtained from Taq polymerase chain reaction amplification and subsequent cycle sequencing of reconfirmed known Dahl salt-sensitive/JR rat mutant T1079 alpha 1 cDNA M13 subclones. This Taq polymerase error results in the reversion of mutant sequence back to the wild-type alpha 1 Na,K-ATPase sequence. This identifies a site- and nucleotide-specific Taq polymerase misincorporation, suggesting that a structural basis might underlie a predisposition to nonrandom Taq polymerase errors.
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PMID:Confirmation of mutant alpha 1 Na,K-ATPase gene and transcript in Dahl salt-sensitive/JR rats. 808 31

Organelles in the axoplasm from the squid giant axon move along exogenous actin filaments toward their barbed ends. An approximately 235-kDa protein, the only band recognized by a pan-myosin antibody in Western blots of isolated axoplasmic organelles, has been previously proposed to be a motor for these movements. Here, we purify this approximately 235-kDa protein (p235) from axoplasm and demonstrate that it is a myosin, because it is recognized by a pan-myosin antibody and has an actin-activated Mg-ATPase activity per mg of protein 40-fold higher than that of axoplasm. By low-angle rotary shadowing, p235 differs from myosin II and it does not form bipolar filaments in low salt. The amino acid sequence of a 17-kDa protein that copurifies with p235 shows that it is a squid optic lobe calcium-binding protein, which is more similar by amino acid sequence to calmodulin (69% identity) than to the light chains of myosin II (33% identity). A polyclonal antibody to this light chain was raised by using a synthetic peptide representing the calcium binding domain least similar to calmodulin. We then cloned this light chain by reverse transcriptase-PCR and showed that this antibody recognizes the bacterially expressed protein but not brain calmodulin. In Western blots of sucrose gradient fractions, the 17-kDa protein is found in the organelle fraction, suggesting that it is a light chain of the p235 myosin that is also associated with organelles.
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PMID:An axoplasmic myosin with a calmodulin-like light chain. 865 Feb 20

The rat nephron displays two ouabain-sensitive K-ATPases: one, which is present in proximal tubules and thick ascending limbs of normal rats, is specifically activated by K+ and is down-regulated by K+ depletion, whereas the other one appears in collecting ducts of K+-depleted rats and is activated by either Na+ or K+. To determine which of these two ATPases is similar to colonic-type H,K-ATPase, we quantitated by reverse transcriptase-polymerase chain reaction (RT-PCR) the mRNAs encoding the colonic H,K-ATPase alpha subunit in microdissected nephron segments. In normal rats, statistically significant amounts of colonic H,K-ATPase mRNAs were detected exclusively in cortical thick ascending limbs and cortical collecting ducts (200-500 copies/mm). Because these levels of expression were low (1-1.2 copies/target cell), they probably have no physiological relevance. In rats fed a K+-depleted diet for 2 weeks, expression of colonic H,K-ATPase was markedly enhanced in cortical and medullary collecting ducts (5000-12,000 copies/mm or 30-40 copies per cell), whereas it remained low in all other nephron segments. Thus, colonic H,K-ATPase alpha subunit is specifically expressed in cortical and outer medullary collecting ducts of K+-depleted rats where it likely accounts for the ouabain-sensitive K-ATPase activity.
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PMID:Quantitative RT-PCR analysis of mRNAs encoding a colonic putative H, K-ATPase alpha subunit along the rat nephron: effect of K+ depletion. 876 9

We report a method that allows accurate, absolute quantification of gene expression in a single reverse transcriptase (RT)-PCR reaction. This method makes use of novel high-performance liquid chromatography (HPLC) technology to resolve and quantify the products of competitive, mutant RNA PCRs. The HPLC technique allows rapid, high resolution of reaction products. On-line UV detection eliminates the need for radiolabel or other tracers. The HPLC technique also demonstrates that these competition reactions readily generate heteroduplex products. The ability of HPLC to resolve and quantify heteroduplex products is fundamental to the accuracy of the technique. Accurate measurements of gene expression have been obtained over four orders of magnitude and experiments employing predetermined quantities of specific native RNA input have demonstrated the ability of the system to provide absolute estimates of gene expression. Large size differences between native and mutant RNA inputs affected reverse transcriptase (RT) efficiency, but not PCR amplification efficiency. However, the magnitude of the RT efficiency effect can be estimated, is reproducible, and can therefore be adjusted by a calculated correction factor. The RT efficiency difference can been eliminated by reduction in the magnitude of the sequence difference between native and mutant RNA so that no correction factor is required. The application of the technique to quantification of expression of the alpha 1 subunit of sodium, potassium-ATPase in microdissected nephron segments is demonstrated.
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PMID:Accurate and absolute quantitative measurement of gene expression by single-tube RT-PCR and HPLC. 880 71

We characterized bradykinin (BK) receptors in a human line of glomerular visceral epithelial cells (hGVEC) transfected by the SV40 virus. [3H]BK bound specifically in a manner consistent with a single high-affinity site. Scatchard analysis yielded dissociation constant and maximum binding values of 0.28 +/- 0.04 nM and 76.6 +/- 4.9 fmol/mg, respectively. Competition binding studies with selective BK type 2 (Hoe-140) receptor antagonist and type 1 ([des-Arg9]BK) receptor agonist showed that hGVEC only expressed type 2 receptors, and this was confirmed by reverse transcriptase-polymerase chain reaction and ribonuclease protection assay. BK stimulated intracellular calcium ion concentration ([Ca2+]i) release in a dose-dependent manner with a threshold at 1 nM. Hoe-140, in contrast with [des-Arg9]BK, abolished this effect. [Ca2+]i stimulation was also inhibited by thapsigargin, an inhibitor of Ca(2+)-adenosinetriphosphatase. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid attenuated but did not suppress the [Ca2+]i peak. These results associated with the stimulatory effect of BK on inositol phosphate production indicated that [Ca2+]i stimulation was produced both by [Ca2+] mobilization from its intracellular stores and by [Ca2+] entry into the cells. In conclusion, hGVEC express specific type 2 BK receptors that enable specific BK-induced responses.
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PMID:Characterization of a B2-bradykinin receptor in human glomerular podocytes. 885 39

The relative mRNA levels corresponding to the different sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase isoforms (SERCA1a, SERCA1b, SERCA2a, SERCA2b and SERCA3) were measured by reverse transcriptase-PCR in rat soleus muscles regenerating after notexin-induced necrosis. The succession of appearance of the different types of SERCA mRNA species in regenerating muscle largely recapitulates those observed during normal ontogenesis. The mRNA levels of the muscle-specific isoforms SERCA1a and SERCA2a became very low on the first and third days after injection of the snake venom. It was only on the fifth day of regeneration that the mRNA of the neonatal variant of the fast-twitch skeletal SERCA1b isoform began to rise, well before the other SERCA transcripts. At 7 and 10 days, i.e. at a time when the new myofibres normally become reinnervated, the mRNA level of SERCA1a and SERCA2a increased markedly, but the fast-twitch skeletal SERCA1a isoform was still the most prominent. On day 21, in the advanced stage of regeneration, a switch in the relative expression levels of SERCA1a and SERCA2a mRNA was observed and the ratio of both isoforms became similar to that found in the normal soleus muscles. This was followed by a decline in the level of all SERCA mRNA species, so that on day 28 the levels of the sarcoplasmic/endoplasmatic-reticulum Ca(2+)-pump RNAs was again lower but their ratio remained similar to that of the untreated control soleus.
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PMID:Changes in mRNA levels of the sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase isoforms in the rat soleus muscle regenerating from notexin-induced necrosis. 894 74

The fluid movements that arise during blastocyst formation (cavitation) are, at least in part, driven by the Na/K-ATPase. In this study, the reverse transcriptase-polymerase chain reaction (RT-PCR) was used to survey bovine pre-attachment embryos for transcripts encoding known isoforms of the Na/K-ATPase alpha- and beta-subunits, including isoforms not previously detected during the first week of mammalian development. Transcripts encoding the Na-K-ATPase alpha 1, alpha 2, alpha 3 and beta 2 isoforms were detected throughout bovine preattachment development. This is the first indication that alpha 2, alpha 3 and beta 2 mRNAs are expressed during this early developmental interval. As in the mouse, beta 1-subunit transcripts were not detected until the morula stage and were also present in blastocysts. Thus, in two mammalian species an increase in abundance of beta 1 isoform transcripts in the morula stage is coincident with the onset of cavitation. Transcripts encoding the recently characterized alpha 4 isoform were not detected. The sensitivity of bovine blastocysts to ouabain (a potent inhibitor of Na/K-ATPase) was determined by assessing the ability of bovine blastocysts to recover in ouabain supplemental culture medium following cytochalasin-induced blastocyst collapse. Re-expansion of bovine blastocysts was inhibited in all ouabain concentrations down to 10(-9) M. Mouse blastocysts, in contrast, were sensitive to ouabain at or above 10(-3)M. These results have established that transcripts encoding multiple isoforms of both the alpha and beta subunits of the Na/K-ATPase are expressed throughout early bovine development and that bovine blastocysts display a greater sensitivity to ouabain than murine blastocysts. Future analysis will determine the possible individual and collective roles of these isoforms during blastocyst formation.
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PMID:Ouabain sensitivity and expression of Na/K-ATPase alpha- and beta-subunit isoform genes during bovine early development. 902 43

The P-type ATPases (e.g., Na+-K+-ATPase and Ca2+-ATPase) occur widely in living cells of fungi, Protozoa, plants, and animals. These ion pumps show a high degree of divergence in their primary structures but share a limited number of common amino acid residues for their ATP-catalytic function. Particularly, the amino acid sequences for the phosphorylation site (DKTGTLT) and the binding site for ATP (and its analogs; GDGVND) are conserved throughout evolution. Using two degenerate oligonucleotides corresponding to these regions, we applied a polymerase chain reaction (PCR) technique to the search for P-type ATPase isoforms, which will provide a clue to the evolutionary mechanisms of ion pumps in Tetrahymena thermophila. A total of 12 distinct P-type ATPase genes were identified. Sequence comparisons revealed that seven of them can be compiled into a multigene family, which is similar to animal Na+-K+- and H+-K+-ATPase genes. One of them is close to the sarco(endo)plasmic reticulum Ca2+-ATPase gene, and the other four share a significant homology with the gene encoding Plasmodium ATPase-1 whose function is unknown. A Northern blot analysis and reverse transcriptase-PCR demonstrated that all identified genes are expressed, but the expression levels vary widely under different culture conditions. A Southern blot analysis after pulse-field gel electrophoresis showed that all of these genes exist in T. thermophila macronuclei. The Na+-K+- and H+-K+-ATPase gene family has a high multiplicity (at least 10 different genes detected on genomic Southern blot analysis) and is distributed on four different macronuclear chromosomes. On the basis of a calculation with the amino acid sequences of the cloned cytoplasmic loop region (between the phosphorylation and the gamma-[4-(N-2-chloroethyl-N-methylamino)]-benzylamido ATP sites), the genes with >80% identity form a cognate linkage group within the same macronuclei chromosome, whereas the genes with <70% identity are separated in different chromosomes. The phylogenetic analysis showed that this multigene family is the result of a series of gene duplications.
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PMID:Primary structure and evolution of the ATP-binding domains of the P-type ATPases in Tetrahymena thermophila. 912 16

Evidence for an H+-K+-adenosinetriphosphatase (HKA) in vascular smooth muscle cells has recently been demonstrated in the rat aorta and a rat smooth muscle cell line (A(7)r(5)) by functional studies. Our laboratory has used reverse transcriptase (RT)-polymerase chain reaction (PCR) and Northern hybridization techniques to define the type of HKA present in canine whole carotid artery and confluent carotid artery cells. PCR primers to detect an HKA message of unknown isoform were designed on the basis of homology to known sequences of gastric, colonic, and toad bladder HKA isoforms and dissimilarity to the Na+-K+-adenosinetriphosphatase isoforms. A PCR product of predicted size was generated from cDNAs of both confluent cells and whole vessel carotid artery that possess 91% nucleotide sequence identity to the canine gastric HKA. Northern hybridization with the PCR product as a probe revealed hybridization to whole vessel carotid artery, cultured confluent carotid artery cell, kidney, and stomach canine total RNA at the known message size for the gastric HKA isoform. These data suggest that vascular smooth muscle cells express an HKA mRNA that may be identical to the gastric isoform.
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PMID:Molecular evidence for a vascular smooth muscle H+-K+-ATPase. 912 50


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