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 describe a new complementation function within the simian virus 40 (SV40) A gene. This function is required for viral DNA replication and virus production in vivo but, surprisingly, does not affect any of the intrinsic enzymatic functions of T antigen directly required for in vitro DNA replication. Other well-characterized SV40 T-antigen mutants, whether expressed stably from integrated genomes or in cotransfection experiments, complement these mutants for in vivo DNA replication and plaque formation. These new SV40 mutants were isolated and cloned from human cells which stably carry the viral DNA. The alteration in the large-T-antigen gene was shown by marker rescue and nucleotide sequence analysis to be a deletion of 322 bp spanning the splice-donor site of the first exon, creating a 14-amino-acid deletion in the large T antigen. The mutant gene was expressed in H293 human cells from an adenovirus vector, and the protein was purified by immunoaffinity chromatography. The mutant protein directs greater levels of DNA replication in vitro than does the wild-type protein. Moreover, the mutant protein reduces the lag time for in vitro DNA synthesis and can be diluted to lower levels than wild-type T antigen and still promote good replication, which is in clear contrast to the in vivo situation. These biochemical features of the protein are independent of the source of the cellular replication factors (i.e., HeLa, H293, COS 7, or CV1 cells) and the cells from which the T antigens were purified. The mutant T antigen does not transform Rat-2 cells. Several different models which might reconcile the differences observed in vivo and in vitro are outlined. We propose that the function of T antigen affected prepares cells for SV40 replication by activation of a limiting cellular replication factor. Furthermore, a link between the induction of a cellular replication factor and transformation by SV40 is discussed.
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PMID:A deletion in the simian virus 40 large T antigen impairs lytic replication in monkey cells in vivo but enhances DNA replication in vitro: new complementation function of T antigen. 131 27

A 6.5-kilobase murine genomic DNA fragment isolated by Levenson et al. (Levenson, R., Racaniello, V., Albritton, L., and Housman, D. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1489-1493) (called the ouabain resistance gene) has been shown to produce ouabain resistance in primate cells. Preliminary sequence information has revealed no homology with the coding sequence of the Na,K-ATPase. We have introduced this murine sequence into monkey and murine cells in an attempt to characterize its mechanism of action. In our experiments, transfection of this DNA fragment is associated with the low frequency (1 in 8 x 10(5) cells) appearance of ouabain-resistant clones of CV1, COS, and NIH 3T3 cells, an event not seen in control transfections. Characterization of a new clone of ouabain-resistant CV1 cells (called OR8 cells) revealed a 5-fold increase in the IC50 for ouabain inhibition of rubidium uptake and a 10-fold increase in cell survival on ouabain. Although the murine sequence was detectable in Southern blots of ouabain-resistant cells soon after transfection, this exogenous DNA was rapidly lost despite continued exposure to ouabain. Furthermore, we were unable to detect message expression by this genomic sequence in any of the three cell types tested. Instead, we found that all three ouabain-resistant cell lines exhibited point mutations in a domain of the alpha-subunit that has been implicated in ouabain sensitivity (H1-H2). One of these mutations (Asp121-Asn121 in OR8 cells) has been previously reported to cause ouabain resistance (Price, E.M., Rice, D.A., and Lingrel, J.B. (1989) J. Biol. Chem. 264, 21902-21906). Other novel mutations in the H2 transmembrane domain were also detected. We postulate that the "ouabain resistance gene" is important in the early selection process on ouabain but that the permanent ouabain-resistant phenotype is due to a stable mutation in one allele of the alpha-subunit of the Na,K-ATPase.
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PMID:Ouabain-resistant transfectants of the murine ouabain resistance gene contain mutations in the alpha-subunit of the Na,K-ATPase. 132 28

Chimeric cDNAs encoding regions of the Na,K-ATPase alpha-subunit and a sarcoplasmic reticulum Ca(2+)-ATPase were constructed and expressed together with the avian Na,K-ATPase beta-subunit cDNA in COS-1 cells to determine which regions of the alpha-subunit are required for assembly with the beta-subunit. Assembly was assayed by immune precipitation of the chimeric subunit with a monoclonal antibody to the avian beta-subunit. A chimera composed of the amino-terminal two-thirds of the Na,K-ATPase and carboxyl-terminal one-third of the Ca(2+)-ATPase did not assemble with the avian beta-subunit. In contrast, the reciprocal chimera, containing the carboxyl-terminal one-third of the Na,K-ATPase, assembled with the beta-subunit. A third chimera, in which 161 amino acids of the Na,K-ATPase carboxyl terminus replaced the corresponding amino acids of the Ca(2+)-ATPase carboxyl terminus, also assembled with the beta-subunit. These results suggest that the aminoacyl residues of the Na,K-ATPase alpha-subunit critical for subunit assembly lie within the carboxyl-terminal 16% of the sequence.
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PMID:The carboxyl-terminal 161 amino acids of the Na,K-ATPase alpha-subunit are sufficient for assembly with the beta-subunit. 132 16

Isoform 4b of the human plasma membrane Ca2+ pump was expressed in COS cells and in the baculovirus system (Sf9 cells). A 105-kDa pump fragment lacking the first two transmembrane domains and the so-called transduction domain was also expressed. The expression level was 2-4 times the background in COS cells and at least 7 times in the baculovirus system. Tests on membranes from both systems showed that the expressed pump was active. The expressed pump and the 105-kDa fragment were isolated from Sf9 cell membranes by calmodulin affinity chromatography. The pump had Ca(2+)-dependent ATPase activity with a calmodulin stimulation factor of 3, formed a La(3+)-stabilized phosphoenzyme, and had a KM (Ca2+) in the presence of calmodulin of about 1 microM. The 105-kDa fragment, assayed by the phosphoenzyme test on COS or Sf9 cell membranes or by ATPase measurements after isolation from Sf9 cells, proved inactive. Laser confocal microscopy on Sf9 cells showed that both the pump and the 105-kDa fragment were apparently associated with the plasma membrane. The expressed pump in COS and Sf9 cells and the endogenous pump in a number of other cell lines had a slower gel mobility (i.e. a higher apparent molecular mass) than the erythrocyte pump.
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PMID:Expression, purification, and properties of the plasma membrane Ca2+ pump and of its N-terminally truncated 105-kDa fragment. 133 59

Site-specific mutagenesis was used to analyse the functional roles of the residues Pro328 and Leu332 located in the conserved PEGLL motif of the predicted transmembrane helix M4 in the alpha 1-subunit of the ouabain resistant rat kidney Na+,K(+)-ATPase. cDNAs encoding either of the Na+,K(+)-ATPase mutants Pro328-->Ala and Leu332-->Ala, and wild type, were cloned into the expression vector pMT2 and transfected into COS-1 cells. Ouabain-resistant clones growing in the presence of 10 microM ouabain were isolated, and the Na+,K+, ATP and pH dependencies of the Na+,K(+)-ATPase activity measured in the presence of 10 microM ouabain were analysed. Under these conditions the exogenous expressed Na+,K(+)-ATPase contributed more than 95% of the Na+,K(+)-ATPase activity. The Pro328-->Ala mutant displayed a reduced apparent affinity for Na+ (K0.5 (Na+) 13.04 mM), relative to the wild type (K0.5 (Na+) 7.13 mM). By contrast, the apparent affinity for Na+ displayed by the Leu332-->Ala mutant was increased (K0.5 (Na+) 3.92 mM). Either of the mutants exhibited lower apparent affinity for K+ relative to the wild type (K0.5 (K+) 2.46 mM for Pro328-->Ala and 1.97 mM for Leu332-->Ala, compared with 0.78 mM for wild type). Both mutants exhibited higher apparent affinity for ATP than the wild type (K0.5 (ATP) 0.086 mM for Pro328-->Ala and 0.042 mM for Leu332-->Ala, compared with 0.287 mM for wild type). The influence of pH was in accordance with an acceleration of the E2 (K)-->E1 transition in the mutants relative to the wild type. These data are consistent with a role of Pro328 and Leu332 in the stabilization of the E2 form and of Pro328 in Na+ binding. The possible role of the mutated residues in K+ binding is discussed.
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PMID:Functional consequences of alterations to Pro328 and Leu332 located in the 4th transmembrane segment of the alpha-subunit of the rat kidney Na+,K(+)-ATPase. 133 48

Several isoforms of organellar Ca(2+)-ATPases have been identified, each of which is expressed in a tissue-specific manner. In order to examine the functional properties of fast-twitch (SERCA 1a), cardiac/slow-twitch (SERCA 2a), and non-muscle (SERCA 3) isoforms of the Ca(2+)-ATPase, cDNAs of each type were expressed transiently in COS-1 cells. A study of the Ca2+ dependence of Ca2+ uptake showed that SERCA 1 and SERCA 2 have identical Ca2+ dependences (K0.5 = pCa 6.87 +/- 0.03 and pCa 6.87 +/- 0.02, respectively), but SERCA 3 has a lower Ca2+ dependence (K0.5 = pCa 6.32 +/- 0.03). A study of the ATP dependence of Ca2+ uptake showed that SERCA 1, 2, and 3 have almost identical ATP dependences. Average Hill coefficients derived from Ca2+ uptake curves ranged from 1.7 to 1.8 for the three isoforms. In order to identify which regions of the linear sequence determine this difference in Ca2+ dependence, chimeric Ca(2+)-ATPases between SERCA 2 and SERCA 3 were constructed. Chimeric Ca(2+)-ATPases containing the nucleotide binding/hinge domain of SERCA 2 had SERCA 2 type Ca2+ dependence, but both nucleotide binding/hinge and COOH-terminal transmembrane domains of SERCA 3 were required for SERCA 3 type Ca2+ dependence. Accordingly, structural interactions between the nucleotide binding/hinge and COOH-terminal transmembrane domains appear to determine isoform-specific Ca2+ dependences.
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PMID:The nucleotide binding/hinge domain plays a crucial role in determining isoform-specific Ca2+ dependence of organellar Ca(2+)-ATPases. 138 16

The cDNA encoding a Ca(2+)-transport ATPase of frog (Rana esculenta) skeletal muscle was isolated and characterized. The deduced amino acid sequence, consisting of 994 residues, showed 89% identity to the fast twitch muscle sarcoplasmic reticulum Ca(2+)-ATPases of chicken and rabbit. Northern blot analysis using a fragment of this cDNA as probe detected a 5.0 kb message in frog skeletal muscle but did not detect any mRNA encoding sarcoplasmic reticulum Ca(2+)-ATPase in frog cardiac muscle. The enzymatic properties of the amphibian skeletal muscle Ca(2+)-ATPase were compared with those of the rabbit fast twitch muscle Ca(2+)-ATPase by functional expression of the cDNAs in COS-1 cells. The amphibian Ca(2+)-ATPase displayed a reduced apparent affinity for Ca2+ and an increased apparent affinity for the inhibitors, vanadate and thapsigargin, relative to the mammalian enzyme. This may be explained by a mechanism in which relatively more of the E2 conformation accumulated in the frog Ca(2+)-ATPase than in the mammalian enzyme.
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PMID:Deduced amino acid sequence and E1-E2 equilibrium of the sarcoplasmic reticulum Ca(2+)-ATPase of frog skeletal muscle. Comparison with the Ca(2+)-ATPase of rabbit fast twitch muscle. 138 27

We have cloned cDNA encoding the Ca2+ ATPase from fast-twitch skeletal muscle and, on the basis of its amino acid sequence and immunological studies of its topology, have made deductions concerning its secondary structure and active sites. These deductions have led us to test models for Ca2+ transport through expression of the protein in functional form in COS-1 cells, mutagenesis, and measurement of altered function. Mutation of about 250 of the 1000 amino acids making up the Ca2+ pump has indicated that the sites of high affinity Ca2+ binding are located in the center of the transmembrane domain and are made up from residues located in transmembrane sequences M4, M5, M6 and M8. The ATP binding site appears to be located in the headpiece and is made up from a series of loop sequences connecting alternating alpha helices and beta strands. Sites of conformational interaction appear in all domains throughout the Ca2+ pump. In our present model, Ca2+ transport occurs through binding to high affinity sites accessible to the cytoplasm in the E1 conformation, followed by release to the lumen from low affinity sites which form during the ATP-induced transition of the protein from the E1 to the E2 conformation.
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PMID:Site-directed mutagenesis of the Ca2+ ATPase of sarcoplasmic reticulum. 144 61

The existence of trans-acting regulatory factors has been demonstrated by in vivo competition with cis-acting sequences from both viral and eukaryotic genomes. Plasmids containing a functional SV40 origin of replication when transfected into permissive SV40 T-antigen producing COS-1 cells will amplify to high copy numbers (5,000 to 10,000) without inflicting toxic effects upon the host cell. This amplification vector (pSVori) has been used to amplify cis-acting regulatory elements which can act as competitors for positive and negative trans-acting factors in vivo. Using this amplification system we conducted experiments to determine whether amplification of alpha-fetoprotein (AFP) and albumin cis-acting promoter sequences could activate a corresponding co-transfected AFP-promoter-CAT or Alb-promoter-CAT expression vector in COS-1 cells. We used pMoMLV(-1009)AFPcat, or p(-308)Albcat-MoMLV as reporter genes and pSVori to amplify specific promoter sequences of the AFP or albumin promoter. Our experiments indicated that amplification of a region from -53 to -202 of the AFP promoter resulted in the activation of the pMoMLV(-1009)AFPcat and p(-308)Albcat-MoMLV expression vectors in COS-1 cells. Surprisingly, amplification of the albumin promoter sequences failed to activate either the pMoMLV(-1009)AFPcat or p(-308)Albcat-MoMLV plasmids.
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PMID:Derepression of a mouse alpha-fetoprotein expression vector in COS-1 cells by amplification of specific cis-acting sequences of the AFP promoter. 170 Dec 43

One of the cell-adhesion molecules (CAMs) responsible for rat hepatocyte aggregation has been described as a glycoprotein having an Mr of 105,000 (cell-CAM105). The Mr and localization of cell-CAM105 in liver membranes are very similar to those of liver ecto-ATPase, an ATPase with its nucleotide-hydrolysing site localized on the outside of the cell membrane. The protein sequence of the ecto-ATPase has been deduced from cDNA cloning. Structural analysis of the sequence indicates that the ecto-ATPase has immunoglobulin-like domains and is a member of the immunoglobulin superfamily. Since a group of proteins in the immunoglobulin superfamily has been shown to have functions related to cell adhesion, the structural characteristics of the ecto-ATPase further led to the possibility that the ecto-ATPase may have functions related to cell adhesion. In this paper, using the cDNA for the ecto-ATPase, the anti-peptide antibodies produced against peptides derived from the ecto-ATPase cDNA sequence and monoclonal antibodies against the cell-CAM105, we present evidence of identity between cell-CAM105 and ecto-ATPase. First, in Western immunoblots, two anti-cell-CAM105 monoclonal antibodies cross-reacted with the purified ecto-ATPase. Secondly, in immunodepletion experiments, antibodies against the ecto-ATPase depleted the same protein recognized by the anti-cell-CAM105 antibodies. Thirdly, in two-dimensional gel-electrophoretic analysis, anti-peptide antibodies generated against an extracellular N-terminal peptide and the intracellular C-terminal peptides of the ecto-ATPase immunoprecipitated proteins of similar isoelectric points and Mr values to those of the cell-CAM105. Fourthly, proteins immunoprecipitated by anti-ecto-ATPase antibodies and anti-cell-CAM105 antibodies have similar V8-proteinase-digest peptide maps. Finally, monoclonal antibodies against the cell-CAM105 specifically recognized the protein expressed in COS cells transfected with the ecto-ATPase cDNA. These results indicate that the ecto-ATPase cDNA codes for a protein that is identical with the cell-CAM105. Since the ecto-ATPase has structural features of immunoglobulin domains, the identity of cell-CAM105 with ecto-ATPase leads to the conclusion that this liver CAM, similarly to neuronal CAM, is also a member of the immunoglobulin supergene family. Furthermore, immunological studies indicate that the cell-CAM105/ecto-ATPase is composed of two isoforms of different C-terminal sequences. The association of ATPase activity with cell-CAM105 raises the possibility that extracellular nucleotides may play important roles in regulating cell adhesion.
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PMID:Immunochemical characterization of two isoforms of rat liver ecto-ATPase that show an immunological and structural identity with a glycoprotein cell-adhesion molecule with Mr 105,000. 183 73


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