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)

BiP possesses ATP binding/hydrolysis activities that are thought to be essential for its ability to chaperone protein folding and assembly in the endoplasmic reticulum (ER). We have produced a series of point mutations in a hamster BiP clone that inhibit ATPase activity and have generated a species-specific anti-BiP antibody to monitor the effects of mutant hamster BiP expression in COS monkey cells. The enzymatic inactivation of BiP did not interfere with its ability to bind to Ig heavy chains in vivo but did inhibit ATP-mediated release of heavy chains in vitro. Immunofluorescence staining and electron microscopy revealed vesiculation of the ER membranes in COS cells expressing BiP ATPase mutants. ER disruption was not observed when a "44K" fragment of BiP that did not include the protein binding domain was similarly mutated but was observed when the protein binding region of BiP was expressed without an ATP binding domain. This suggests that BiP binding to target proteins as an inactive chaperone is responsible for the ER disruption. This is the first report on the in vivo expression of mammalian BiP mutants and is demonstration that in vitro-identified ATPase mutants behave as dominant negative mutants when expressed in vivo.
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PMID:In vivo expression of mammalian BiP ATPase mutants causes disruption of the endoplasmic reticulum. 761 64

Although many in vitro gene transfer methods already exist, such as calcium phosphate precipitation, electroporation, or cationic liposomes, these methods cause significant cell injury and cell death. The study of the biology of endogenous autocrine-paracrine vasoactive systems such as the renin-angiotensin system in vascular cells is limited by the lack of a suitable gene transfer method with high efficiency of transfection and expression that will permit cell biology studies. Recently, the Sendai virus (hemagglutinating virus of Japan, HVJ)-liposome-mediated gene transfer method has been shown to be an efficient and nontoxic method of gene transfer. In this study, we characterized the efficiency and suitability of the HVJ method for vascular biology research. Using SV40 T-antigen complementary DNA (cDNA), we initially compared the efficiency of the HVJ method and lipofection for transfection of cultured vascular smooth muscle cells (VSMCs). We observed that after 35 minutes of incubation, the HVJ method exhibited a 10-fold higher efficiency of transfection than lipofection. We used this method to study vascular angiotensin converting enzyme (ACE) expression in cultured VSMCs and cultured rat carotid arteries in vitro. The HVJ method of transfection of human ACE cDNA into VSMCs and COS cells was significantly more efficient than lipofection. Using this method, we demonstrated that transfection of ACE cDNA resulted in increased DNA synthesis, which was inhibited by the specific angiotensin II receptor antagonist DuP 753 (10(-6) M).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Novel in vitro gene transfer method for study of local modulators in vascular smooth muscle cells. 768 4

Site-specific mutagenesis was used to replace Asn326 in transmembrane segment M4 of the ouabain-insensitive alpha 1-isoform of rat kidney Na+, K(+)-ATPase. Mutant Asn326-->Leu was functional as demonstrated by the ability of COS cells expressing the mutant enzyme to grow in the presence of ouabain. In three independent assays encompassing Na+ titrations of Na+,K(+)-ATPase activity, Na(+)-ATPase activity, and phosphorylation from ATP, the Asn326-->Leu mutant displayed a reduced apparent affinity for Na+. By contrast, this mutant exhibited a slightly increased apparent affinity for K+ relative to the wild-type enzyme. In the presence of Na+ without K+, the Asn326-->Leu mutant hydrolyzed ATP at a high rate corresponding to 32% of the maximal Na+,K(+)-ATPase activity, and the rate of dephosphorylation of the phosphoenzyme intermediate was enhanced in the mutant relative to that of the wild-type enzyme. Oligomycin, known to stabilize the Na(+)-occluded phosphoenzyme intermediate, reduced the dephosphorylation rate of the mutant and increased the steady-state phosphoenzyme level formed by the mutant at least 3-fold, whereas an increase in the steady-state phosphoenzyme level of only 10-15% was determined for the wild-type enzyme. The molecular turnover number for the Na+,K(+)-ATPase reaction, calculated when the steady-state phosphoenzyme level obtained in the presence of oligomycin was taken as a measure of the concentration of active sites, was slightly reduced relative to that of the wild-type enzyme. The data are discussed in terms of a role for Asn326 in binding of cytoplasmic Na+ and in mediation of inhibition of dephosphorylation.
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PMID:Functional consequences of mutation Asn326-->Leu in the 4th transmembrane segment of the alpha-subunit of the rat kidney Na+, K(+)-ATPase. 772 43

ATP-powered Ca2+ pumps are located in the plasma membrane (PMCA) and the sarco(endo)plasmic reticulum (SERCA). The two pump types share numerous structural and functional features; nevertheless, they are strictly targeted to different cell compartments. Chimeric SERCA/PMCA pumps were constructed to investigate the structural determinants responsible for their specific cellular location. The level of expression of the chimeric constructs and of the wild-type pumps in COS-7 and Sf9 cells was the same and so was their stability. One exception was chimera D, which showed special propensity to degradation. The chimeric constructs had no Ca(2+)-dependent ATPase activity, although in one trace amounts of the Ca(2+)-dependent phosphoenzyme intermediate were detected. Thus, the exchange of the regions encompassing the (NH2-terminal) transmembrane domains apparently is incompatible with the activity of the pump. The immunofluorescence experiments showed that the 85 NH2-terminal residues of the SERCA pump, encompassing the first transmembrane domain, contain a signal-promoting retention of chimeric constructs otherwise consisting of the PMCA pump structure in the endoplasmic reticulum. Additional structural determinants most likely also contribute to the retention of the SERCA pump in the endoplasmic reticulum: one chimeric construct (E) composed of the first two transmembrane domains of the PMCA pump, followed by the remainder of the SERCA pump structure, was still retained, even if not completely, in the endoplasmic reticulum.
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PMID:Subcellular targeting of the endoplasmic reticulum and plasma membrane Ca2+ pumps: a study using recombinant chimeras. 776 60

Positive and negative effects of DNA replication on gene transcription have been documented in a variety of systems. We examined the effects of the simian virus 40 (SV40) origin of replication on transcription from the human immunodeficiency virus type 1 (HIV-1) promoter, using a transient expression assay in COS-1 cells. The basal activity and Tat transactivation of the HIV promoter were greatly stimulated by the SV40 origin of replication independent of its position relative to the long terminal repeat. These effects were abolished by mutational inactivation of the SV40 origin and were reduced by a DNA replication inhibitor. The magnitude of promoter activation exceeded the increment expected from the increase in template number resulting from DNA replication. The SV40 T-antigen-induced DNA replication augmented the generation of both processive and nonprocessive HIV long terminal repeat-directed transcripts, and Tat primarily enhanced the initiation of those transcripts that were destined to be efficiently elongated. Our data suggest that the HIV promoter displays greater transcriptional activity on replicative DNA templates. This property may influence the activity of integrated HIV provirus and its transition from latency to productive infection.
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PMID:Effects of the simian virus 40 origin of replication on transcription from the human immunodeficiency virus type 1 promoter. 781 9

Site-specific mutagenesis was used to replace Glu329, Glu781, Asp806, Thr809, and Asp810 in the transmembrane domain of the ouabain-insensitive alpha 1-isoform of rat kidney Na+,K(+)-ATPase. cDNAs encoding any of the mutants Glu329-->Ala, Glu781-->Ala, Asp806-->Asn, Thr809-->Ala, and Asp810-->Asn were transfected into COS cells, and transfectants were grown in the presence of ouabain to inhibit the endogenous COS cell Na+,K(+)-ATPase. Mutants Glu781-->Ala and Thr809-->Ala were functional as evidenced by their ability to confer ouabain resistance to the cells, whereas mutants Glu329-->Ala, Asp806-->Asn, and Asp810-->Asn were inactive. The apparent Na+ affinities determined by titrations of Na+,K(+)-ATPase activity, Na(+)-ATPase activity, and phosphorylation from ATP in mutants Glu781-->Ala and Thr809-->Ala were strongly reduced relative to the affinity of the wild type (6-8-fold increase in K0.5 for Na+ in the phosphorylation assay for both mutants). The Glu781-->Ala mutant displayed a 3-4-fold reduction in the apparent affinity for K+ and was able to hydrolyze ATP at a high rate in the absence of K+ (Vmax for Na(+)-ATPase activity 5-fold higher than that of the wild-type enzyme). The steady-state phosphoenzyme level formed by the Glu781-->Ala mutant was increased 3-fold by addition of oligomycin, whereas only a slight effect of oligomycin was observed for mutant Thr809-->Ala and the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mutant Glu781-->Ala of the rat kidney Na+,K(+)-ATPase displays low cation affinity and catalyzes ATP hydrolysis at a high rate in the absence of potassium ions. 782 94

An allelic variant of the ouabain-insensitive rat kidney Na+,K(+)-ATPase alpha 1-isoform was identified by chance in a cDNA library. The variant differed from the wild-type rat kidney Na+,K(+)-ATPase by a single G-to-C base substitution in the cDNA, which on amino acid level gave rise to a glutamine in place of the glutamate residue Glu329 previously suggested as a likely donator of oxygen ligands for Na+ and K+ binding. The variant cDNA was transfected into COS-1 cells and the transfectants expanded with success into stable cell lines that were able to grow in the presence of a concentration of ouabain highly cytotoxic to the parental cells containing only the endogenous COS-1 cell Na+,K(+)-ATPase. Under these conditions, the viability of the cells depended on the cation transport mediated by the ouabain-insensitive Glu329-->Gln variant, whose cDNA was shown by polymerase chain reaction amplification to be stably integrated into the COS-1 cell genome. The maximum specific ATP hydrolysis activity of isolated plasma membranes of the Glu329-->Gln variant did not differ significantly from that of plasma membranes containing the wild type. A method was established for measurement of the phosphorylation capacity of the expressed Glu329-->Gln variant and wild-type enzyme, and it was thereby demonstrated that the variant had a turnover number similar if not identical to that of the wild-type.
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PMID:A Glu329-->Gln variant of the alpha-subunit of the rat kidney Na+,K(+)-ATPase can sustain active transport of Na+ and K+ and Na+,K(+)-activated ATP hydrolysis with normal turnover number. 790 Oct 51

Na,K-ATPase is a potential target for regulatory phosphorylation by protein kinase A and C (PKA and PKC). To identify the phosphorylation sites, we have mutated the alpha 1-subunit of Bufo marinus in a highly conservative PKA and in 20 different PKC consensus sequences. The mutants were expressed in Xenopus oocytes and their phosphorylation capacity tested in homogenates upon stimulation of PKA or PKC. While serine 943 (Ser-943) was identified as a unique target site for PKA, none of the PKC consensus serine or threonine residues are implicated in PKC phosphorylation. Controlled trypsinolysis of phosphorylated alpha-subunits of various purified enzyme preparations and of alpha/beta complexes from oocyte homogenates revealed that PKC phosphorylation was exclusively associated with the N terminus. A fusion protein containing the first 32 amino acids of the Bufo alpha-subunit was phosphorylated in vitro and serine and threonine residues (Thr-15 and Ser-16) in this region were identified by site-directed mutagenesis as the PKC phosphorylation sites. Finally, the Bufo alpha-subunit was phosphorylated by protein kinases in transfected COS-7 cells. In intact cells, PKA stimulation induced phosphorylation exclusively on Ser-943 and PKC stimulation mainly on Thr-15 and Ser-16, which are contained in a novel PKC phosphorylation motif.
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PMID:Phosphorylation of the Na,K-ATPase alpha-subunit by protein kinase A and C in vitro and in intact cells. Identification of a novel motif for PKC-mediated phosphorylation. 792 6

Five different chimeric fusion proteins between the sarcoplasmic reticulum Ca(2+)-ATPase and the rat kidney alpha 1 isoform of the Na+,K(+)-ATPase were expressed in COS-1 cells and analyzed functionally. In two chimeras, the only Na+,K(+)-ATPase-derived part consisted of the region corresponding to the third transmembrane segment. These proteins phosphorylated from ATP in the presence of a high Ca2+ concentration and from Pi in the absence of Ca2+, but the phosphorylation displayed strongly reduced sensitivity to inhibition by thapsigargin. In two other chimeras, the N-terminal two-thirds of the molecule were derived from Na+,K(+)-ATPase, except the region containing the fourth transmembrane segment. These proteins were unable to phosphorylate from ATP but phosphorylated from Pi, the latter reaction being insensitive to inhibition by thapsigargin. In the last chimera, the N-terminal two-thirds of the molecule were derived from Na+,K(+)-ATPase, except the region corresponding to both the third and the fourth transmembrane segments. This chimera phosphorylated from Pi in the reaction that was fully sensitive to inhibition by thapsigargin. It can be concluded that the third transmembrane segment is indispensable to thapsigargin sensitivity of the Ca(2+)-ATPase and most likely, therefore, constitutes a major binding region for thapsigargin.
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PMID:Transmembrane segment M3 is essential to thapsigargin sensitivity of the sarcoplasmic reticulum Ca(2+)-ATPase. 792 87

An allelic variant of the ouabain-insensitive rat kidney Na+,K(+)-ATPase alpha 1-isoform was identified by chance in a cDNA library. The variant differed from the wild-type rat kidney Na+,K(+)-ATPase by a single G to C base substitution in the cDNA, which on the amino acid level gave rise to a glutamine in place of the glutamate residue Glu329, previously suggested as a likely donator of oxygen ligands for Na+ and K+ binding. The variant cDNA was transfected into COS-1 cells and the transfectants expanded with success into stable cell lines that were able to grow in the presence of a concentration of ouabain highly cytotoxic to the parental cells containing only the endogenous COS-1 cell Na+,K(+)-ATPase. Under these conditions, the viability of the cells depended on the cation transport mediated by the ouabain-insensitive Glu329-->Gln variant, whose cDNA was shown by polymerase chain reaction amplification to be stably integrated into the COS-1 cell genome. Functional analysis on isolated plasma membranes demonstrated that the Glu329-->Gln variant was able to catalyze Na(+)- and K(+)-activated ATPase activity with a maximum turnover number similar if not identical to that of the wild type, but the variant exhibited a reduced affinity for both cations corresponding to a 2-fold increase in K0.5 for Na+ and a 6-fold increase in K0.5 for K+. Moreover, the apparent affinity for ATP was increased 15-fold in the variant relative to wild-type Na+,K(+)-ATPase. The Na+,K(+)-ATPase activity of the variant displayed an anomalous pH dependence with a down-shift of the pH optimum and a nearly constant rate in the range between pH 7.0 and 8.7.
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PMID:Glutamate 329 located in the fourth transmembrane segment of the alpha-subunit of the rat kidney Na+,K+-ATPase is not an essential residue for active transport of sodium and potassium ions. 824 Nov 90


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