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)

We have reconstituted an ATP-dependent protein folding machinery using purified yeast cytosolic proteins. The S. cerevisiae Hsp70 Ssa1p and the DnaJ homolog Ydj1p refolded denatured firefly luciferase. In E. coli, efficient refolding of luciferase requires the Hsp70 DnaK and two modulators, DnaJ and GrpE, that synergistically stimulate its ATPase activity. Exchanging DnaJ homologs between the S. cerevisiae and E. coli systems revealed that their ability to stimulate Hsp70 ATPase activity was conserved. In contrast, GrpE further stimulated only DnaK's ATPase activity. Efficient refolding of luciferase by Ssa1p and DnaJ, but not by DnaK and Ydj1p, suggests that a compatible Hsp70/DnaJ homolog pair can act as a protein folding machinery.
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PMID:Conserved ATPase and luciferase refolding activities between bacteria and yeast Hsp70 chaperones and modulators. 763 93

Monocyte-macrophage differentiation was used as a model system for studying gene regulation of the human vacuolar H(+)-ATPase (V-ATPase). We examined mRNA levels of various V-ATPase subunits during differentiation of both native monocytes and the cell line THP-1, and found that transcriptional and post-transcriptional mechanisms could account for increases in cell V-ATPase content. From nuclear runoff experiments, we found that one subunit in particular, the B2 isoform (Mr = 56,000), was amplified primarily by transcriptional means. We have begun to examine the structure of the B2 subunit promoter region. Isolation and sequencing of the first exon and 5'-flanking region of this gene reveal a TATA-less promoter with a high G + C content. Primer extension and ribonuclease protection analyses indicate a single major transcriptional start site. We transfected promoter-luciferase reporter plasmids into THP-1 cells to define sequences that mediate transcriptional control during monocyte differentiation. We found that sequences downstream from the transcriptional start site were sufficient to confer increased expression during THP-1 differentiation. DNase I footprinting and sequence analysis revealed the existence of multiple AP2 and Sp1 binding sites in the 5'-untranslated and proximal coding regions.
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PMID:Transcriptional regulation of the vacuolar H(+)-ATPase B2 subunit gene in differentiating THP-1 cells. 770 73

Biologically active amidated gastrin is synthesized by carboxyl-terminal alpha-amidation of a glycine-extended progastrin post-translational processing intermediate (G-Gly). Although plasma levels of G-Gly are equivalent to those of gastrin, G-Gly has essentially no acute effect on gastric acid secretion. However, we have observed that inhibition of gastrin amidation leads to increased plasma concentrations of G-Gly and enhanced gastric acid secretion. We hypothesized, therefore, that G-Gly might have a chronic effect to increase H+,K(+)-ATPase expression in gastric parietal cells. In the present studies, we observed that a 2-day preincubation with G-Gly significantly enhanced histamine-stimulated [14C]aminopyrine uptake by isolated canine gastric parietal cells but acutely administered G-Gly had no effect. On Northern blot analysis, both G-Gly and gastrin dose-dependently increased H+,K(+)-ATPase alpha-subunit gene expression with maximal induction (225 +/- 35 and 170 +/- 29% of basal, mean +/- S.E.) achieved at concentrations of 10(-9) M G-Gly and 10(-8) M gastrin, respectively. Using an H+,K(+)-ATPase alpha-subunit gene-luciferase chimeric reporter construct transfected into primary cultured parietal cells, we observed that both G-Gly and gastrin increased luciferase activity in a manner similar to that obtained by Northern blot analysis. L365,260, a specific gastrin/CCKB receptor antagonist, completely reversed the stimulation of luciferase activity induced by gastrin but had no effect on G-Gly-stimulated activity. Gastrin increased [Ca2+]i, although G-Gly did not, however, genistein (a tyrosine kinase inhibitor) significantly reduced induction of luciferase activity by both G-Gly and gastrin. Specific binding of 125I-Leu15-G2-17-Gly to gastric parietal cells was dose-dependently displaced by G2-17-Gly but not by gastrin nor L365,260. Gastrin peptides truncated at the carboxyl- (G1-13) and amino terminus (G5-17-Gly) both induced H+,K(+)-ATPase alpha-subunit gene expression and inhibited 125I-Leu15-G2-17-Gly binding, but were less potent than G2-17-Gly. These data indicate that G-Gly may have a functional role in potentiating gastric acid secretagogue action via enhanced expression of the gene responsible for H+ generation through action at a novel receptor that can be distinguished from the gastrin/CCKB receptor. Thus, both the substrate and product of the terminal progastrin processing reaction appear to have complementary functions in regulation of gastric acid secretion.
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PMID:Glycine-extended progastrin processing intermediates induce H+,K(+)-ATPase alpha-subunit gene expression through a novel receptor. 774 46

The actin Ser14 hydroxyl is one of a number of ligands that binds to the gamma-phosphate of ATP thereby stabilizing the actin.ATP complex. In yeast actin, conversion of Ser14 to Ala (S14A), causes a temperature-sensitive phenotype in vivo and temperature-sensitive polymerization defects in vitro (Chen, X., and Rubenstein, P. A. (1995) J. Biol. Chem. 270, 11406-11414). Here, using a new luciferase-based procedure, we show that the mutation results in a 40-60-fold decrease in actin's affinity for ATP. The mutation causes a decrease in the intrinsic ATPase activity of both Ca- and Mg-G-actin at 30 degrees C and alters the protease susceptibility of sites on subdomain 2. Ca-S14A-actin but not Mg-S14A-actin binds etheno-ATP at 37 degrees C. Intrinsic tryptophan fluorescence measurements show that at 37 degrees C, Mg-S14A-actin but not the calcium form unfolds. CD measurements show the mutation causes a decrease in the apparent denaturation temperature for Ca-actin from 57 to 45 degrees C and for the magnesium form a decrease from 52 to 40 degrees C. Based on a re-examination of actin's crystal structure coordinates, we propose that the Ser14 hydroxyl forms a polar bridge between the ATP gamma-phosphate and the amide nitrogen of Gly74, thus conferring additional stability on the actin small domain.
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PMID:The effect of the S14A mutation on the conformation and thermostability of Saccharomyces cerevisiae G-actin and its interaction with adenine nucleotides. 774 78

Thyroid hormone (T3) stimulates Na,K-ATPase activity and alpha and beta subunit mRNA abundances in myocardial cells in vivo and in vitro. In this study, we used transient transfection and nuclear run-on assays to determine whether T3 regulates the transcription rate of the Na,K-ATPase alpha 2 subunit gene. Primary cultures of neonatal rat cardiac myocytes were incubated with 100 nM T3 for 1, 3, and 6 d, and alpha 2 mRNA levels were measured by Northern blot hybridization analysis. There was no change in the abundance of alpha 2 mRNA by 1 d of T3 treatment, whereas a two- and threefold increase in alpha 2 mRNA was evident when cells were exposed to T3 for 3 and 6 d, respectively. A portion of the rat alpha 2 gene containing 1700 base pairs (bp) of 5'-flanking DNA sequence was isolated and fused to the firefly luciferase gene. Transient transfection experiments utilizing this chimeric gene showed no T3 trans-activation of reporter gene activity either in the absence or presence of cotransfected beta 1 or alpha 1 isoforms of rat T3 receptor (T3R). In contrast, cotransfection of T3R facilitated a strong stimulation of luciferase activity driven by a construct containing a single copy of a palindromic T3 response element (TRE). Nuclear run-on analysis indicated that the rate of transcription of the endogenous alpha 2 gene was enhanced 1.2-fold at 3 d of T3 treatment, and was not regulated at either 1 or 6 d. These results indicate that the T3-dependent increase in alpha 2 mRNA content at 6 d is mediated at a post-transcriptional level. Unexpectedly, we observed a T3-dependent three-to sixfold repression of alpha 2/luciferase expression in cardiac myocytes cotransfected with T3R. Deletion analysis of the 5' end of the alpha 2 gene revealed a negative TRE between nucleotides -354 and -100.
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PMID:Thyroid hormone regulation of Na,K-ATPase alpha 2 gene expression in cardiac myocytes. 780 25

Central to the chaperone function of Hsp70 stress proteins including Escherichia coli DnaK is the ability of Hsp70 to bind unfolded protein substrates in an ATP-dependent manner. Mg2+/ATP dissociates bound substrates and, furthermore, substrate binding stimulates the ATPase of Hsp70. This coupling is proposed to require a glutamate residue, E175 of bovine Hsc70, that is entirely conserved within the Hsp70 family, as it contacts bound Mg2+/ATP and is part of a hinge required for a postulated ATP-dependent opening/closing movement of the nucleotide binding cleft which then triggers substrate release. We analyzed the effects of dnaK mutations which alter the corresponding glutamate-171 of DnaK to alanine, leucine or lysine. In vivo, the mutated dnaK alleles failed to complement the delta dnaK52 mutation and were dominant negative in dnaK+ cells. In vitro, all three mutant DnaK proteins were inactive in known DnaK-dependent reactions, including refolding of denatured luciferase and initiation of lambda DNA replication. The mutant proteins retained ATPase activity, as well as the capacity to bind peptide substrates. The intrinsic ATPase activities of the mutant proteins, however, did exhibit increased Km and Vmax values. More importantly, these mutant proteins showed no stimulation of ATPase activity by substrates and no substrate dissociation by Mg2+/ATP. Thus, glutamate-171 is required for coupling of ATPase activity with substrate binding, and this coupling is essential for the chaperone function of DnaK.
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PMID:The chaperone function of DnaK requires the coupling of ATPase activity with substrate binding through residue E171. 790 76

The two major molecular chaperone families that mediate ATP-dependent protein folding and refolding are the heat shock proteins Hsp60s (GroEL) and Hsp70s (DnaK). Clp proteins, like chaperones, are highly conserved, present in all organisms, and contain ATP and polypeptide binding sites. We discovered that ClpA, the ATPase component of the ATP-dependent ClpAP protease, is a molecular chaperone. ClpA performs the ATP-dependent chaperone function of DnaK and DnaJ in the in vitro activation of the plasmid P1 RepA replication initiator protein. RepA is activated by the conversion of dimers to monomers. We show that ClpA targets RepA for degradation by ClpP, demonstrating a direct link between the protein unfolding function of chaperones and proteolysis. In another chaperone assay, ClpA protects luciferase from irreversible heat inactivation but is unable to reactivate luciferase.
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PMID:A molecular chaperone, ClpA, functions like DnaK and DnaJ. 799 9

Previous work in our laboratories has shown that, amongst other effects, irradiation of frog skin with low intensity ultrasound causes reductions in the chemical driving force of the short-circuit current. This indicated that either the Na/K dependent ATPase or ATP availability were being reduced. We measured the effect of ultrasound irradiation on ATP and NA/K-dependent ATPase from inverted erythrocyte ghosts and on firefly luciferin and luciferase activity. Our findings demonstrate that ultrasonic cavitation-induced sonochemical reactions were responsible for irreversible inactivation of luciferase and ATPase but had little or no effect on ATP and luciferin. We measured the levels of hydrogen peroxide generated by ultrasound under the conditions of our experiments and found that it could account for only part of the enzyme inactivation observed. Free radical scavengers/antioxidants were capable of fully protecting the enzymes from ultrasound-induced inactivation. These findings demonstrate that, in addition to hydrogen peroxide, free radicals generated by ultrasound are responsible for the effects.
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PMID:Inactivation of firefly luciferase and rat erythrocyte ATPase by ultrasound. 800 40

A cDNA clone encoding a zinc finger protein (AREB6) was isolated from a HeLa cell expression library using a positive regulatory element (-102 to -58) of rat Na,K-ATPase alpha 1 subunit gene (Atp1a1) as a probe. The clone is apparently an extended one of Nil-2-a originally isolated as a negative regulator of interleukin 2 gene [Williams, T.M. et al. (1991) Science 254, 1791-1794]. The open reading frame encodes 1,124 amino acids. It contains 7 zinc-finger motifs arranged in two widely separated clusters. A glutamic acid-rich region is observed at the C terminus from residues 989 to 1123. Co-transfection of the AREB6 cDNA with Atp1a1 fused to a reporter luciferase gene indicated that the AREB6 protein enhances or represses the promoter activity of the gene depending on the quantity of cDNA and on the cell type. The mRNA of AREB6 is expressed in heart and skeletal muscle, but not in liver, spleen, or pancreas. Genomic Southern analysis indicated that the gene encoding AREB6 is present as only one copy or two at most. Another cDNA clone obtained by using the same probe was identified as HEB [Hu, J.S., Olson, E.N., & Kingston, R.E. (1992) Mol. Cell. Biol. 12, 1031-1042]. Co-transfection of the cDNA enhanced or repressed the promoter activity of Atp1a1 depending on the cell type.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Transcription factors positively and negatively regulating the Na,K-ATPase alpha 1 subunit gene. 813 42

We have carried out a detailed analysis of viral mRNAs and proteins produced in cultured cells infected with a temperature-sensitive vaccinia virus mutant (ts36) containing a modified nucleoside triphosphate phosphohydrolase I (NPH-I), a nucleic acid-dependent ATPase. Using a recombinant virus (ts36LUC) which expresses the luciferase marker, we showed in seven different cell lines that early expression of the receptor gene is strongly inhibited (73.8 to 98.7%) at the nonpermissive temperature. The steady-state levels of different early viral polypeptides were also severely reduced. Analysis of steady-state mRNA levels for two early genes (DNA polymerase and D5) showed that inhibition of early polypeptide synthesis correlated with a reduction in the levels of mRNA accumulated at the nonpermissive temperature. Analysis of steady-state levels of late viral polypeptides and of mRNAs indicated that NPH-I regulation of intermediate and late gene expression is direct and not simply a consequence of its role in inhibiting early gene expression. Characterization of a rescued virus (R36) demonstrated that the temperature-sensitive phenotype of ts36 is due solely to the point mutation in the NPH-I gene. The mutant phenotype is not due to reduced levels of NPH-I present in ts36 virions or to the differential stability of this enzyme in cells infected at the nonpermissive temperature but to inhibition of normal enzymatic activity for this protein. Measurement of viral transcriptional activity in permeabilized purified virions demonstrated that NPH-I is required for normal rates of transcription in vaccinia virus. Our findings show ts36 to be a strongly defective early mutant of vaccinia virus and prove that NPH-I plays a key role in the control of early and late virus gene expression, possibly by way of an auxiliary function which regulates mRNA transcription during the virus growth cycle.
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PMID:Vaccinia virus nucleoside triphosphate phosphohydrolase I controls early and late gene expression by regulating the rate of transcription. 823 Apr 76


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