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)

Resistance to toxic oxyanions of arsenic and antimony in Escherichia coli is conferred by the conjugative R-factor R773, which encodes an ATP-driven anion extrusion pump. The ars operon is composed of three structural genes, arsA, arsB, and arsC. Although transcribed as a single unit, the three genes are differentially expressed as a result of translational differences, such that the ArsA and ArsC proteins are produced in high amounts relative to the amount of ArsB protein made. Consequently, biochemical characterization of the ArsB protein, which is an integral membrane protein containing the anion-conducting pathway, has been limited, precluding studies of the mechanism of this oxyanion pump. To overexpress the arsB gene, a series of changes were made. First, the second codon, an infrequently used leucine codon, was changed to a more frequently utilized codon. Second, a GC-rich stem-loop (delta G = -17 kcal/mol) between the third and twelfth codons was destabilized by changing several of the bases of the base-paired region. Third, the re-engineered arsB gene was fused 3' in frame to the first 1458 base pairs of the arsA gene to encode a 914-residue chimeric protein (486 residues of the ArsA protein plus 428 residues of the mutated ArsB protein) containing the entire re-engineered ArsB sequence except for the initiating methionine. The ArsA-ArsB chimera has been overexpressed at approximately 15-20% of the total membrane proteins. Cells producing the chimeric ArsA-ArsB protein with an arsA gene in trans excluded 73AsO2- from cells, demonstrating that the chimera can function as a component of the oxyanion-translocating ATPase.
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PMID:Construction of a chimeric ArsA-ArsB protein for overexpression of the oxyanion-translocating ATPase. 128 74

The energy requirement for protein translocation across membrane was studied with inverted membrane vesicles from an Escherichia coli strain that lacks all components of F1F0-ATPase. An ompF-lpp chimeric protein was used as a model secretory protein. Translocation of the chimeric protein into membrane vesicles was totally inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or valinomycin and nigericin and partially inhibited when either valinomycin or nigericin alone was added. Depletion of ATP with glucose and hexokinase resulted in the complete inhibition of the translocation process, and the inhibition was suppressed by the addition of ATP-generating systems such as phosphoenolpyruvate-pyruvate kinase or creatine phosphate-creatine kinase. These results indicate that both the proton motive force and ATP are required for the translocation process. The results further suggest that both the membrane potential and the chemical gradient of protons (delta pH), of which the proton motive force is composed, participate in the translocation process.
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PMID:In vitro translocation of protein across Escherichia coli membrane vesicles requires both the proton motive force and ATP. 302 75

Cytogeneticists recognize that karyotypic abnormalities are associated with specific malignancies. In 1960, Nowell described the Philadelphia chromosome (Ph) and its relationship to chronic myelogenous leukemia (CML). Subsequent work in molecular genetics and biology has revealed that the Ph is a translocation that causes fusion of gene sites that code for the break cluster region (BCR) and the avian blastic leukemia (ABL) proteins. This so-called fusion protein is present in a large percentage of the patients who have CML. A related fusion protein is seen in about one third of patients with acute lymphoblastic leukemia. The BCR-ABL fusion protein results in increased tyrosine kinase activity. The mechanism of action is thought to be via signal transduction related to guanosine triphosphatase activating protein which interacts with a ras-p21 binding protein. Acute promyelocytic leukemia (APL) is associated with the cytogenetic abnormality of t(15;17). This alters the promyelocytic leukemia (PML) and the retinoic acid receptor alpha (RARA) gene sites. Two fusion proteins are the result of this cytogenetic abnormality. They are termed PML-RARA and RARA-PML. Only one, the PML-RARA, is associated with APL. The PML-RARA chimeric protein has two zinc finger-like regions. It retains the ligand binding domain of RARA. The protein called PML has some similarities with a family of proteins which are thought to fuse to proto-oncogenes and to act as transforming proteins. The role of classical cytogenetics and the added capability of molecular biology has helped to elucidate some of the potential mechanisms for the development of cancer and provided additional understanding of neoplasia. (ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cytogenetics, gene fusions, and cancer. 748 13

In an attempt to understand the mechanisms of sorting of mitochondrial inner membrane proteins, we have analyzed the import of subunit 9 of the mitochondrial F1F0-ATPase (Su9) from Neurospora crassa, an integral inner membrane protein. A chimeric protein was used consisting of the presequence and the first transmembrane domain of Su9 fused to mouse dihydrofolate reductase (preSu9(1-112)-DHFR). This protein attains the correct topology across the inner membrane (Nout-Cin) following import. The transmembrane domain becomes first completely imported into the matrix, where after processing of the presequence, it mediates membrane insertion and export of the N-terminal tail. Import and export steps can be experimentally dissected into two distinct events. Translocation of the N-terminal hydrophilic tail out of the matrix was blocked when the presequence was not processed, indicating an important role of the sequences and charges flanking the hydrophobic domain. Furthermore, export was supported by a delta pH and required matrix ATP hydrolysis. Thus the hydrophobic transmembrane domain operates as a membrane insertion signal and not as a stop-transfer signal. Our findings suggest that several aspects of this sorting process have been conserved from their prokaryotic ancestors.
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PMID:Conservative sorting of F0-ATPase subunit 9: export from matrix requires delta pH across inner membrane and matrix ATP. 762 45

Chimeric proteins consisting of parts from the alpha-subunit of Torpedo californica (Na, K) ATPase (N) and the rabbit sarcoplasmic reticulum Ca-ATPase (C) were expressed in Xenopus oocytes by injecting the respective chimeric cRNA in combination with cRNA for the beta-subunit of Torpedo (Na, K) ATPase. The chimeric protein (NCN) that consisted of the NH2-terminal and COOH-terminal one-thirds of the alpha-subunit of the (Na, K) ATPase and the central one-third of the Ca-ATPase was able to assemble with the beta-subunit in the same fashion as the wild-type alpha-subunit of the (Na, K) ATPase (NNN). On the other hand, chimeric proteins in which the COOH-terminal one-third was derived from the Ca-ATPase (NNC and NCC) were unable to form stable complexes with the beta-subunit. These results suggest that the COOH-terminal one-third of the (Na, K) ATPase alpha-subunit is required for the assembly with the beta-subunit.
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PMID:Expression of (Na, K) ATPase/Ca-ATPase chimeric proteins in Xenopus oocytes revealed that the COOH-terminal one-third of the (Na, K) ATPase alpha-subunit is involved in assembly with the beta-subunit. 782 18

The plasmid-borne arsenical resistance (ars) operon encodes an arsenical-translocating ATPase and confers resistance to antimonials and arsenicals in Escherichia coli by extrusion of the toxic compounds from the cytosol. The trans-acting regulatory ArsR protein was shown to bind to a fragment of DNA containing the ars promoter. Hybrid formation of the ArsR protein with a ArsR-beta-lactamase chimeric protein suggested that the active form of the ArsR repressor is a dimer. From footprinting analysis the binding site was defined as a region of imperfect dyad symmetry just upstream of the -35 site. In vivo the operon was derepressed by oxyions of +III oxidation state of arsenic, antimony, and bismuth, as well as arsenate (As(V)), whereas in vitro ArsR protein-operator interaction was reduced by each of those compounds except arsenate, as determined by gel retardation and DNase I and hydroxyl radical footprinting experiments. This indicates that arsenate is not a true inducer and must be reduced to arsenite in vivo to induce. An operator mutant obtained by deletion of the in vitro ArsR-protected DNA sequence exhibited constitutive ars promoter activity, demonstrating that the binding site is the functional target for the ArsR repressor in vivo.
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PMID:Metalloregulated expression of the ars operon. 841 57

One of the strategies used by Gram-negative bacteria to secrete proteins across the two membranes which delimit the cells, is sec independent and dedicated to proteins lacking an N-terminal signal peptide. It depends on ABC protein-mediated exporters, which consist of three cell envelope proteins, two inner membrane proteins, an ATPase (the ABC protein), a membrane fusion protein (MFP) and an outer membrane polypeptide. Erwinia chrysanthemi metalloproteases B and C and Serratia marcescens hemoprotein HasA are secreted by such homologous pathways and interact with the ABC protein. Using as protein substrates HasA and GST-PrtC, a chimeric protein which has a glutathione S-transferase moiety fused to a large C-terminal domain of protease C, we developed a simple system to identify proteins bound to the substrate based on substrate affinity-chromatography using heme- or glutathione-agarose. We show an ordered association between the protein substrates and the three exporter components: the substrate recognizes the ABC protein which interacts with the MFP which in turn binds the outer membrane component. Substrate binding is required for assembly of the three components.
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PMID:Protein secretion in gram-negative bacteria: assembly of the three components of ABC protein-mediated exporters is ordered and promoted by substrate binding. 891 58

The ATP-binding-cassette (ABC) protein LacK of Agrobacterium radiobacter displays high sequence similarity to the MalK subunit of the Salmonella typhimurium maltose-transport system (MalFGK2). We have used LacK as a tool to identify sites of interaction of MalK with the membrane-integral components MalF and MalG. Small amounts of LacK, resulting from the expression of the plasmid-borne lacK gene, proved to be sufficient for partial restoration of growth of a malK strain of S. typhimurium on maltose. LacK failed to substitute for MalK in regulating the expression of maltose-inducible genes but the hybrid complex MalFGLacK2 was sensitive to inducer exclusion. The lacK gene also complemented a ugpC mutant of Escherichia coli to growth on sn-glycerol-3-phosphate as the phosphate source. Partially purified LacK exhibited a spontaneous ATPase activity comparable to that of MalK. A MalK"-'LacK chimeric protein was isolated (by in vivo recombination) in which the N-terminal 140 amino acids of MalK are fused to residues 141-363 of LacK. The protein substituted for MalK in maltose transport considerably better than LacK. Furthermore, random mutagenesis of the plasmid-borne lacK gene yielded three clones that were superior to wild-type lacK in complementing a malK mutation. Single mutations (V114M or L123F) substantially improved the growth of a malK strain on maltose, whereas a double mutation (L123F, S295N) resulted in growth and transport rates that were indistinguishable from those obtained with MalK. In contrast, the introduction of the single change S295N into LacK had no effect but combination with the V114M mutation led to a further twofold increase in transport activity. These results indicate that a putative helical domain in MalK, encompassing residues 89-140, is crucial for a functional, high-affinity interaction with MalF and MalG.
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PMID:A putative helical domain in the MalK subunit of the ATP-binding-cassette transport system for maltose of Salmonella typhimurium (MalFGK2) is crucial for interaction with MalF and MalG. A study using the LacK protein of Agrobacterium radiobacter as a tool. 895 13

The DnaK, DnaJ, and GrpE proteins of Escherichia coli have been universally conserved across the biological kingdoms and work together to constitute a highly efficient molecular chaperone machine. We have examined the extent of functional conservation of Saccharomyces cerevisiae Ssc1p, Mdj1p, and Mge1p by analyzing their ability to substitute for their corresponding E. coli homologs in vivo. We found that the expression of yeast Mge1p, the GrpE homolog, allowed for the deletion of the otherwise essential grpE gene of E. coli, albeit only up to 40 degrees C. The inability of Mge1p to substitute for GrpE at very high temperatures is consistent with our previous finding that it specifically failed to stimulate DnaK's ATPase at such extreme conditions. In contrast to Mge1p, overexpression of Mdj1p, the DnaJ homolog, was lethal in E. coli. This toxicity was specifically relieved by mutations which affected the putative zinc binding region of Mdj1p. Overexpression of a truncated version of Mdj1p, containing the J- and Gly/Phe-rich domains, partially substituted for DnaJ function at high temperature. A chimeric protein, consisting of the J domain of Mdj1p coupled to the rest of DnaJ, acted as a super-DnaJ protein, functioning even more efficiently than wild-type DnaJ. In contrast to the results with Mge1p and Mdj1p, both the expression and function of Ssc1p, the DnaK homolog, were severely compromised in E. coli. We were unable to demonstrate any functional complementation by Ssc1p, even when coexpressed with its Mdj1p cochaperone in E. coli.
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PMID:Structure-function analyses of the Ssc1p, Mdj1p, and Mge1p Saccharomyces cerevisiae mitochondrial proteins in Escherichia coli. 932 54

Arachidonic acid, phosphatidic acid, and other lipids inhibit the catalytic fragment of neurofibromin more potently than that of p120 guanosine triphosphatase-activating protein (GAP). The effects of fatty acids other than arachidonic acid on full-length neurofibromin and p120 GAP, to our knowledge, have not been studied. In this study, we analyzed the effects of eight nutritionally relevant fatty acids on guanosine triphosphatase (GTPase) stimulatory activity of full-length neurofibromin and p120 GAP. The fatty acids tested were saturated stearic acid, monounsaturated oleic acid, and three n-6 and three n-3 polyunsaturated fatty acids. Analysis was performed by Ras immunoprecipitation GTPase assay. The full-length p120 GAP expressed in insect Sf9 cells and immunoaffinity-purified full-length neurofibromin were used. In contrast to neurofibromin, which was readily inhibited by stearic and oleic acid, p120 GAP was only weakly inhibited even at high concentrations (> 80 microM). Neurofibromin was also two- to threefold more sensitive to inhibition by other fatty acids tested. A chimeric protein in which the neurofibromin catalytic domain was fused to the NH2-terminal sequences of p120 GAP was used to determine that differential sensitivity to fatty acid inhibition maps to the catalytic domain of the proteins. These results indicate that nutritionally relevant fatty acids can modulate the GTPase function of c-Ha-Ras protein by inhibiting GTPase stimulatory activity of two Ras regulators, full-length neurofibromin and p120 GAP, at physiologically relevant concentrations in vitro.
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PMID:Differential regulation of neurofibromin and p120 GTPase-activating protein by nutritionally relevant fatty acids. 958 27


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