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)

By combining the tools of site-directed mutagenesis and [3H]ouabain binding, the functional role of glutamic acid 327 in the fourth transmembrane domain of the sheep alpha 1 isoform of Na+,K(+)-ATPase was examined with respect to its interactions with ouabain, Na+,K+,Mg2+, and inorganic phosphate. Using site-directed mutagenesis, this glutamic acid was substituted with alanine, aspartic acid, glutamine, and leucine. The mutant proteins were constructed in a sheep alpha 1 protein background such that [3H]ouabain binding could be utilized as a highly specific probe of the exogenous protein expressed in NIH 3T3 cells. Na+ competition of [3H]ouabain binding to the mutant forms of Na+,K(+)-ATPase revealed only slight alterations in their affinities for Na+ and in their abilities to undergo Na(+)-induced conformational changes which inhibit ouabain binding. In contrast, K+ competition of [3H]ouabain binding to all four mutant forms of Na+,K(+)-ATPase displayed severely altered interactions between these proteins and K+. Interestingly, [3H]ouabain binding to the mutant E327Q was not inhibited by the presence of K+. This mutant was previously reported to be functionally able to support cation transport with a 5-fold reduced K0.5 for K(+)-dependent ATPase activity (Jewell-Motz, E. A., and Lingrel, J.B. (1993) Biochemistry 32, 13523-13530; Vilsen, B. (1993) Biochemistry 32, 13340-13349). Thus, it appears that this glutamic acid in the fourth transmembrane domain may be important for stabilizing a K(+)-induced conformation within the catalytic cycle of Na+,K(+)-ATPase that is not rate-limiting in the overall ATPase cycle but that displays a greatly reduced affinity for ouabain.
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PMID:Glutamic acid 327 in the sheep alpha 1 isoform of Na+,K(+)-ATPase stabilizes a K(+)-induced conformational change. 785 79

The 16K subunit of the vacuolar H(+)-ATPase binds specifically to the bovine (BPV) and human (HPV) papillomavirus E5 oncoproteins, and it has been suggested that this interaction may contribute to cell transformation (Goldstein, D. J., and Schlegel, R. (1990) EMBO J. 9, 137-146; Goldstein, D. J., Finbow, M. E., Andresson, T., McLean, P., Smith, K., Bubb, V. J., and Schlegel, R. (1991) Nature 352, 347-349; Conrad, M., Bubb, V. J., and Schlegel, R. (1993) J. Virol. 67, 6170-6178; Goldstein, D. J., Toyama, R., Schlegel, R., and Dhar, R. (1992) Virology 190, 889-893). We generated mutations within the 16K protein to define binding domains for BPV-1 E5 as well as to characterize the role of 16K in cell transformation. 16K consists predominantly of 4 transmembrane (TM) domains. We showed that mutations within the TM4 domain severely inhibited E5 binding. More specifically, conversion of glutamic acid 143 to arginine within TM4 severely reduced 16K/E5 binding, suggesting that charged interactions facilitated efficient binding. This hypothesis was confirmed by demonstrating that binding to the defective 16K arginine mutant could be restored by complementary charge mutations in E5; conversion of E5 glutamine 17 to glutamic acid or aspartic acid enhanced interactions with the 16K arginine mutant. Surprisingly, mutants in TM4 not only bound poorly to wild-type E5 but were converted into an oncoprotein and induced anchorage-independent growth of NIH 3T3 cells. These data define glutamic acid 143 in the 16K TM4 domain and glutamine 17 within E5 as important contributors to E5/16K binding and suggest a role for the 16K protein in the regulation of cell proliferation.
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PMID:Vacuolar H(+)-ATPase mutants transform cells and define a binding site for the papillomavirus E5 oncoprotein. 789 30

The effect of the argemone alkaloid, sanguinarine, was studied on the active transport of D-glucose and some of the L-amino acids in everted sacs of the small intestine of rats. Sanguinarine (1.0 mumole) was found to inhibit (61%) the transport of D-glucose, while an alkaloid concentration of 0.1 mumole was ineffective. Both 0.1 and 1.0 mumole of sanguinarine had no effect on the transport of the L-amino acids including aspartic acid, lysine, and tyrosine. Sanguinarine showed a dose dependent inhibition of intestinal and hepatic Na+,K(+)-ATPase in a non-competitive manner. The inhibition of Na+,K(+)-ATPase by sanguinarine may in turn inhibit the active transport of D-glucose which requires a sodium pump.
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PMID:Effect of sanguinarine on the transport of essential nutrients in an everted gut sac model: role of Na+,K(+)-ATPase. 816 40

During germination spores of Streptomyces viridochromogenes NRRL B-1551 excrete a compound, germicidin, which has an inhibitory effect on the germination of its own arthrospores at a concentration as low as 200 pM (40 pg/ml). At higher concentrations germicidin inhibits porcine Na+/K(+)-activated ATPase and retards the germination of the cress Lepidium sativum. Germicidin is the first known autoregulative inhibitor of spore germination in the genus Streptomyces and was isolated from the supernatant of germinated spores, but also from the supernatant of the submerged culture. Spectroscopic analysis and derivatization reactions revealed germicidin to be 6-(2-butyl)-3-ethyl-4-hydroxy-2-pyrone (C11H16O3). Crude isolates of germicidin from the supernatant of submerged culture, but not from the spores, contained a second, structurally very similar compound (C10H14O3), in which in contrast to germicidin a 2-propyl instead of the 2-butyl chain was bound to C-6 and which did not show any activity in the germination and ATPase assay. The germination assay was evaluated as a new screening model for specifically active compounds.
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PMID:Germicidin, an autoregulative germination inhibitor of Streptomyces viridochromogenes NRRL B-1551. 836 Jan 9

Photodynamic treatment of yeast with the sensitizer Toluidine blue results in loss of cell viability. In previous investigations it was suggested that plasma membrane damage might be responsible for the loss of colony forming capacity. In this context the influence of photodynamic treatment on transmembrane transport systems was studied. It appeared that the uptake of the sugars glucose, lactose and galactose, the amino acids arginine, phenylalanine, glycine and aspartic acid and of the inorganic compound phosphate was inhibited by photodynamic treatment. The different elements of the energy providing system necessary for active transport, viz. the plasma membrane ATPase and the protonmotive force, were not significantly affected by Toluidine blue and light, indicating that inhibition of transport is not caused by a reduction of the membrane potential or the transmembrane pH gradient. These observations suggest that the transport carriers themselves were damaged by treatment with Toluidine blue and light. This could be confirmed in experiments, in which the lactose and galactose transport proteins of treated and untreated cells were reconstituted in plasma membrane vesicles. It appeared that the carriers, obtained from photodynamically treated Kluyveromyces marxianus cell, had lost their transport capacity.
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PMID:Inhibition of transport systems in yeast by photodynamic treatment with toluidine blue. 837 89

Gastric H+,K(+)-ATPase was functionally expressed in the human kidney HEK293 cell line. The expressed enzyme catalyzed ouabain-resistant K(+)-dependent ATP hydrolysis. The K(+)-ATPase activity was inhibited by SCH 28090, a specific inhibitor of gastric proton pump, in a dose-dependent manner. By using this functional expression system in combination with site-directed mutagenesis, we investigated effects of mutations in the putative cation binding site and the catalytic center of the gastric H+,K(+)-ATPase. In Na+,K(+)-ATPase, the glutamic acid residue in the 4th transmembrane segment is regarded as one of the residues responsible for the K(+)-induced conformational change (Kuntzweiler, T. A., Wallick, E. T., Johnson, C. L., and Lingrel, J. B. (1995) J. Biol. Chem. 270, 2993-3000). When the corresponding glutamic acid (Glu-345) of H+,K(+)-ATPase was mutated to aspartic acid, lysine, or valine, the SCH 28080-sensitive K(+)-ATPase activity was abolished. However, when this residue was replaced by glutamine, about 50% of the activity was retained. This mutant showed a 10-fold lower affinity for K+ (Km = 2.6 mM) compared with the wild-type enzyme (Km = 0.24 mm). Thus, Glu-345 is important in determining the K+ affinity of H+,K(+)-ATPase. When the aspartic acid residue in the phosphorylation site was mutated to glutamic acid, this mutant showed no SCH 28080-sensitive K(+)-ATPase activity. Thus, amino acid replacement of the phosphorylation site is not tolerated and a stringent structure appears to be required for enzyme activity. When the lysine residue in the fluorescein isothiocyanate binding site (part of ATP binding site) was mutated to arginine, asparagine, or glutamic acid, the SCH 28080-sensitive K(+)-ATPase activity was eliminated. However, the mutant in which this residue was changed to glutamine had about 30% of the activity, suggesting that amino acid replacement of this site is tolerated to a certain extent.
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PMID:Functional expression of gastric H+,K(+)-ATPase and site-directed mutagenesis of the putative cation binding site and catalytic center. 857 49

Semliki Forest virus-specific polypeptide nsP2 is a nonstructural protein involved in multiple steps during viral RNA replication. It was recently shown to possess single-stranded RNA-stimulated ATPase and GTPase activities. Replacement of the highly conserved lysine (Lys-192) within the classical nucleotide-binding motif A/GXXGXGKS/T with asparagine abolished its NTP-hydrolyzing activity. Also, about half of nsP2 is transported into the nucleus during viral infection. Substitution of the second arginine in its nuclear localization signal (P648RRRV) with aspartic acid rendered nsP2 totally cytoplasmic. To assess the functional importance of these sequence motifs, the same mutations were introduced into a cDNA clone of Semliki Forest virus, from which infectious RNA can be produced in vitro. Transfection of an RNA encoding Lys-192 --> Asn mutation into BHK cells did not promote viral infection. However, revertants encoding the wild-type amino acid were obtained. Cells transfected with RNA coding for Arg-649 --> Asp mutation gave rise to infectious virus termed SFV-RDR. Indirect immunofluorescence and subcellular fractionation of SFV-RDR-infected cells confirmed the cytoplasmic localization of nsP2. Measurement of host DNA synthesis late in infection revealed that infection with the parental virus inhibited DNA synthesis to 10% of control cells. In contrast, infection with SFV-RDR led only to a partial shutoff of cellular DNA synthesis. Mice experiments indicated that the pathogenicity of SFV-RDR was attenuated.
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PMID:Functional significance of the nuclear-targeting and NTP-binding motifs of Semliki Forest virus nonstructural protein nsP2. 861 Apr 62

Terminase the DNA packaging enzyme of bacteriophage chi, is a heteromultimer of gpNul (21 kDa) and gpA (74 kDa) subunits, encoded by the chi Nul and A genes, respectively. Sequence comparisons indicate that both gpNu1 and gpA have a match to the P-loop motif of ATPase centers, which is a glycine-rich segment followed by a lysine. By site-specific mutagenesis, we changed the lysines of the putative P-loops of gpNul (k35) and gpA (K497) to arginine, alanine, or aspartic acid, and studied the mutant enzymes by kinetic analysis and photochemical cross-linking with 8-azido-ATP. Both the gpNul and gpA subunits of wild-type terminase were covalently modified with 8-N3[32P] ATP in the presence of UV light. Saturation occurred with apparent dissociation constants of 508 and 3.5 microM for gpNul and gpA, resepctively. ATPase assays showed two activities: a low-affinity activity (Km=469 microM), and a high-affinity activity (Km=4.6 microM). The gpNul K35A and gpNul K35D mutant terminases showed decreased activity in the low-affinity ATPase activity. The reduced activities of these enzymes were recovered when 10 times more DNA was added, suggesting that the primary defect of the enzymes is alteration of the nonspecific, double-stranded DNA binding activity of terminase. ATPase assays and photolabeling of the gpA K497A and gpA K497D mutant terminases showed reduced affinity for ATP at the high-affinity site which was not restored by increased DNA. In summary, the results indicate the presence of a low-affinity, DNA-stimulated ATPase center in gpNul, and a high-affinity site in gpA.
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PMID:Kinetic and mutational dissection of the two ATPase activities of terminase, the DNA packaging enzyme of bacteriophage Chi. 861 86

Site-directed mutagenesis was used to investigate the roles of a short series of hydrophobic amino acids in the b-subunit of the Escherichia coli F0F1-ATPase. A mutation affecting one of these, G131D, had been previously characterized and was found to interrupt assembly of the F0F1-ATPase (Jans, D. A., Hatch, L., Fimmel, A. L., Gibson, D., and Cox, G. B. (1985) J. Bacteriol. 162, 420-426). To extend this work, aspartic acid was substituted for each one of the residues from positions 124 to 132. The properties of mutants in this series are consistent with the region from Val124 to Gly131 forming an alpha-helix. Two of the mutations, V124D and A128D, resulted in a similar phenotype to the G131D mutation. This suggested that Val124, Ala128, and Gly131 form a helical face which may have a role in inter- or intrasubunit interactions. This was tested by overexpressing and purifying the cytoplasmic domains of the wild type and A128D mutant b-subunits. Sedimentation equilibrium centrifugation indicated that the wild type domain formed a dimer whereas the mutant was present as a monomer.
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PMID:A mutation in which alanine 128 Is replaced by aspartic acid abolishes dimerization of the b-subunit of the F0F1-ATPase from Escherichia coli. 863 35

To address the functional significance of motif III in Escherichia coli DNA helicase II, the conserved aspartic acid at position 248 was changed to asparagine. UvrDD248N failed to form stable binary complexes with either DNA or ATP. However, UvrDD248N was capable of forming an active ternary complex when both ATP and single-stranded DNA were present. The DNA-stimulated ATPase activity of UvrDD248N was reduced relative to that of wild-type UvrD with no significant change in the apparent Km for ATP. The mutant protein also demonstrated a reduced DNA unwinding activity. The requirement for high concentrations of UvrDD248N to achieve unwinding of long duplex substrates likely reflects the reduced stability of various binary and ternary complexes that must exist in the catalytic cycle of a helicase. The data suggest that motif III may act as an interface between the ATP binding and DNA binding domains of a helicase. The uvrDD248N allele was also characterized in genetic assays. The D248N protein complemented the UV-sensitive phenotype of a uvrD deletion strain to levels nearly equivalent to wild-type helicase II. In contrast, the mutant protein only partially complemented the mutator phenotype. A correlation between the level of genetic complementation and the helicase activity of UvrDD248N is discussed.
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PMID:A partially functional DNA helicase II mutant defective in forming stable binary complexes with ATP and DNA. A role for helicase motif III. 881 Mar 1


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