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)

Methanospirillum hungatei strains GP1 and JF1 when exposed to the adenosinetriphosphatase inhibitor N,N'-dicyclohexylcarbodiimide experienced a marked decline in growth rates, methane synthesis activities, and intracellular ATP concentrations. Although growth was inhibited, the intracellular ATP concentrations of all other methanogens tested were little affected by high concentrations of dicyclohexylcarbodiimide (500 microM). Thus, for studies of ATP synthesis, or for ATP depletion in whole cell suspensions of methanogens, the use of dicyclohexylcarbodiimide appears limited to M. hungatei.
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PMID:Sensitivity of methanogenic bacteria to dicyclohexylcarbodiimide. 713 10

When melanin absorbs light energy, it can produce potentially damaging active oxygen species. There is little doubt that constitutive pigment in dark-skinned individuals is photoprotective against skin cancer, but induced pigment-as in tanning-may not be. The first step in cancer induction is mutation in DNA. The most suitable systems for evaluating the role of melanin are those in which pigment can be varied and mutations can be measured. Several cell lines from Cloudman S91 mouse melanoma can be induced to form large quantities of melanin pigment after treatment with a number of different agents enabling comparison of mutant yields in the same cells differing principally in pigment concentration. In these studies, melanin was induced with synthetic alpha-melanocyte-stimulating hormone and with isobutyl methyl xanthine in the cell line S91/mel. The former inducer produced about 50% more pigment than the latter. Survival and mutation induction at the Na+/K(+)-ATPase locus were studied using ethyl methane sulfonate (EMS), a standard mutagen and five UV lamps emitting near monochromatic and polychromatic UV light in the three wave-length ranges of UV. There was greater protection against killing and mutation induction in the more heavily pigmented cells after exposure to EMS and after irradiation with monochromatic UVC and UVB. There was significant protection against killing by polychromatic UVB + UVA (FS20), but the small degree of protection against mutation was not significant. No significant change in killing and mutation using the same protocol was seen in S91/amel, a related cell line that does not respond to these inducers. No mutants were produced by either monochromatic or polychromatic UVA at doses that killed 50% of the cells. Our results show that induced pigment-shown earlier to be eumelanin (K. A. Cieszka et al., Exp. Dermatol. 4, 192-198, 1995)-is photo- and chemoprotective, but it is less effective in protection against mutagenesis by polychromatic UVB + UVA in a spectrum that more nearly approximates the solar spectrum.
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PMID:Induced melanin reduces mutations and cell killing in mouse melanoma. 907 36

A mutant of Methanobacterium thermoautotrophicum with a lesion in membrane Na+-translocating ATPase (synthase) was isolated. The total ATPase activity in permeabilized cells of this mutant was elevated three-fold as compared with the wild-type strain. In contrast to wild-type cells, mutant ATPase was neither inhibited by DCCD nor stimulated by Na+ ions. The methane formation orate of the mutant cells at pH 7.5 under non-growing conditions was nearly twice that of the wild-type strain and was stimulated by sodium ions. On the other hand, the ATP synthesis driven by methanogenesis under the same conditions was lower that of the wild-type under the same conditions, and contrary to the wild-type was not stimulated by Na+ ions. ATP synthesis driven by a potassium diffusion potential in the presence of sodium ions was markedly diminished in the mutant cells. The membrane potential values of the wild-type and the mutant cells in the presence of 10 mM NaCl at pH 7.0 were comparable at energized conditions (-223 mV and -230 mV respectively). The Mg2+-dependent ATPase activity of the 10(5) x g supernatant of broken cells from the mutant cells was 30% higher than in the wild-type. On the other hand, two bands with Mg2+-dependent ATPase activity were identified by native PAGE in this fraction in both wild-type as well as in mutant. These data suggest that the binding of Na+-translocating ATPase (synthase) to the membrane spanning part is changed in the mutant strain.
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PMID:Isolation and characterization of a neomycin-resistant mutant of Methanobacterium thermoautotrophicum with a lesion in Na+-translocating ATPase (synthase). 945 May 56

The RAD51 gene is a eukaryotic homolog of rec A, a critical component in homologous recombination and DNA repair pathways in Escherichia coli . We have cloned the RAD51 homolog from Tetrahymena thermophila , a ciliated protozoan. Tetrahymena thermophila RAD51 encodes a 36.3 kDa protein whose amino acid sequence is highly similar to representative Rad51 homologs from other eukaryotic taxa. Recombinant Rad51 protein was purified to near homogeneity following overproduction in a bacterial expression system. The purified protein binds to both single- and double-stranded DNA, possesses a DNA-dependent ATPase activity and promotes intermolecular ligation of linearized plasmid DNA. While steady-state levels of Rad51 mRNA are low in normally growing cells, treatment with UV light resulted in a >100-fold increase in mRNA levels. This increase in mRNA was time dependent, but relatively independent of UV dose over a range of 1400-5200 J/m2. Western blot analysis confirmed that Rad51 protein levels increase upon UV irradiation. Exposure to the alkylating agent methyl methane sulfonate also resulted in substantially elevated Rad51 protein levels in treated cells, with pronounced localization in the macronucleus. These data are consistent with the hypothesis that ciliates such as T.thermophila utilize a Rad51-dependent pathway to repair damaged DNA.
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PMID:Identification and characterization of the RAD51 gene from the ciliate Tetrahymena thermophila. 962 14

The proliferating cell nuclear antigen (PCNA) is a highly conserved protein required for the assembly of the DNA polymerase delta (pol delta) holoenzyme. Because PCNAs from Saccharomyces cerevisiae and human do not complement each other using in vitro or in vivo assays, hybrids of the two proteins would help identify region(s) involved in the assembly of the pol delta holoenzyme. Two mutants of human PCNA, HU1 (D21E) and HU3 (D120E), and six hybrids of human and S. cerevisiae PCNA, HC1, HC5, CH2, CH3, CH4, and CH5, were prepared by swapping corresponding regions between the two proteins. In solution, all PCNA assembled into trimers, albeit to different extents. These PCNA variants were tested for stimulation of pol delta and in vitro replication of M13 and SV40 DNA as well as to stimulate the ATPase activity of replication factor C (RF-C). Our data suggest that in addition to the interdomain connecting loop and C terminus, an additional site in the N terminus is required for pol delta interaction. PCNA mutants and hybrids that stimulated pol delta and RF-C were deficient in M13 and SV40 DNA replication assays, indicating that PCNA-induced pol delta stimulation and RF-C-mediated loading are not sufficient for coordinated DNA synthesis at a replication fork.
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PMID:Human-Saccharomyces cerevisiae proliferating cell nuclear antigen hybrids: oligomeric structure and functional characterization using in vitro DNA replication. 1109 57

The membrane potential (delta psi) of whole cells of Methanobacterium thermoautotrophicum strain delta H was estimated under different external conditions using a TPP(+)-sensitive electrode. The results show that the delta psi values of M. thermoautotrophicum at alkaline pHout (8.5) are comparable with delta psi values under slightly acidic conditions (pH 6.8; 230 and 205 mV, respectively). On the other hand, the size of colonies on Petri dishes was remarkably smaller at pH 8.5 than at 6.8. The delta psi was insensitive to relevant ATPase inhibitors. At pH 6.8, the protonophore 3,3',4',5-tetrachlorosalicylanilide (TCS) strongly inhibited delta psi formation and ATP synthesis driven by methanogenic electron transport. On the other hand, at pH 8.5 the CH4 formation and ATP synthesis were insensitive to TCS and a protonophore-resistant delta psi of approximately 150 mV was determined. The finding of a protonophore-resistant delta psi at pH 8.5 indicates that at alkaline pHout these cells can switch from H(+)-energetics to Na(+)-energetics, when the delta [symbol: see text] H+ becomes limited. The results strongly support the hypothesis that at alkaline pHout Na+ ions might fully substitute for H+ in these cells as the coupling ions.
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PMID:The membrane potential of Methanobacterium thermoautotrophicum under different external conditions. 1127 16

In Methanobacterium thermoautotrophicum, the protonmotive force for the H+-translocating ATPase consists mainly of a transmembrane electrical gradient (Deltapsi). These cells do not establish a significant transmembrane pH gradient (inside alkaline) and, in fact, if the suspending medium is of pH >/= 7.0, the pH gradient may be reversed-i.e., inside acid with respect to the extracellular pH. These studies show by both 23Na NMR and 22Na+ distribution that Na+ extrusion with the generation of Deltapsi precedes methanogenesis in Mb. thermoautotrophicum. It is calculated that the newly established Na+ gradients increase Deltapsi by approximately 50 mV (inside negative). There is no detectable H+ extrusion during methane synthesis; instead there is a high rate of H+ consumption for methane synthesis and an increase in internal pH. This was supported by 31P NMR experiments, which showed an internal pH shift from 6.8 to 7.6. With the cells maintained at an external pH of 7.2, the initial transmembrane pH gradient of -0.4 (inside acid) at 60 degrees C is equivalent to Deltapsi of + 27 mV (inside positive); after 20 min of incubation, the transmembrane pH gradient is + 0.4 (inside alkaline), which at 60 degrees C is equivalent to Deltapsi of -27 mV (inside negative). Actively respiring cells generated a protonmotive force of -198 mV. It is proposed that energy for CO2 reduction to the level of formaldehyde (the first step in methane synthesis) in Mb. thermoautotrophicum is derived from the Deltapsi generated by electrogenic Na+ extrusion. The protonmotive force required for ATP synthesis consists primarily of Deltapsi and appears to be the result of both an electrogenic Na+ extrusion and a pH gradient (inside alkaline) which develops during methanogenesis.
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PMID:Ion transport and methane production in Methanobacterium thermoautotrophicum. 1160 73

NS-398 (N-(2-cyclohexyloxy-4-nitrophenyl)-methane sulfonamide) is a selective inhibitor of the cyclooxygenase-2 isozyme in vitro and in vivo. This study reports on acute inhibition of receptor-mediated contractions of isolated rat aorta by NS-398 and its modulation by endothelium-derived nitric oxide. NS-398 (1-10 microM) blocked norepinephrine, and 5-hydroxytryptamine (5-HT) evoked contractions and suppressed E(max) responses for both agonists. E(max) changes occurred in endothelium-intact vessel rings and in the absence, as well as in the presence of cycloheximide or dexamethasone in the physiological salt solution (PSS) bathing the tissues. NS-398 altered contractions to these receptor agonists in denuded rings only at 10 microM, and did not significantly alter contractions to KCl and sodium fluoride in all situations. NS-398 (3 and 10 microM) reduced aortic contractions initiated by cyclopiazonic acid (CPA), a sarcoplasmic reticulum Ca(2+)-ATPase blocker, in endothelium intact rings bathed with PSS with/without nitro-D-arginine methyl ester (D-NAME; 100 microM), but did not alter contractions to the compound in endothelium-denuded aortic rings and in vessel rings bathed with PSS+L-NAME (100 microM). Western blot analyses reveal significantly denser cyclooxygenase-2 protein expressions in freshly isolated endothelium-intact, compared to, denuded vessel segments. We conclude that: (1) cyclooxygenase-2 is constitutively expressed in rat aortic endothelial and smooth muscle cells, and (2) NS-398 modulates aortic contractions principally through an action on endothelial cyclooxygenase-2. Our data strongly suggest that cyclooxygenase-2 and/or its product(s), in concert with endothelium-derived nitric oxide, regulates the sarcoplasmic reticulum Ca(2+) pump activity in rat aorta.
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PMID:NS-398, a selective cyclooxygenase-2 blocker, acutely inhibits receptor-mediated contractions of rat aorta: role of endothelium. 1249 19

The human multidrug resistance P-glycoprotein (P-gp, ABCB1) actively extrudes a broad range of potentially cytotoxic compounds out of the cell. Key steps in understanding the transport process are binding of drug substrates in the transmembrane domains, initiation of ATPase activity, and subsequent drug efflux. We used cysteine-scanning mutagenesis of the transmembrane segment residues and reaction with the thiol-reactive drug substrate analog of rhodamine, methane-thiosulfonate-rhodamine (MTS-rhodamine), to test whether P-gp could be trapped in an activated state with high levels of ATPase activity. The presence of such an activated P-gp could be used to further investigate P-gp-drug substrate interactions. Single cysteine mutants (149) were treated with MTS-rhodamine, and ATPase activities were determined after removal of unreacted MTS-rhodamine. One mutant, F343C(TM6), showed a 5.8-fold increase in activity after reaction with MTS-rhodamine. Pre-treatment of mutant F343C with rhodamine B protected it from activation by MTS-rhodamine, indicating that residue Cys-343 contributes to the rhodamine-binding site. The ATPase activity of MTS-rhodamine-treated mutant F343C, however, was not stimulated further by colchicine or calcein-AM. By contrast, verapamil and Hoechst 33342 stimulated and inhibited, respectively, the ATPase activity of the MTS-rhodamine-treated mutant F343C. These results indicate that the MTS-rhodamine binding site overlaps that of colchicine and calcein-AM but not that of verapamil and Hoechst 33342 within the common drug-binding pocket.
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PMID:Methanethiosulfonate derivatives of rhodamine and verapamil activate human P-glycoprotein at different sites. 1452 74

The sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitors thapsigargin (0.1-1 microM) and cyclopiazonic acid (10 microM), failed to affect resting [Ca(2+)] in human spermatozoa. Slow progesterone-induced [Ca(2+ i)](i) oscillations in human spermatozoa, which involve cyclic emptying-refilling of an intracellular Ca(2+) store were also insensitive to these inhibitors. Non-selective doses of thapsigargin (5-30 microM, 50-300 times the saturating dose for SERCA inhibition), caused elevation of resting [Ca(2+)](i) and partial, dose-dependent disruption of oscillations. A 10-40 microM concentration of bis(2-hydroxy-3-tert-butyl-5-methyl-phenyl)methane (bis-phenol), which inhibits both thapsigargin-sensitive and -insensitive microsomal Ca(2+) ATPases, caused elevation of resting [Ca(2+)](i) and inhibition of [Ca(2+)](i) oscillations at doses consistent with inhibition of thapsigargin-resistant, microsomal ATPase and liberation of stored Ca(2+). Low doses of bis-phenol had marked effects on [Ca(2+)](i) oscillation kinetics. Application of the drug to cells previously stimulated with progesterone had effects very similar to those observed when it was applied to unstimulated cells, suggesting that the sustained Ca(2+) influx induced by progesterone is not mediated via mobilisation of Ca(2+) stores. Western blotting for human sperm proteins showed expression of secretory pathway Ca(2+) ATPase (SPCA1). Immunolocalisation studies revealed expression of SPCA1 in all cells in an area behind the nucleus, extending into the midpiece. Staining for SERCA, carried out in parallel, detected no expression with either technique. We conclude that: (1) intracellular Ca(2+) store(s) and store-dependent [Ca(2+)](i) oscillations in human spermatozoa rely primarily on a thapsigargin/cyclopiazonic acid-insensitive Ca(2+) pump, which is not a SERCA as characterised in somatic cells; (2) effects of high-dose thapsigargin on spermatozoa primarily reflect non-specific actions on non-SERCAs and; (3) secretory pathway Ca(2+) ATPases contribute at least part of this non-SERCA Ca(2+) pump activity.
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PMID:Secretory pathway Ca(2+)-ATPase (SPCA1) Ca(2)+ pumps, not SERCAs, regulate complex [Ca(2+)](i) signals in human spermatozoa. 1581 49


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