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 report a procedure for the large-scale purification of the Escherichia coli Rep protein, a helicase that is involved in the replication of the E. coli chromosome as well as a number of single-stranded bacteriophages. The procedure starts with E. coli cells harboring an overproducing plasmid, pRepO, in which the E. coli rep gene is under transcriptional control of the inducible lambda PL promoter (Colasanti, J., and Denhardt, D. T. (1987) Mol. Gen. Genet. 209, 382-390). The purification procedure results in greater than 98% pure Rep protein, which is free of contaminating nuclease activity, with yields of 40-50 mg of Rep protein/50 g of induced MZ-1/pRepO cells. We also show that cell death occurs upon inducing such a large overproduction of the E. coli Rep protein in MZ-1/pRepO. The Rep protein purified by this procedure has high specific single-stranded DNA-dependent ATPase activity, as well as helicase activity, with an apparent 3' to 5' directionality. The extinction coefficient of purified E. coli Rep protein is epsilon 280 = 1.16 +/- 0.04 ml mg-1 cm-1 (8.47 +/- 0.28 X 10(4) M-1 cm-1) in 10 mM Tris (pH 7.5), 20% (v/v) glycerol, 0.10 M NaCl at 25 degrees C. The solubility properties of the purified Rep protein have been examined as a function of glycerol, NaCl, MgCl2, ATP, and ADP concentrations at 25 and 37 degrees C (pH 7.5). Rep protein solubility decreases significantly with decreasing concentrations of glycerol and monovalent salt and increasing temperature; however, the presence of 1.5 mM ATP or ADP or MgCl2 at low NaCl concentrations increases the solubility. At 4 degrees C, in the presence of 20% glycerol and greater than or equal to 50 mM NaCl, the free Rep protein exists as a stable monomer under all conditions examined (+/- ATP and +/- MgCl2). The single-stranded DNA-dependent ATPase activity decreases with increasing glycerol concentration, such that in 25% (v/v) glycerol it has approximately 40% of its activity as compared to solutions that contain no glycerol. The dependence of the single-stranded DNA-dependent ATPase activity on salt concentration for a series of monovalent salts indicates the presence of both cation and anion effects, with decreasing activity in the order glutamate greater than acetate greater than chloride. The ability to obtain highly purified E. coli Rep protein in large quantities with relative ease will greatly facilitate physical characterizations of the protein and its interactions with DNA.
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PMID:Large-scale purification and characterization of the Escherichia coli rep gene product. 252 89

Termination of transcription at tR1, the rho-dependent terminator between genes cro and cII of bacteriophage lambda, is mediated by interactions between rho protein and an RNA sequence element called rut. We show, using a filter retention assay technique, that rho protein binds with about 10-fold lower affinity to variants of cro RNA lacking both parts of rut or to normal cro RNA having one or the other part of rut bound to a complementary DNA oligonucleotide than it binds to unmodified cro RNA. These same variant and modified forms are nearly devoid of the strong rho ATPase cofactor activity of cro RNA. Estimates of binding energies of the rho-cro RNA interaction under different conditions reveal that termination function correlates with about 12.6 kcal of binding energy, of which two-thirds is due to nonelectrostatic interactions. The rut segment is shown to contribute about 1 kcal, nearly all to nonelectrostatic interactions. KCl is found to be more effective than potassium glutamate as a competitive counterion, and a decrease in 1.4 kcal of binding energy due to counterion competition correlates with a loss of termination and ATPase activities. In sum, the results indicate that the rut sequence contributes substantially to the overall binding affinity, that ionic interactions are also important, and that mere binding of rho to RNA is not sufficient for rho ATPase activation.
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PMID:Thermodynamic and enzymological characterization of the interaction between transcription termination factor rho and lambda cro mRNA. 252 25

The effect of dimethyl sulfoxide (DMSO) on the structure of sarcoplasmic reticulum was analyzed by Fourier transform infrared (FTIR) and fluorescence spectroscopy. Exposure of sarcoplasmic reticulum vesicles to 35% DMSO (v/v) at 2 degrees C for several hours in a D2O medium produced no significant change in the phospholipid and protein Amide I regions of the FTIR spectra, but the intensity of the Amide II band decreased, presumably due to proton/deuterium exchange. At 40% to 60% DMSO concentration a shoulder appeared in the FTIR spectra at 1630 cm-1, that is attributed to the formation of new beta or random coil structures; irreversible loss of ATPase activity accompanied this change. At 70% DMSO concentration the intensity of the main Amide I band at 1639 cm-1 decreased and a new band appeared at 1622 cm-1, together with a shoulder at 1682 cm-1. These changes indicate an abrupt shift in the conformational equilibrium of Ca2+-ATPase from alpha to beta structure or to a new structure characterized by weaker hydrogen bonding. Decrease of ionization of aspartate and glutamate carboxyl groups in the presence of DMSO may also contribute to the change in intensity at 1622 cm-1. The changes were partially reversed upon removal of DMSO. Exposure of sarcoplasmic reticulum vesicles to 1.5 kbar pressure for 1 h at 2 degrees C in an EGTA-containing (low Ca2+) medium causes irreversible loss of ATPase activity, with the appearance of new beta structure, and abolition of the Ca2+-induced fluorescence response of FITC covalently bound to the Ca2+-ATPase; DMSO (35%) stabilized the Ca2+-ATPase against pressure-induced changes in structure and enzymatic activity, while urea (0.8 M) had the opposite effect.
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PMID:Correlation of structure and function in the Ca2+-ATPase of sarcoplasmic reticulum: a Fourier transform infrared spectroscopy (FTIR) study on the effects of dimethyl sulfoxide and urea. 252 64

The transmembrane segments predicted for the Neurospora H-ATPase are laid out diagrammatically in Figure 10. Although the eight segments have arbitrarily been compressed into rectangles of the same size, they range in length from 20 residues (II) to 30 residues (IV and VI), so the corresponding helices must vary in length as well. Notable features of the model include the charged residues located just outside the plane of the membrane, with a clear excess of negative charges (5-, 1+) at the extracellular surface and a slight excess of positive charges (4+, 3-) at the cytoplasmic surface. There are also a conspicuous number of bulky residues (tryptophan, phenylalanine, and tyrosine) just inside the plane of the membrane. Within the bilayer, most of the helices are noticeably amphipathic, consistent with the expectation that at least some of them stack together to form a channel-like structure with a hydrophobic surface and a hydrophilic core. The charged residues predicted to lie within the membrane are listed in Table 2, which is a summary of data from eight of the P-type ATPases; the S. cerevisiae and S. pombe enzymes have not been included because they are nearly identical in this respect to the Neurospora enzyme. Interestingly, all of the ATPases have more membrane-embedded negative charges (5 to 8) than positive ones (0 to 4), a pattern that may be connected with their role as cation transporters. Certainly, other unrelated transport proteins have a rather different pattern of positive and negative charges: for example, the mammalian glucose transporter (1+, 2-), Na-glucose transporter (3+, 3-), and the E. coli lac permease (11+, 7-). The actual positioning of the negative charges in the P-type ATPases does not make it easy to single out the functionally important ones, however. The glutamyl residue in segment I is present in the fungal, plant, and Leishmania H-ATPases but not in the gastric H,K-ATPase. The same is true for the aspartate in segment II, except that it also appears in the muscle and brain Ca-ATPases. A glutamate is found at one end of segment III in the E. coli and fungal enzymes and at the other end in Arabidopsis; in segment IV, another glutamate appears in a well-conserved region in the Leishmania and mammalian enzymes but not in the bacterial, fungal, or plant ones.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Transmembrane segments of the P-type cation-transporting ATPases. A comparative study. 256 19

In submerged grown hyphae of Penicillium cyclopium the activities of seven transport systems could be distinguished which share in the uptake of L-arginine, L-glutamic acid, L-phenylalanine and L-leucine. They include the specific systems a (accepting L-arginine and L-lysine), b (L-phenylalanine, L-tyrosine), c (L-glutamic acid) and d (L-leucine), system I (a 'general amino-acid permease') and the low-affinity systems II and III, which accept acidic or basic amino acids, respectively, but also L-phenylalanine. In nutrient-sufficient cells, systems I, II and III remain repressed; uptake is dominated by the specific systems b, c, d and a, the latter reaching its maximum activity. Nitrogen starvation is the most powerful signal for the development of systems I, II and III, whereas, in carbon-starved cells, systems b, c and d reach maximum activities. The development of the general amino-acid permease in nitrogen-starved cells requires both translational and--with a few hours delay--transcriptional events as indicated by the influence of cycloheximide and 5-fluorouracil. The uptake of all amino acids is accompanied by a transient acidification of the cellular interior. Short-time preaccumulation of several anions, such as citrate, alpha-oxo-glutarate, glutamate (but not glutamine), increases the initial rate of amino-acid uptake at a pH above the optimum. Uncouplers inhibit the uptake not only under aerobic but also under anaerobic conditions, where the ATP content is not influenced by these compounds. These findings point to an H+/amino acid symport, which is tightly connected with the recycling of the incoming protons by the plasmalemma H+-ATPase.
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PMID:Kinetic properties, nutrient-dependent regulation and energy coupling of amino-acid transport systems in Penicillium cyclopium. 256 28

Measurements have been made of the ATP-dependent membrane potential (delta psi) and pH gradient (delta pH) across the membranes of the synaptic vesicles purified from bovine cerebral cortex, using the voltage-sensitive dye bis[3-propyl-5-oxoisoxazol-4-yl]pentamethine oxanol and the delta pH-sensitive fluorescent dye 9-aminoacridine respectively. A pre-existing small delta pH (inside acidic) was detected in the synaptic vesicles, but no additional significant contribution by MgATP to delta pH was observed. In contrast, delta psi (inside positive) increased substantially upon addition of MgATP. This ATP-dependent delta psi was reduced by thiocyanate anion (SCN-), a delta psi dissipator, or carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), a protonmotive-force dissipator. Correspondingly, a substantially larger glutamate uptake occurred in the presence of MgATP, which was inhibited by SCN- and FCCP. A nonhydrolysable analogue of ATP, adenosine 5'-[beta gamma-methylene]triphosphate, did not substitute for ATP in either delta psi generation or glutamate uptake. The results support the hypothesis that a H+-pumping ATPase generates a protonmotive force in the synaptic vesicles at the expense of ATP hydrolysis, and the protonmotive force thus formed provides a driving force for the vesicular glutamate uptake. The delta psi generation by ATP hydrolysis was not affected by orthovanadate, ouabain or oligomycin, but was inhibited by N-ethylmaleimide, quercetin, trimethyltin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid. These results indicate that the H+-pumping ATPase in the synaptic vesicle is similar to that in the chromaffin granule, platelet granule and lysosome.
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PMID:Glutamate uptake into synaptic vesicles of bovine cerebral cortex and electrochemical potential difference of proton across the membrane. 256 9

Synaptic vesicles contain a H+-ATPase that generates a proton electrochemical gradient (delta mu H+) required for the uptake of neurotransmitters into the organelles. In this study, the synaptic vesicle H+-ATPase was examined for structural and functional similarities with other identified ATPases that generate a delta mu H+ across membranes. The synaptic vesicle H+-ATPase displayed immunological similarity with the 115-, 72-, and 39-kDa subunits of a vacuolar-type H+-ATPase purified from chromaffin granules. Functionally, the ATP-dependent H+ pumping across synaptic vesicles and ATP hydrolysis were sensitive to the sulfhydryl-modifying reagents, N-ethylmaleimide and 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, at concentrations known to affect vacuolar-type H+-ATPases. In addition, as with vacuolar-type H+-ATPases, the presence of NO3-, SO4(2-), or F- inhibited the generation of a delta mu H+, but addition of vanadate or oligomycin had no effect. The delta mu H+ is a function of the pH gradient (delta pH) and membrane potential (delta psi sv) across the synaptic vesicle. Acidification (delta pH) of the synaptic vesicle interior was enhanced in the presence of permeant anions, such as Cl-, or the K+ ionophore, valinomycin. In the absence of permeant anions, the H+-ATPase generated a delta psi sv that effected the transport of L-glutamate into the synaptic vesicles. Dissipation of delta psi sv by incubation with increased external Cl- or nigericin resulted in the abolition of glutamate uptake, despite the continued maintenance of a delta mu H+ across the synaptic vesicle as a substantial delta pH. The results suggest that the synaptic vesicle H+-ATPase is of a vacuolar type and energizes the uptake of anionic glutamate by virtue of the delta psi sv component of the delta mu H+ it generates.
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PMID:Characterization of a H+-ATPase in rat brain synaptic vesicles. Coupling to L-glutamate transport. 256 4

Synaptosomes were isolated from cerebrums of rats fed standard (20% protein) or protein-free diets for 30 days. Arrhenius plots of their (Na+/K+)ATPase activities revealed a transition temperature of 25.5 degrees C for control rats and 23.4 degrees C for rats on protein-free diet, indicating that the latter increases synaptosomal membrane fluidity. The only change observed in the composition of the synaptosomal membranes was a 26% decrease of sialic acid. In synaptosomes from rats on protein-free diet the uptake of tyrosine was slightly reduced while that of glutamate was not affected. However, the exit of glutamate was reduced.
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PMID:A protein-free diet changes synaptosomal membrane fluidity and tyrosine and glutamate transport. 256 92

Effect of methotrexate (MTX) on mitochondrial oxygen uptake, oxidative phosphorylation and on the activity of several enzymes linked to respiratory chain was studied. MTX was able to inhibit state III respiration activated by ADP and to decrease the respiratory coefficient with the substrates alpha-ketoglutarate and glutamate; these effects became pronounced when mitochondria were pre-incubated with MTX for 10 min. No effect was observed on ATPase activity of undamaged or broken mitochondria; the same was true for NADH-oxidase, NADH-dehydrogenase, NADH-cytochrome c reductase, succinate oxidase, and cytochrome c oxidase activity. The effect on the steady-state of cytochrome b, as well as, the inhibitory effect on state III of respiration with NAD+-linked substrates, offers a reasonable possibility to suggesting that the inhibition site of MTX could be in a place anterior to cytochrome b region, and not linked to respiratory chain.
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PMID:Methotrexate: studies on the cellular metabolism. I. Effect on mitochondrial oxygen uptake and oxidative phosphorylation. 283 95

Following cryogenic lesions of the brain in the rabbit, ictal activity appears within min with a maximum at 2 h. Brain edema increases rapidly between 2-4 h with a maximum at 8 h. The glutamate concentration reaches 209% of control in the perilesional area at 2 h and the time course of glutamate/GABA ratio parallels the time course of epileptic activity. The impairment of Na+-K+-ATPase activity (rise of KMapp for K+) in the glial fraction coincides with the increase of edema. A positive correlation is found between the total amount of ictal activity and the total amount of edema in individual animals, suggesting that epilepsy may enhance edema formation.
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PMID:Relationship between epileptic activity and edema formation in the acute phase of cryogenic lesion. 284 Jun 11


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