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)

The H(+)-ATPase from chloroplasts CFoF1, was brought into the active, reduced state by illumination of thylakoids in the presence of thioredoxin and dithiothreitol. Uni-site ATP synthesis was initiated by the addition of 20 nM [alpha-32P]ADP, and enzyme-bound and free nucleotides were separated by a pressure column. The ratio of enzyme-bound ADP to ATP was 0.55 +/- 0.05. In a second experiment, uni-site ATP hydrolysis under energized conditions was initiated by the addition of 36 nM [alpha-32P]ATP; enzyme-bound and free nucleotides were separated by a pressure column. Both procedures were carried out under continuous illumination. The ratio of enzyme-bound ADP to ATP was 0.46 +/- 0.04. In a third experiment, uni-site ATP hydrolysis under de-energized conditions was initiated by the addition of 39 nM [alpha-32P]ATP and NH4Cl/valinomycin in the absence of illumination. Free and enzyme-bound nucleotides were separated also by a pressure column. The ratio of enzyme-bound ADP to ATP was 0.43 +/- 0.02. This ratio was always the same irrespective of whether the reaction runs in the synthesis or the hydrolysis direction. Furthermore, the ratio does not depend on the membrane energization. We conclude, therefore, that the protons are not directly involved in the reaction at the catalytic site.
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PMID:Transport protons do not participate in ATP synthesis/hydrolysis at the nucleotide binding site of the H(+)-ATPase from chloroplasts. 133 Jul 4

ATPase activity of the coupling factor 1, CF1, isolated from spinach chloroplasts, was enhanced by reduction with dithiothreitol. Reduced thioredoxins from spinach chloroplasts, Escherichia coli and human lymphocytes replaced dithiothreitol as reductant and activator of the ATPase. CF1 must be in an oxidized activated state to be further activated by reduced thioredoxin. This state was obtained either by heating CF1 or removing the inhibitory intrinsic epsilon subunit from CF1. Efficiency and primary structure of the different thioredoxins were compared. The progressive addition of KCl during ATPase activation by reduced thioredoxin increases then decreases this process. We proposed that three basic amino acids corresponding to arginine 73 and lysines 82 and 96 in Escherichia coli thioredoxin play an important role in the anchorage of the thioredoxin to the negatively charged surface of the CF1 and are involved in the dual effect of KCl. The variations in the screening effect of the negative charges of the CF1 surface by K+ ions can indeed explain the changes in the anchorage of these 3 basic amino acids with concomitant variation in ATPase activity. Human thioredoxin must be 10 times more concentrated than Escherichia coli or spinach chloroplast thioredoxin to exhibit the same activation effect on the ATPase. This fact was related to the properties of a sequence equivalent to the part from amino acid 59 to 72 in Escherichia coli thioredoxin. This part which joins the two lobes of the thioredoxin is more hydrophilic and more negatively charged in human thioredoxin than in Escherichia coli or spinach chloroplast thioredoxin. Although ATPase activation was obtained at a very low concentration of the reduced spinach chloroplast thioredoxin, the thioredoxin formed only a loose complex with CF1.
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PMID:Induction by different thioredoxins of ATPase activity in coupling factor 1 from spinach chloroplasts. 214 Feb 77

A dormant enzyme, nucleoside triphosphate hydrolase (EC 3.6.1.3) purified from the tachyzoite of Toxoplasma gondii, was activated by the treatment with dithiothreitol. The catalytic activity remained after exclusion of dithiothreitol from the enzyme solution with a Sephadex G-25 column. This activity was completely blocked by the additional treatment with N-ethylmaleimide. It was concluded that the activation occurred through the reductive cleavage of disulfide bond on the enzyme. The reduced type of thioredoxin, partially purified from mouse liver, could replace the effect of dithiothreitol. These results strongly suggest that the enzyme activity is regulated by the oxido-reduction change in the enzyme molecule.
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PMID:Possible regulation mechanism of potent nucleoside triphosphate hydrolase in Toxoplasma gondii. 282 9

The membrane-bound H(+)-ATPase from chloroplasts, CF0F1, was brought into the active, reduced state by illumination in the presence of thioredoxin and dithiothreitol. The endogenous nucleotides were removed by a washing procedure so that the active, reduced enzyme contained one tightly bound ATP per CF0F1. When [14C]ADP was added in substoichiometric amounts during continuous illumination, ADP was bound to the enzyme, phosphorylated and released as [14C]ATP, i.e., the tightly bound ATP was not involved in the catalytic turnover ('uni-site ATP-synthesis'). The rate constant for ADP binding was k = (2.0 +/- 0.5) x 10(6) M-1 s-1. The rate of ATP synthesis was measured as a function of the ADP concentration from 8 nM up to 1 mM in the presence of 2 mM phosphate during continuous illumination. A linear increase of the rate was observed up to 100 nM. Above this concentration a supralinear increase was found, indicating the occupation of a second ADP-binding site. A plateau was reached between 1.5 microM and 2.3 microM ADP with a rate of vpl = 3.7 s-1. The half-maximal rate from this plateau was observed at 780 nM. Above 2.3 microM ADP up to 1 mM ADP the data were described by Michaelis-Menten kinetics (vmax = 80 s-1; apparent KM = 32 microM). These results indicated the participation of at least two different ADP binding sites in ATP synthesis catalyzed by the membrane-bound CF0F1.
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PMID:From uni-site to multi-site ATP synthesis in thylakoid membranes. 836 35

Fluorescence changes attributed to state transitions have been shown to exist in phycobilisome-containing organisms. Contradictory conclusions have been derived from studies about the mechanism of state transitions carried out either in cyanobacteria or in red algae. In this paper, fluorescence changes induced by light 1 and light 2 are reinvestigated in a unicellular red alga, Rhodella violacea, by performing 77 K fluorescence spectra and fluorescence yield measurements at room temperature in the presence of uncouplers and inhibitors of the electron transfer. We show that transfer of light 1-adapted cells to light 2 (green light) induces a large quenching of photosystem II which is suppressed by subsequent incubation in light 1 (far-red or blue light). The level of the photosystem I-related fluorescence does not change during these transfers. We demonstrate that the large quenching of photosystem II induced by low intensities of green light is completely suppressed by addition of NH4Cl, an uncoupler that inhibits ATP synthesis by canceling the delta pH across the membrane. DCCD, which is an inhibitor of the ATPase that swells the delta pH, maintains the quenched state even under light 1 illumination. The opposite effects of DCMU and DBMIB on state transitions are demonstrated to be due to a suppression (by DCMU) or maintenance (by DBMIB) of the delta pH and not to change in the redox state of the plastoquinone. We conclude that, in R. violacea, the fluorescence change commonly associated with state 2 transition is in fact a delta pH-dependent quenching. This type of quenching has always been associated with near-saturating light intensities. Here, we show that very low intensities of a light that activates only the photosystem II induce a delta pH across the membrane that is not dissipated since the ATPase is not activated. The delta pH is dissipated only under conditions in which the photosystem I turns, confirming that the thioredoxin must be reduced to activate the ATPase. We suggest that the fluorescence changes, induced by various light conditions, in cyanobacteria and red algae could be associated with different phenomena.
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PMID:State transitions or delta pH-dependent quenching of photosystem II fluorescence in red algae. 875 22

Analysis of 76 kb of newly sequenced DNA, located between map positions 182 and 258 kb in the 330-kb chlorella virus PBCV-1 genome, revealed 175 open reading frames (ORFs) of 65 codons or longer. One hundred and five of these 175 ORFs were considered major ORFs. Twenty-one of the 105 major ORFs resembled proteins in databases including ribonucleotide reductase small subunit, RNase III, thioredoxin, glutaredoxin, protein disulfide isomerase, deoxynucleoside kinase, frog virus 3 ATPase, Acetobacter cellulose synthase, a bacteriophage encoded endonuclease, and two C-5 cytosine DNA methyltransferases. One of the ORFs was the PBCV-1 major capsid protein. The 105 major ORFs were evenly distributed along the genome. One set of ORFs was separated by 543 nucleotides whereas 75 of the ORFs were separated by fewer than 100 nucleotides. Nineteen of the 175 ORFs resembled other PBCV-1 ORFs, suggesting that they represent either gene duplications or gene families.
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PMID:Analysis of 76 kb of the chlorella virus PBCV-1 330-kb genome: map positions 182 to 258. 880 66

Two thioredoxin cDNAs from soybean were isolated by screening an expression library using an anti-(plasma membrane) serum. The nucleotide sequences of the two cDNAs were found to be 89% identical. The polypeptides encoded by the two cDNAs, designated TRX1 and TRX2, contain a disulfide active site, as found in other thioredoxins. TRX1 was expressed as a fusion protein in Escherichia coli and shown to possess thiol-disufide interchange activity. Unlike other eukaryotic thioredoxins, these two soybean thioredoxins contain a putative transmembrane domain in their N-terminal regions. To determine subcellular location, the TRX1 was fused with a reporter epitope at its C-terminus and expressed in transgenic tobacco plants. The fusion protein was co-purified with plasma membrane markers 1,3 beta-glucan synthase and vanadate-sensitive ATPase, indicating the plasma membrane location of TRX1. When the reporter epitope was inserted between the start codon and the transmembrane domain in the N-terminus, the fusion protein was found in the soluble fraction, possibly due to disruption of the transmembrane domain by the highly hydrophilic epitope sequence. Taken together, our results demonstrate that soybean TRX1 is a plasma membrane-bound thioredoxin, which is most likely anchored to the membrane through the N-terminal transmembrane domain. It is known that plant plasma membranes contain various proteins with thiol-disulfide interchange activity. The soybean thioredoxins reported here are the first group of such proteins to be characterized at the molecular level. However, the biological function of the plasma membrane-bound thioredoxin remains to be determined.
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PMID:A novel plasma membrane-bound thioredoxin from soybean. 898 May 17

The kinetics of thiol modulation of the chloroplast H+-ATPase (CF0CF1) in membrana were analyzed by employing thioredoxins that were kept reduced by 0.1 mM dithiothreitol. The kinetics of thiol modulation depend on the extent of the proton gradient. The process is an exponential function of the thioredoxin concentration and reaction time and can be described by an irreversible second order reaction. The results indicate that the formation of the complex between thioredoxin and CF0CF1 is slow compared with the subsequent reduction step. Furthermore we have compared the efficiencies of the Escherichia coli thioredoxin Trx and the two chloroplast thioredoxins Tr-m and Tr-f. The second order rate constants are 0.057 (Tr-f), 0.024 (Trx), and 0.010 s-1 microM-1 (Tr-m) suggesting that Tr-f rather than Tr-m is the physiological reductant for the chloroplast ATPase. The often employed artificial reductant dithiothreitol exhibits a second order rate constant in thiol modulation of 1.02.10(-6) s-1 microM-1.
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PMID:Kinetics and thioredoxin specificity of thiol modulation of the chloroplast H+-ATPase. 920 2

Vacuole fusion requires Sec18p (NSF), Sec17p (alpha-SNAP), Ypt7p (GTP binding protein), Vam3p (t-SNARE), Nyv1p (v-SNARE), and LMA1 (low Mr activity 1, a heterodimer of thioredoxin and I(B)2). LMA1 requires Sec18p for saturable, high-affinity binding to vacuoles, and Sec18p "priming" ATPase requires both Sec17p and LMA1. Either the sec18-1 mutation and deletion of I(B)2, or deletion of both I(B)2 and p13 (an I(B)2 homolog) causes a striking synthetic vacuole fragmentation phenotype. Upon Sec18p ATP hydrolysis, LMA1 transfers to (and stabilizes) a Vam3p complex. LMA1 is released from vacuoles in a phosphatase-regulated reaction. This LMA1 cycle explains how priming by Sec18p is coupled to t-SNARE stabilization and to fusion.
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PMID:LMA1 binds to vacuoles at Sec18p (NSF), transfers upon ATP hydrolysis to a t-SNARE (Vam3p) complex, and is released during fusion. 965 46

Many membrane proteins that belong to the ATP-binding cassette (ABC) superfamily are clinically important, including the cystic fibrosis transmembrane conductance regulator, the sulphonylurea receptor and P-glycoprotein (multidrug resistance gene product; MDR1). These proteins contain two multispanning transmembrane domains, each followed by one nucleotide-binding domain (NBD) and a linker region distal to the first NBD. ATP hydrolysis by the NBDs is critical for ABC protein function; the linker region seems to have a regulatory role. Previous attempts to express soluble NBDs and/or linker regions without detergent solubilization, or to purify NBDs at high yields as soluble fusion proteins, have been unsuccessful. Here we present a system for the expression in Escherichia coli of the first NBD of MDR1 followed by its linker region (NBD1MLD). A comparison of the expressions of NBD1MLD fused to glutathione S-transferase, thioredoxin and maltose-binding protein (MBP) shows that a high level of expression in the soluble fraction (approx. 8% of total E. coli protein) can be achieved only for MBP-NBD1MLD. The addition of a proteolytic thrombin site just proximal to the N-terminal end of NBD1MLD allows the cleavage of NBD1MLD from MBP, which can be easily purified with retention of its ATPase activity. In summary, success was obtained only when using an MBP fusion protein vector containing a thrombin proteolytic site between MBP and NBD1MLD. The approach described here could be generally applicable to solving the problems of expression and purification of NBDs/linker regions of ABC proteins.
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PMID:Expression and purification of the first nucleotide-binding domain and linker region of human multidrug resistance gene product: comparison of fusions to glutathione S-transferase, thioredoxin and maltose-binding protein. 993 1


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