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)

Incubation in the dark of 32P-labeled 2-azido-adenine nucleotides with submitochondrial particles from beef heart led to tight binding of the label by membrane-bound F1. That is, the label remained with the particles following two passages through centrifuge columns. After removal of free nucleotides and ultraviolet irradiation, the radioactive label was covalently bound exclusively to the beta subunit of the ATPase. Extraction of the modified enzyme from the membrane with chloroform followed by tryptic digestion and separation of peptides by reverse-phase high-pressure liquid chromatography indicated that the radioactive label had been inserted into a peptide fragment that included part of the catalytic site. Covalent modification of catalytic sites by 2-azido-ADP was accompanied by parallel inhibition of both ATP synthesis and ATP hydrolysis by submitochondrial particles. Estimation of the likely amount of F1 participating in the reaction and extrapolation to complete inhibition suggested that modification of no more than a single site was sufficient to block both reactions. The results support suggestions of cooperative interactions between catalytic sites as well as a single catalytic pathway for both enzymic reactions.
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PMID:Covalent modification of catalytic sites on membrane-bound beef heart mitochondrial ATPase by 2-azido-adenine nucleotides. 792 3

A small proteolipid called PMP1 is associated with yeast plasma membrane H(+)-ATPase (Navarre, C., Ghislain, M., Leterme, S., Ferroud, C., Dufour, J.-P., and Goffeau, A. (1992) J. Biol. Chem. 267, 6425-6428). We have identified a second Saccharomyces cerevisiae plasma membrane proteolipid gene by hybridization with a PMP1 probe. The sequence of the corresponding gene, called PMP2, is 92% identical to the PMP1 gene sequence. PMP2 encodes a 43-amino acid polypeptide that can be extracted from the membrane with chloroform/methanol. The two proteolipids differ at residue 21, which is an alanine in PMP1 and a serine in PMP2. The two PMP genes are similarly expressed in the wild-type strain, and no modification of the level of transcription of one PMP gene is detected in a strain deleted of the other. A regulatory function of the proteolipids is indicated by the observation that a strain lacking both PMP genes and no longer containing plasma membrane proteolipids displays a lower Vmax of the plasma membrane H(+)-ATPase activity.
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PMID:Two distinct genes encode small isoproteolipids affecting plasma membrane H(+)-ATPase activity of Saccharomyces cerevisiae. 806 50

Following the earlier observation that inhalation of volatile lipid solvents and of narcotic gases causes cholestasis, we studied the effects of various organic solvents on bile flow, plasma membrane fluidity and potassium movement in rat liver. Both in vivo and in the isolated perfused liver, applications of CCl4, CHCl3, dichloromethane, trichloroethylene, halothane, benzene and cyclohexane elicited rapid and sustained but reversible cholestasis. A transient phase of choleresis was observed prior to and after cholestasis, during the increase and fall in liver tissue solvent concentrations, respectively. Tissue concentrations required to produce cholestasis were lower the higher the lipophilicity of the solvent. Membrane fluidity was measured in isolated basolateral liver cell membranes by fluorescence polarization. Fluidity increased with increasing solvent concentration, the increase being associated with either biphasic stimulation and inhibition of membrane enzymes (Na+,K(+)-ATPase, 5'nucleotidase) or with inhibition alone (Mg(2+)-ATPase). In the isolated perfused liver, application of organic solvents caused hepatic uptake of K+ that was followed by K+ release upon withdrawal of the solvent. The magnitude of K+ uptake elicited by the solvent was comparable with the effect of blocking K+ channels with 2 mM Ba2+, but Ba2+ was ineffective in the presence of the solvent. In contrast, application of ouabain caused K+ release in equal amounts in the absence and presence of the solvent, indicating that K+ uptake elicited by the solvent results from inhibition of K+ efflux through K+ channels rather than stimulation of the Na+,K+ pump. The data show that cholestasis elicited by lipid solvents is associated with an increase in membrane fluidity and with disturbance of liver K+ homeostasis. The significance of these observations is discussed with respect to other models of experimental cholestasis.
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PMID:Organic solvents increase membrane fluidity and affect bile flow and K+ transport in rat liver. 821 71

Interactions between Escherichia coli membrane phospholipids and the hydrophobic c subunit of the F1F0 proton-translocating ATPase were characterized. Extraction of E. coli membranes with a neutral mixture of chloroform and methanol and subsequent separation steps produced several protein-containing fractions. The protein-containing fraction most soluble in organic solvents contained subunit c and a lipid fraction enriched in phosphatidylglycerol compared to total E. coli membrane phospholipids. Other ATPase subunits and some additional proteins extracted from the membranes by this procedure could be separated from the c subunit by subsequent extraction. The purified and delipidated c subunit contained fatty acids which were released upon treatment with boron trifluoride methanol. Furthermore, deleting and restoring the genes for the F0 subunits changed the composition of extractable membrane phospholipid and fatty acids, indicating that the F0 plays a significant structural role in the membrane.
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PMID:Protein-lipid interactions of the proteolipid c subunit of the Escherichia coli proton-translocating adenosinetriphosphatase. 834 58

The membrane-intrinsic protein phospholamban (PLN), the regulatory protein of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, was chemically synthesized. The synthesis was accomplished by double couplings and efficient capping procedures, thus eliminating hydrophobic failure sequences. The crude peptide was purified by high-performance liquid chromatographic ion exchange and gel permeation chromatography in chloroform-methanol mixtures. Ion spray mass spectroscopy showed that the product had the correct molecular mass. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis runs produced the typical monomer-pentamer structural pattern. A predominantly helical CD spectrum was obtained in 0.075% C12E8 (67.9% helix, 1.8% beta, 12.2% turn, 18.1% random coil). Synthetic PLN was phosphorylated in detergent solutions by protein kinase A with a stoichiometry close to 1:1 (Pi to PLN monomer). Reconstitution of the isolated skeletal muscle SR Ca2+ ATPase in phosphatidylcholine membranes in the presence of PLN using the freezing and thawing technique yielded a preparation with lower Ca(2+)-dependent ATPase activity. The inhibition was mainly due to a decrease in the affinity (Km(Ca)) of the ATPase for Ca2+ and was partially reversed by PLN phosphorylation with protein kinase A. By contrast, addition of PLN to diluted intact SR vesicles uncoupled the Ca(2+)-transport reaction, suggesting an ionophoric effect of PLN. Because this effect was observed at very high PLN-to-SR vesicle ratios and was not influenced by PLN phosphorylation, its biological function is doubtful.
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PMID:Total synthesis and functional properties of the membrane-intrinsic protein phospholamban. 838 40

Vasoconstrictor and Na/K pump inhibitory properties of a bufodienolide Na/K-ATPase inhibitor, marinobufagenin, were studied in isolated rings of 2 to 3 order branches of human pulmonary arteries respectively. Marinobufagenin displayed concentration-dependent vasoconstrictor activity (0.01 to 10 mmol/L). In sarcolemma membranes prepared from pulmonary artery marinobufagenin inhibited Na/K-ATPase (IC50 = 50 nmol/L). In eight healthy male Caucasians, concentrations of marinobufagenin-like immunoreactive material in C-18 extracted plasma were 1.38 +/- 0.60 nmol/L. Twenty-four-hour urinary release of marinobufagenin-like immunoreactive material in eight healthy males was 1.20 +/- 0.95 nmol/day. Chloroform extract of human urine was fractionated using reverse-phase high-performance liquid chromatography (32% acetonitrile, Deltapak). The HPLC fraction coeluting with marinobufagenin in 7 min, cross reacted with antimarinobufagenin and antidigoxin, but not antiouabain antibody. These results demonstrate that human plasma and urine contains a bufodienolide vasoconstrictor EDLF, marinobufagenin-like immunoreactive Na,K pump inhibitor.
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PMID:Endogenous marinobufagenin-like immunoreactive substance. A possible endogenous Na, K-ATPase inhibitor with vasoconstrictor activity. 889 50

A method has been evolved toward the aim of getting suitable crystals for high resolution of structural analysis of F1-ATPase by X-ray crystallography. The different conditions for crystal growth of ATPase that were isolated and purified by different methods from pig heart mitochondrial ATP synthase had been compared and screened. A simple method for purification of F1-ATPase was adopted. The F1-ATPase is released with chloroform from submitochondrial particles. Then it was treated with fractional precipitation of (NH4)2SO4 and finally was further purified by employing the sephadex G 200 column. The crystals of F1-ATPase were usually obtained after a few months. They appeared to have uniform morphology of tetrahedron. They diffracted to a resolution of 7A. The diffraction data were collected on the XRD-100 Siemens Area Detector. According to a total of 240 frames, the cell parameters obtained are a = b = 147 A, c = 208 A, alpha = beta = gamma = 90 alpha, the probable space group is P4 or its antipode. The reproducibility of this method for crystallization of F1-ATPase is good.
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PMID:Purification and crystal growth of F1-ATPase from pig heart mitochondria. 890 56

The isolation and properties of F1-mitochondrial ATPase from rat testis are described. The isolation medium involves a chloroform extraction, and it is suitable even with small amounts of starting material that have a relatively low specific activity as in the case of rat testis submitochondrial particles. The isolated enzyme from rat testis had a specific activity of 30-45 mumol Pi/min/mg protein, which could be increased up to 90 mumol Pi/min/mg protein only in the presence of bicarbonate and maleate. The isolated enzyme represented less than 0.6% of the initial membrane proteins. It exhibited a typical five-band pattern in sodium dodecyl sulfate gel electrophoresis. However, it showed a ratio of subunits alpha:beta higher than the heart enzyme; its significance is unknown. The purified enzyme was cold labile and inhibited by natural ATPase inhibitor protein from bovine heart mitochondria and by dicyclohexylcarbodiimide. The results presented suggest that the low ATPase activity of testis submitochondrial particles is due to a reduced content of the F1-ATPase.
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PMID:Isolation and comparative studies of mitochondrial F1-ATPase from rat testis and beef heart. 911 89

Escherichia coli strain PEF42 produces a sodium-ion-dependent hybrid F1F0-ATPase consisting of the Propionigenium modestum subunits a, b, c and delta, of a hybrid alpha subunit and of the E. coli subunits beta, gamma and epsilon. The gene encoding subunit c of the P. modestum F1F0-ATPase was cloned into the pT7-7 expression vector to yield plasmid pT7c. E. coli PEF42 was transformed with plasmid pT7c together with plasmid pGP1-2, which harbours the gene for the T7 RNA polymerase. The production of the P. modestum subunit c was induced by a temperature shift from 30 degrees C to 42 degrees C for 30 min and led to an increased concentration of this protein in the membrane of the host strain. The c subunit produced in E. coli moved as a monomer in dodecylsulfate electrophoresis. The protein was extracted from the cells with chloroform/methanol, purified and incorporated into sodium dodecylsulfate micelles. Circular dichroism of subunit c in sodium dodecylsulfate showed a temperature-stable spectrum (between 20-60 degrees C) with a high proportion of alpah-helical structure. Upon incubation of subunit c with [14C]dicyclohexylcarbodiimide the protein became labelled in a sodium-ion-dependent manner, similar to the labelling observed if the purified F1F0-ATPase of P. modestum, was treated with the radioactive carbodiimide. The Na+-specific site was therefore retained in the isolated c subunit dissolved in dodecylsulfate.
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PMID:Subunit c from the sodium-ion-translocating F1F0-ATPase of Propionigenium modestum--production, purification and properties of the protein in dodecylsulfate solution. 928 3

A mini review of the properties of the natural phytotoxin, tentoxin, is proposed. In particular, the biological activities of tentoxin on the chloroplast F0F1 proton ATPase, which realizes the synthesis of ATP at the expense of an electrochemical gradient of protons, are discussed. In this respect, structure-activity relationships of tentoxin have been re-examined in the light of the recent developments obtained by two-dimensional proton nuclear magnetic resonance (81). The conformations of the cyclic tetrapeptide [cyclo-(L-MeAla1-L-Leu2-MePhe[(Z) delta]3-Gly4)] have been studied in aqueous solution at various temperatures. Contrary to what was observed in early studies in chloroform, tentoxin was proved to exhibit multiple exchanging conformations in water. Four conformations with different proportions (51, 37, 8 and 4%) were found. Models were derived from nuclear magnetic resonance parameters and restrained molecular dynamics simulations. They confirmed that the four conformers exhibited the cis-trans-cis-trans configuration of the amide bond sequence. The conversion from one form to another is accomplished by a conformational peptide flip consisting of a 180 degrees rotation of a non-methylated peptide bond. In addition, important aggregation phenomena were observed. These effects have also been evidenced in chloroform, and compared to results derived from experiments carried out in the presence of DPC micelles. The tentoxin molecule was found self-associated in solution in a micellar-like organization. On the basis of these observations, we propose to design new analogues, with the intention of elucidating the mode of action of tentoxin in plants on the molecular level, especially under the aspect of its interaction with the chloroplast ATPase.
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PMID:[Tentoxin: structure-activity relationship. Application to the study of its action on chloroplast ATP-synthase]. 929 66


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