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 purification of the Escherichia coli dnaB protein by affinity chromatography on nucleotides bound to agarose is described. The dnaB protein, which contains an associated ribonucleoside triphosphatase activity (Wickner, S., Wright, M., and Hurwitz, J. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 783-787) binds to immobilized ATP, ADP, and UDP, but not to AMP. The type of linkage of ATP to agarose influences the adsorption, elution, and purification of the enzyme. Optimal purification is achieved using ATP bound to agarose via its oxidized ribose moiety. By this means, the dnaB protein can be obtained at least 95% electrophoretically pure after only three purification steps. The enzyme can be eluted from immobilized nucleoside-5'-di- and -triphosphates by ATP, ADP, and pyrophosphate, but not by AMP or orthophosphate. ADP and pyrophosphate, as well as the substrate ATP in high concentration are at the same time inhibitors of the ribonucleoside triphosphatase. The dnaB complementing and ribonucleoside triphosphatase activities could not be separated from each other by affinity chromatography, supporting the finding of others that they both reside on the same protein complex, namely a dnaB multimer. The results indicate that the dnaB protein binds to immobilized nucleotides by means of its ribonucleoside triphosphatase, and that at least the pyrophosphate moiety is essential for adsorption as well as elution of the enzyme.
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PMID:Escherichia coli dnaB protein. Affinity chromatography on immobilized nucleotides. 35 57

Preparations of intestinal epithelial cell basal lateral plasma membranes were analyzed with free flow electrophoresis and density perturbation with digitonin. The initial basal lateral membrane preparations were obtained by equilibrium density gradient centrifugation after two different schemes of homogenization and differential sedimentation (A.K. Mircheff, C.H. van Os, and E.M. Wright. 1978. Membr. Biochem. 1:177, and A.K. Mircheff, S.D. Hanna, M.W. Walling, and E.M. Wright. 1979. Prep. Biochem. 9:33. In these preparations, Na,K-ATPase, a marker for the basal lateral mambrane, was purified 16- to 18-fold over the initial homogenate. The preparations were also enriched in NADPH-cytochrome c reductase, alkaline phosphatase, acid phosphatase, and galactosyltransferase. Both free-flow electrophoresis, which separates on the basis of surface charge, and density perturbation with digitonin, which depends on a specific interaction of digitonin with cholesterol-rich membranes, resolved the preparation into three populations of particles. The major population, which represented basal lateral membranes purified 20- to 32-fold with respect to the initial homogenate, contained Na,K-ATPase, alkaline phosphatase, adenylate cyclase, and acid phosphatase. A second population was defined by its content of NADPH-cytochrome c reductase, and the third was defined by its content of galactosyltransferase. Guanylate cyclase appeared to be partitioned between the Na,K-ATPase-rich and NADPH-cytochrome c reductase-rich populations. Galactosyltransferase is also present in fractions which contain the Na,K-ATPase-rich membranes, but the present data cannot exclude the possibility of spillover by the adjacent, galactosyltransferase-rich population. This work emphasizes the importance of multiple, physical criteria for purity in the isolation of subcellular components.
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PMID:Highly purified basal lateral plasma membranes from rat duodenum. Physical criteria for purity. 51 18

Treatment of the solubilized and purified Ca(2+)-translocating ATPase (Ca(2+)-ATPase) (136 kDa) from human erythrocyte plasma membranes with endoproteinase Glu-C from Staphylococcus aureus strain V8 (V8 protease) yielded transient fragments of 96 kDa and 76 kDa and more stable fragments of 60 kDa and 37/36 kDa (doublet). The presence of calmodulin did not alter the fragmentation pattern. The 60 kDa fragment contains the protein kinase C (bovine brain) phosphorylation site(s), which we previously localized in the C-terminal region [Wang, Wright, Machan, Allen, Conigrave & Roufogalis (1991) J. Biol. Chem. 266, 9078-9085]. On the other hand, the 37/36 kDa fragments possess the ability to form an acyl-phosphate intermediate. Furthermore, the presence of the 60 kDa and 37/36 kDa fragments together results in expression of calmodulin-sensitive Ca(2+)-ATPase activity. However, further degradation of the 60 kDa fragment was coupled with the appearance of calmodulin-independent activity, whereas the 37/36 kDa fragment doublet remained stable. It was concluded that the 60 kDa and the 37/36 kDa fragments: (a) together represent the C-terminal two-thirds of the enzyme, which is functional as an Ca(2+)-ATPase, (b) were produced by a single cleavage near the C-terminal side of the cytosolic catalytic domain, and (c) probably remain physically and functionally associated even after cleavage has occurred. At the C-terminus, the basic calmodulin-binding domain is flanked by two highly acidic regions (domains A and B). Our results indicate that domains A and B, despite containing many Asp and Glu residues, were not readily cleaved by V8 protease, which is known to cleave selectively peptide bonds at the C-terminal side of Asp and Glu. However, if the Ca(2+)-ATPase were pre-digested with calpain I from human erythrocytes, which removed its calmodulin-binding domain (along with domain B), multiple cleavages by V8 protease in domain A were then readily observed. We propose that the calmodulin-binding domain is closely associated with the acidic domains A and B and that these acidic domains might help to co-ordinate the stimulation of the enzyme by calmodulin.
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PMID:Structure--function relationship of the human erythrocyte plasma membrane Ca(2+)-ATPase revealed by V8 protease treatment. 183 79

To determine whether thiophosphorylation of the 20-kDa myosin light chain activates each head of smooth muscle myosin independently of the head with which it is paired, chicken gizzard smooth muscle myosin was randomly thiophosphorylated, producing a mixture of unphosphorylated and singly and doubly thiophosphorylated myosin. Thiophosphorylation levels were measured by glycerol-urea gels, and the activity of this myosin was determined by actin-activated adenosinetriphosphatase measurements and in an in vitro motility assay, where the velocity of actin filaments moving over a myosin-coated surface is measured. Activity at each thiophosphorylation level was similar to that previously observed for mixtures of unphosphorylated and doubly thiophosphorylated myosin (D. E. Harris, S. S. Work, R. K. Wright, N. R. Alpert, and D. M. Warshaw. J. Muscle Res. Cell Motil. 15: 11-19, 1994). All doubly thiophosphorylated myosin was then formed into filaments and removed from randomly thiophosphorylated myosin by centrifugation. The remaining myosin (mixture of unphosphorylated and singly phosphorylated myosin), which could not polymerize because of their conformation, retained approximately 70% activity compared with mixtures of unphosphorylated and doubly thiophosphorylated myosin. Thus a thiophosphorylated smooth muscle myosin head can produce substantial biochemical and mechanical activity, even when it is paired with an unphosphorylated partner.
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PMID:Thiophosphorylation independently activates each head of smooth muscle myosin in vitro. 749 5

We have previously shown in intact human erythrocytes that both the plasma membrane Ca2+ pump activity and its phosphorylation can be increased by phorbol-12-myristate 13-acetate (PMA), a known stimulator of protein kinase C. These effects were inhibited by high doses of adriamycin (L. C. Wright et al., 1993, Arch. Biochem. Biophys. 306, 277-284). We now show that low doses of adriamycin (ADR) (maximum effect at 10 microM for 1-6 min) decrease the amplitude of the intracellular calcium ([Ca2+]i) transient induced by 2.5 microM CaCl2 and 10 microM A23187 in intact human erythrocytes. This is reflected by a parallel increase in Ca(2+)-ATPase activity in plasma membranes isolated from pretreated intact cells. When 10 microM ADR and 1 microM PMA were combined the effects were additive, with a maximum decrease in the Ca2+ transient amplitude of 50%. A similar effect was seen on the Ca(2+)-ATPase activities in isolated membranes. In erythrocytes labeled with [32P]orthophosphate 10 microM ADR induced a 1.5-fold increase in the phosphorylation of the Ca2+ pump and when combined with 1 microM PMA the phosphorylation was greatly enhanced (2.3 times that induced by PMA alone). ADR alone and in combination with PMA was found to decrease both 32P labeling and lipid phosphate content of phosphatidylinositol 4,5-bisphosphate (PIP2). This was accompanied by an increase in the amount of 1,2-diacylglycerol formed in response to 10 microM ADR. We conclude that low doses of ADR are able to stimulate the breakdown of 6-13% of erythrocyte PIP2 by phospholipase C at an intracellular calcium concentration of 2.5 microM, normally regarded as below threshold for phospholipase C activation in erythrocytes. The diacylglycerol formed appears to stimulate protein kinase C to activate the Ca2+ pump and enhance its phosphorylation and Ca2+ efflux in intact human erythrocytes.
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PMID:Regulation of the activity and phosphorylation of the plasma membrane Ca(2+)-ATPase by adriamycin in intact human erythrocytes. 764 72

A vacuolar type H(+)-ATPase inhibitor, concanamycin A (CMA), raised the pH and induced morphologic changes such as vacuolation in lytic granules in the CD8+ cytotoxic T lymphocyte clone OE4. Here we measured the pH in lytic granules present in OE4 by immunoelectron microscopic observation using (3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP) as a pH probe. In control OE4, only the peripheral region, but not cores, were heavily stained with DAMP. Exposure to 100 nM CMA for 60 min raised the pH in the lytic granules from 5.7 +/- 0.2 to 6.8 +/- 0.2. Furthermore, the number of lytic granules in OE4 was estimated under phase contrast microscopy after Wright staining. About 20 lytic granules in average were detected in control cells, while CMA induced vacuolation and decreased their number by 20 to 30%.
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PMID:Estimation of pH and the number of lytic granules in a CD8+ CTL clone treated with an inhibitor of vacuolar type H(+)-ATPase concanamycin A. 898 77

The uptake of sucrose into isolated discs cut from sink (growing) and source (sprouting) potato (Solanum tuberosum L.) tuber tissue was studied. The uptake of sucrose into sink-tuber discs demonstrated biphasic kinetics. The large saturable component was inhibited by incubation of the discs with p-chloromercuribenzene sulfonic acid (PCMBS) whilst both the saturable and linear components were inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP). By contrast, in source-tuber discs, the linear component represented the majority of sucrose taken up, the saturable component playing only a minor role. In source discs, only the saturable component of uptake was inhibited by either PCMBS or CCCP. A large proportion (up to 25%) of sucrose taken up into sink-tuber discs was converted to starch but as the tubers aged the proportion of sucrose converted to starch decreased to the level found in source-tuber discs (approx. 3%). By contrast with sink-tuber discs (see Oparka and Wright, 1988b, Planta 175, 520-526) sucrose uptake into source discs was insensitive to turgor and demonstrated an uptake pattern similar to that of CCCP-treated sink tissue. It is proposed that exogenous sucrose is taken into the storage parenchyma of sink-tuber discs by both a carrier-mediated and a diffusional process. By contrast, uptake into the storage parenchyma of source-tuber discs appears to be essentially diffusional. The turgor sensitivity of sucrose uptake into sink-tissue discs may be mediated via the plasmalemma H(+)-ATPase. As the tuber ages the sucrose-uptake activity decreases and the capacity of the storage parenchyma to synthesise starch is lost. The data are discussed in relation to the in-vivo mechanisms of sucrose transport in storage tissues.
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PMID:Sucrose uptake and partitioning in discs derived from source versus sink potato tubers. 2421 46

The Na(+)-glucose cotransporter is a key transport protein that is responsible for absorbing Na(+) and glucose from the luminal contents of the small intestine and reabsorption by the proximal straight tubule of the nephron. Robert K. Crane originally described the cellular model of absorption of Na(+) and glucose by a "cotransport process" in 1960. Over the past 50+ yr, numerous groups have tested and verified Crane's hypothesis. Eventually, Wright and colleagues cloned the Na(+)-glucose cotransporter (SGLT1; the product of the SLC5A1 gene) in 1987. This article provides a "hands-on" laboratory exercise using the everted mouse jejunal preparation (everted sac) that allows students to investigate various components of the Na(+)-glucose cotransport absorptive cell model (e.g., Na(+) dependence of SGLT1, inhibition of SGLT1, and inhibition of Na(+)-K(+)-ATPase). Additionally, the laboratory exercise includes a case-based study of glucose-galactose malabsorption in which the students conduct an internet search and participate in a small-group discussion during the laboratory period to better understand the basic principles and functions of the Na(+)-glucose absorptive process of the small intestine. This laboratory exercise was introduced into the second-year undergraduate physiology curriculum in 2008, and >850 physiology students have participated in this laboratory exercise. The students have produced very robust and reproducible data that clearly illustrate the theory of the cellular model for Na(+)-glucose absorption by the jejunum.
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PMID:Glucose transport into everted sacs of the small intestine of mice. 2429 21