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 Ca2+-binding component of troponin (TnC) and its proteolytic fragments containing Ca2+-binding sites I-III (TH1) or sites III and IV (TR2C) have been labeled with the fluorescent probes dansylaziridine (DANZ) at methionine 25 or 5-(iodoacetamidoethyl)amino-naphthalene-1-sulfonic acid (AEDANS) at cysteine-98. These probes report binding of Ca2+ to the low and high affinity sites, respectively. Fluorescence changes as a function of [Ca2+] were measured for the free peptides, their complexes with troponin I + troponin T, and these complexes bound to actin-tropomyosin in the presence of Mg2+ and ATP with and without myosin. An apparent Hill coefficient of 1.0-1.1 has been obtained for the Ca2+-induced fluorescence changes in TnC, its fragments, and their ternary complexes regardless of the label used. When a ternary complex containing appropriately labeled TnC or its fragment is bound to the actin-tropomyosin complex, the Hill coefficient for the titration of the low affinity sites increases to 1.5-1.6 and further increases to greater than 2 in the presence of myosin. To interpret the apparent Hill coefficients, we used a model containing two binding sites and a single reporter of the conformational change. Hill coefficients between 1.0 and 1.2 can be obtained for the fluorescence change without true cooperativity in metal binding, depending on the mechanism of the fluorescence change; i.e. the contribution of the singly or doubly occupied species to the fluorescence change. A Hill coefficient between 1.2 and 2, however, always indicates cooperativity in binding independently of the mechanism. Thus, our finding that fluorescence titrations of Ca2+ binding to TnCDANZ bound to actin-tropomyosin exhibit a Hill coefficient of 1.5 in the absence of myosin and 2.4 in its presence indicates the existence of true positive cooperativity in metal binding to sites I and II. No cooperativity was observed for AEDANS-labeled complexes that reflect Ca2+-binding to the high affinity sites. Plots of the Ca2+ dependence of myosin ATPase activity activated by actin-tropomyosin in the presence of any of the troponin complexes used had apparent Hill coefficients of approximately 4. The higher value suggests cooperative interactions in the activation of ATPase beyond those involved in Ca2+-binding to the Ca2+-specific sites.
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PMID:Cooperative binding to the Ca2+-specific sites of troponin C in regulated actin and actomyosin. 664 69

Some lipopolysaccharide-defective mutants of Escherichia coli showed, without ethylenediaminetetraacetic acid treatment, a quick and high uptake of lipophilic cations such as triphenylmethylphosphonium and tetraphenylphosphonium. The rate and amount of uptake were comparable to those of an ethylenediaminetetraacetic acid-treated wild type. Transmembrane electrical potential, which was calculated from the distribution of these lipophilic cations between the inside and outside of the mutant cells, was about -150 mV at pH 7.5 and showed a strong dependency on the external pH. One of the E. coli mutants, the acrA mutant, was found to be also permeable to dicyclohexylcarbodiimide, an H+-adenosine triphosphatase inhibitor, and 1-anilino-8-naphthalene sulfonate, a fluorescent dye. The acrA mutant was vigorously motile and highly sensitive to many bacteriophages and colicins. Thus, the acrA mutant is quite useful for the quantitative measurement of transmembrane electrical potential by lipophilic cations in intact and metabolizing cells especially in relation to motility and actions of colicins and bacteriophages.
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PMID:Use of lipophilic cation-permeable mutants for measurement of transmembrane electrical potential in metabolizing cells of Escherichia coli. 679 76

The activity and stability of carbamoyl-phosphate synthetase (EC 6.3.4.16) may involve hydrophobic and ionic bonds within the enzyme. The 1-anilino-8-naphthalene sulfonate (ANS) equilibrium binding method with hydrophobic and ionic sites in enzymes, therefore, seemed suitable for the study of the acetylglutamate activation and ATP binding of the enzyme. The enzyme had a high affinity for the dye but low fluorescent yields. The enzyme had 32-88 ANS binding sites, depending on combination with ATP and acetylglutamate, and individual affinity constants for each combination. Despite the large number of binding sites, the acetylglutamate and ATP concentrations for half-maximal fluorescent change (10-40 microM) corresponded to the high-affinity bound ATP (ATPB) and acetylglutamate Kd values. In kinetic studies, ANS competed with ATP or acetylglutamate. The extrapolated ANS Ki values for ATP or acetylglutamate were both 35 microM. This value agreed with the ANS Kd value of the enzyme X ATP conformation, indicating that this was the conformation competed for by ANS. Since ANS did not influence the HCO3-dependent ATPase, ANS was concluded to compete with the ATPB binding conformation and transitional changes. This study suggests that part of the activator role of acetylglutamate may be to change the tertiary structure of the enzyme to induce hydrophobic sites which are accessible to ANS and possibly at the ATPB site.
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PMID:Fluorescent probe study with 1-anilino-8-naphthalene sulfonate on acetylglutamate activation, ATP binding and conformational changes of the rat liver carbamoyl-phosphate synthetase. 687 Dec 32

Carbonic anhydrase (CA) activity was localized in the salivery glands of the cockroach, Periplaneta americana, by (1) Hansson's histochemical technique, and (2) the use of the fluorescent sulphonamide, 5-dimethyl-amino-naphthalene-1-sulphonamide (DNSA). Both techniques reveal the same distribution pattern of CA in the four morphologically different cell types of the glands: peripheral cells, central cells, inner acinar duct cells, and distal duct cells. Positive reactions with Hansson's cobalt/phosphate technique were found in the apical regions of the peripheral cells and the distal duct cells, and were inhibited by 10(-5) M acetazolamide in control experiments. No staining could be detected in the central cells and the inner acinar duct cells. The fluorescent CA inhibitor DNSA (10(-4)M) specifically stained the peripheral cells and the distal duct cells in methanol-fixed cryostat sections, whereas the central cells and the inner acinar duct cells remained unstained. The role of CA in the peripheral cells is not clear. CA activity in the distal duct cells may provide the protons needed to run the vacuolar-type H(+)-ATPase on the apical infoldings of the cells. This ATPase may be involved in modification of the primary saliva.
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PMID:Localization of carbonic anhydrase in the salivary glands of the cockroach, Periplaneta americana. 784 90

The excised rat crystalline lens opacified when incubated aerobically with phenazine methosulfate, but no opacification was observed under anaerobic conditions. Morphological studies revealed development of opacification in the cortex. The opacification resembled that often seen in the early period of senile cataract as well as in naphthalene-induced and UV cataract. Both an increase in hydration and in electrolyte imbalance accompanied this opacification. Na,K-ATPase activity of the opacified lens was found to decrease. In order to investigate if activated oxygen is involved in these processes, we conducted an electron spin resonance study by means of a spin trapping technique. When the lens homogate was incubated with phenazine methosulfate, OH radicals were generated under aerobic but not under anaerobic conditions. Reduced pyridine nucleotides must be involved in the process, because the mixture of nicotinamide adenine dinucleotide phosphate [NAD(P)] and phenazine methosulfate did not generate OH radicals, but the mixture of NAD(P)H and phenazine methosulfate generates OH radicals, indicating that reduced phenazine methosulfate was involved in the OH radical generation. Probably, the generated OH radicals inactivated Na,K-ATPase residing in the epithelium of the lens, which eventually caused opacification of the lens. The present experiment system may be used for the elucidation of lens opacification (cataract) involved with reactive oxygen species.
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PMID:Reactive oxygen species involved in phenazine-methosulfate-induced rat lens opacification. An experimental model of cataract. 813 88

Renal basolateral membranes contain protein kinase A (PKA) and Ca-dependent protein kinases. We studied the effect of cyclic adenosine monophosphate (cAMP), the active phorbol ester phorbol 12-myristate 13-acetate (PMA) and calmodulin on Na-phosphate cotransporter. Rabbit renal basolateral membranes, enriched 15-fold in Na-K-ATPase activity, were phosphorylated with 50 microM ATP, and 32P uptake was measured in the presence of Na or K. 32P uptake was greater in the presence of Na than in the presence of K, indicating the existence of Na-dependent phosphate uptake, i.e., Na-phosphate cotransporter. cAMP, 1 microM, and the catalytic subunit of cAMP-dependent protein kinase (PKA-CSU, 15 mU/ml) inhibited Na-phosphate cotransporter activity by 30-50%, respectively. The effect of CSU was prevented by the PKA inhibitor (1 microgram/ml). Calmodulin, 1 microM, also inhibited Na-phosphate cotransporter by 48% (p < 0.05), and this effect of calmodulin was prevented by the inhibitor naphthalene sulfonamide W-13 (100 microM). In contrast, the active phorbol ester PMA, 1 microM, increased the Na-phosphate cotransporter by 62%, while the inactive analog 4-alpha phorbol failed to elicit such a stimulation. The results demonstrate the presence of Na-dependent phosphate transport in rabbit renal basolateral membranes which is modulated by PKA and by Ca-dependent protein kinases.
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PMID:Modulation of renal basolateral Na-phosphate cotransporter by protein kinase A and Ca-dependent protein kinases. 816 19

The Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum can be labelled at Cys-670 and Cys-674 with 5-[[2-[(iodoacetyl) amino]ethyl]amino]naphthalene-1-sulphonic acid (IAEDANS). Resonance energy transfer has been used to measure the distance between Cys-670/Cys-674 and Glu-439 labelled with 5-(bromomethyl)fluorescein as 40 A. The height of Cys-670/Cys-674 above the phospholipid/water interface has been measured by resonance energy transfer between IAEDANS-labelled ATPase and fluorescein-labelled phosphatidylethanolamine as 54 A. This locates the hinge region of the ATPase close to the mouth of the pore observed in the cytoplasmic region of the ATPase in electron micrographs. No significant changes in these distances can be detected by resonance energy transfer on binding Ca2+ or vanadate. The height of the IAEDANS label above the phospholipid/water interface is the same for bilayers of dimyristoleoylphosphatidylcholine and dioleoylphosphatidylcholine. Conformation changes on the Ca(2+)-ATPase appear to be localised to small regions of the ATPase.
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PMID:Localization of the hinge region of the Ca(2+)-ATPase of sarcoplasmic reticulum using resonance energy transfer. 820 50

Fluorescence energy transfer measurements have been carried out to estimate intramolecular distances between probes bound to Ca(2+)-transporting ATPase (Ca(2+)-ATPase) as well as distances between these probes and the phospholipid headgroup. The nucleotide binding site was monitored by using 1,N6-ethenoadenosine 5'-triphosphate, a fluorescent analogue of ATP, and also by labelling Lys515 with fluorescein 5'-isothiocyanate. Three different cysteine residues were individually labelled using the following probes: 5-[(2-iodoacetyl)aminoethyl]amino-naphthalene-1-sulfonic acid (I-AEDANS), 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl) and fluorescent maleimides. The surface of the membrane was labelled by reconstitution with fluorescent phospholipids (fluorescein and rhodamine derivatives). We found a distance of 4.1 nm from the nucleotide binding site to NBD (at Cys344), and the same distance to fluorescent maleimides (at Cys364). The AEDANS label (at Cys670,672) was found separated 3.5 nm from NBD, 4.4 nm from fluorescent maleimides, and 3.9 nm from the lipid matrix. The NBD label was 3.2 nm apart from fluorescent maleimides and 2.2 nm from the lipid matrix. Finally, fluorescent maleimides were found to be located 4.2 nm above the membrane surface. All these distances agree with a molecular model in which NBD is located in the stalk portion of the Ca(2+)-ATPase, near the surface of the membrane, and the rest of the probes are above it, in the globular domain of the protein.
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PMID:Intramolecular distances within the Ca(2+)-ATPase from sarcoplasmic reticulum as estimated through fluorescence energy transfer between probes. 822 16

The oxyanion-translocating ATPase encoded by the plasmid-borne ars operon catalyzes extrusion of antimonials and arsenicals from cells of Escherichia coli, thus providing resistance to those toxic oxyanions. The purified catalytic subunit of the ATPase, the ArsA protein, exhibits oxyanion-stimulated ATPase activity. The nature of the oxyanion binding site was probed by reaction with the fluorescent sulfhydryl probe 2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS). Our results indicate that MIANS reacts with the ArsA protein in an antimonite-dependent manner. After the protein had been modified with MIANS, two of four cysteines in the ArsA protein had reacted with the probe in the absence of the oxyanionic substrate, and three in the presence of antimonite. The quantum yield of the MIANS-ArsA protein adduct was significantly higher if modification of the protein had occurred in the presence of oxyanionic substrates. Thus binding of the anionic substrate of the pump produces a conformational change in the ArsA protein such that a single additional cysteinyl residue reacts more readily with the sulfhydryl probe.
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PMID:Reaction of the ArsA adenosinetriphosphatase with 2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid. 824 Nov 93

Recently, it was reported that muscarinic-type cholinergic receptors coupled to the phosphoinositide messenger system are present in the rabbit inner medullary collecting duct and Madin-Darby canine kidney (MDCK) cells. The receptor density in MDCK cells is 50 times more than that in inner medullary collecting duct cells. To examine if muscarinic receptor activation influences Na-K-ATPase, the effects of a cholinergic agonist, carbachol, on Na-K-ATPase activity in MDCK cells were measured. Carbachol inhibited Na-K-ATPase activity in a time- and concentration-dependent manner. A maximum of approximately 80% of the enzyme activity was inhibited in 160 min with an EC50 of 5 microM carbachol. The inhibition of Na-K-ATPase activity was reversible; up to 80% of the enzyme activity was recovered within 4 h after carbachol was removed. The inhibitory effect of carbachol was blocked by a muscarinic antagonist atropine and by inhibitors of protein kinase C (PKC), 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine HCl, and N-(2-(methylamino)ethyl)-5-isoquinoline sulfonamide HCl. Direct activators of PKC, phorbol 12-myristate 13-acetate, N(n-heptyl)-5-chloro-1-naphthalene sulfonamide, and phosphatidyl serine, also inhibited Na-K-ATPase activity in MDCK cells, and their effect was also blocked by PKC inhibitors. These results indicate that cholinergic agonists inhibit Na-K-ATPase activity in MDCK cells by the activation of PKC. It is concluded that the inhibition of Na-K-ATPase by PKC may, in part, be responsible for the natriuretic action of cholinergic agonists, which have been shown to stimulate phosphoinositide hydrolysis in renal collecting duct cells.
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PMID:Cholinergic inhibition of Na-K-ATPase via activation of protein kinase C in Madin-Darby canine kidney cells. 840 83


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