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)

Nucleoside diphosphokinase (NDK) of human platelets has been purified by chromatography on Blue Sepharose CL-6B gel (purification factor of 950) and shown to be free of adenylate kinase, ATPase and adenylate cyclase. The molecular weight was 70,000 with subunits of 17,000. The pH optimum was 8.0 Km values for ATP and dTDP were determined in two ways using the pyruvate kinase-lactate dehydrogenase coupled enzyme assay. Values of 0.38 and 0.20 mM were obtained for ATP and 0.29 and 0.21 mM for dTDP. Km values for ADP (0.024 mM) and GTP (0.12 mM) were determined with the hexokinase-glucose-6-phosphate dehydrogenase coupled enzyme assay. These values are in agreement with those reported for NDK from other sources. Theophylline, which inhibits the NDK activity of intact platelets and platelet membrane preparations and inhibits the ADP-induced shape change of platelets, was shown to be a competitive inhibitor of both the free and phosphorylated forms of NDK with competitive inhibition constants (Kic) of 9.3 and 9.6 mM respectively. Papaverine, another cAMP phosphodiesterase inhibitor, which also inhibits the ADP-induced shape change of platelets, had no inhibitory effect on platelet NDK. It was concluded that the inhibitory effect of theophylline on the activity of the purified enzyme was due to the structural similarity between the methylxanthine and the adenine moiety of ADP.
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PMID:Isolation and kinetic studies of nucleoside diphosphokinase from human platelets and effects of cAMP phosphodiesterase inhibitors. 302 50

This paper describes characterization of the reaction of calmodulin with a series of nitrosoureas which are capable of releasing amine-reactive isocyanates of varying hydrophobic character. The site of calcium-dependent carbamoylation on calmodulin by the antineoplastic agent 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (methyl CCNU) was determined to be Lys-75 as demonstrated using [ring-14C]methyl CCNU and sequence analysis of the sole labeled peptide obtained from tryptic digestion of reversed-phase high pressure liquid chromatography (HPLC)-purified radiolabeled calmodulin. CCNU, the 4-desmethylcyclohexyl derivative of methyl CCNU, and its reactive hydrolysis product, cyclohexyl isocyanate, were also determined to modify calmodulin in a similar manner and at the same site, as demonstrated by specific blockade of modification by the calmodulin antagonist calmidazolium. Nitrosoureas which release the less hydrophobic 4-hydroxy- and 4-carboxycyclohexyl isocyanates are unable to modify calmodulin at 25-fold higher concentrations than those required for modification with methyl CCNU, CCNU, or cyclohexyl isocyanate. With this monomodified Lys-75 derivative, purified to homogeneity by HPLC, differential effects of modification on the activation of bovine brain 3',5'-cyclic nucleotide phosphodiesterase (phosphodiesterase) and human erythrocyte Ca2+,Mg2+-ATPase were observed. Compared to the amounts of native calmodulin needed, phosphodiesterase required 7-fold higher amounts of this derivative to reach maximal activation, whereas the activation of the ATPase was unaffected. Clearly, different regions of calmodulin are responsible for the activation of phosphodiesterase and the ATPase. We conclude that Lys-75 is not essential for the function of calmodulin but is in a region of the molecule involved in interaction with phosphodiesterase as well as the binding of certain hydrophobic calmodulin antagonists.
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PMID:Modification of calmodulin on Lys-75 by carbamoylating nitrosoureas. 313 56

Neuropeptide Y (NPY), which co-exists with noradrenaline (NA) in postganglionic sympathetic nerves, was able to potentiate NA-evoked constriction in certain isolated rabbit blood vessels. The phenomenon was observed in the femoral, the gastroepiploic and the pulmonary arteries but not in the femoral or the gastroepiploic veins or in the aorta. Thus, NPY potentiated NA-evoked vasoconstriction predominantly in muscular arteries with alpha-1 adrenoceptors. NPY-related peptides, such as peptide YY and to some extent pancreatic polypeptide shared this ability, whereas calcitonin gene-related peptide or LPLRFamide did not. The mode of action by which NPY potentiates NA-evoked vasoconstriction was analyzed using the femoral artery. Pretreatment of the vessel with cocaine, a blocker of amine re-uptake, or rolipram, an inhibitor of phosphodiesterase, left the potentiation unaffected, whereas Na+ deficiency or ouabain, an inhibitor of Na+/K+-adenosine triphosphatase, abolished this effect of NPY. Nifedipine, a blocker of Ca++ entry, or removal of extracellular Ca++ shortly before the application of NPY had little effect. After prolonged exposure to a Ca++-free medium (with ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid) the maximum response to NA was greatly reduced and the potentiating effect of NPY was abolished. Thus, the potentiation of NA-evoked vasoconstriction by NPY seems to depend upon the presence of Na+ but not upon a Ca++ influx. An intracellular sequestered Ca++ pool appears to play a critical role.
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PMID:Neuropeptide Y potentiates noradrenaline-evoked vasoconstriction: mode of action. 392 74

We describe an abrupt increase (at 32 degrees ) in the energy of activation for the reaction of hepatic adenylyl cyclase in the presence of glucagon or epinephrine. This increase is not seen in the presence of fluoride, prostaglandin E(1), or 1-propanol, or in the absence of cyclase stimulators. The change in energy of activation found with hormones is abolished by 1-propanol. This change does not represent differences in hormone or substrate binding at different temperatures, but seems to reflect interactions among elements of the cyclase stimulation sequence. Similar changes in energy of activation were not observed for alkaline phosphatase, cyclic AMP-phosphodiesterase, 5'-nucleotidase, or ouabain-sensitive ATPase. Since the mole fraction of cholesterol in liver membranes is sufficiently high to preclude a phase change in bulk membrane lipids, our observation suggests either that cyclase is restricted to cholesterol-poor membrane regions or that the change in its energy of activation is largely restricted to protein components of the cyclase apparatus. The data are compatible with fundamental differences in the stimulation process(es) for the hormones (glucagon and epinephrine) as compared with those for fluoride and prostaglandin E(1).
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PMID:A temperature-sensitive change in the energy of activation of hormone-stimulated hepatic adenylyl cyclase. 435 55

The phosphohydrolase activity of the membrane-associated (Ca2+ + Mg2+)-dependent adenosine triphosphatase (ATPase) of the human erythrocyte can be inhibited by micromolar of nanomolar concentrations of cyclic AMP. Millimolar concentrations of cyclic AMP are less effective. The inhibitory effect of cyclic AMP is potentiated in the presence of the phosphodiesterase inhibitor, theophylline.
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PMID:The influence of adenosine 3',5'-monophosphate upon the activity of the membrane-associated (Ca+ + Mg2+)-dependent adenosine triphosphatase of the human erythrocyte. 610 81

The effect of Ca2+ and calmodulin on (CaM) on the activation of Ca2+-dependent Mg2+-activated ATPase (Ca2+,Mg2+-ATPase; ATP phosphohydrolase, EC 3.6.1.3) has been carried out because of the finding that the CaM dependence of the activation varies with the concentration of free Ca2+, similarly to brain phosphodiesterase and adenylate cyclase. The study was carried out in the absence of chelating agents because they strongly interfere in the enzyme kinetics. Three main conclusions can be drawn (i) CaM-Ca3 and CaM-Ca4 together are the biochemically active species in vitro. (ii) These species bind in a non-cooperative way to the CaM-binding site of the enzyme with a dissociation constant of 6 x 10(-10) M or 1.1 x 10(-8) M, depending on whether Ca2+ saturates the substrate binding site of the enzyme or not. (iii) The binding of CaM-Ca3 to the enzyme lowers the dissociation constant of the enzyme for Ca2+ at the substrate binding site from 51.5 to 2.8 microM. Contrary to general belief, CaM does not induce pronounced positive cooperativity in the binding of Ca2+ to the enzyme. Such a cooperativity is seen only when the enzyme is incompletely saturated with the activator, but it disappears in the presence of saturating concentrations of CaM-Ca3. The rate equation proposed here accurately predicts the extent of enzyme activation over a wide range of Ca2+ and CaM concentration. In healthy erythrocytes the concentrations of Ca2+ and CaM are such that the Ca pump works with a minimal dissipation of energy, but a small increase in the intracellular Ca2+ concentration leads to a strong amplification of the pumping activity.
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PMID:Activation of human erythrocyte Ca2+-dependent Mg2+-activated ATPase by calmodulin and calcium: quantitative analysis. 612 73

Pyrophosphate, p-nitrophenyl phosphate and a variety of pyrimidine and purine nucleotides are hydrolyzed by the solubilized membrane-bound enzymes of the brush border plasma membrane of Hymenolepis diminuta. The pH optima (or ranges) for hydrolysis of substrates are 8.0 (pyrophosphate), 8.8 (p-nitrophenyl phosphate), 8.4-8.9 (nucleoside monophosphates), and 7.1-8.1 (nucleoside triphosphates); all substrates, with the exception of nucleoside triphosphates, have a higher affinity for the solubilized enzyme at pH 7.4 than at their optimal pH for hydrolysis. ATP is degraded completely by the enzyme preparation to adenosine and inorganic phosphate, but since neither ADP nor ATP accumulate in the incubation medium it is not known whether ATP hydrolysis involves the sequential hydrolysis of terminal phosphate groups. Isoelectric focusing and various chromatographic procedures (gel permeation, ion-exchange and hydrophobic interaction chromatography) fail to separate the alkaline phosphatase, phosphodiesterase, 5'-nucleotidase, adenosine triphosphatase and ribonuclease activities associated with the solubilized membrane preparation. Additionally, inhibitor studies indicate that only a single enzyme with low substrate specificity is involved in the hydrolysis of nucleotides, p-nitrophenyl phosphate, pyrophosphate and hexose phosphate esters. Purines and pyrimidines and their nucleosides interact with the active site, and in some instances activity of the enzyme is stimulated by an unknown mechanism.
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PMID:Nucleotide hydrolysis by solubilized membrane-bound enzymes of the brush border plasma membrane of Hymenolepis diminuta. 613 88

Sarcolemma (SL) vesicles, isolated from pig heart, contain both a Ca2+-calmodulin-dependent protein kinase (CaM-PK) and a Ca2+-dependent Mg2+-ATPase (Ca2+/Mg2+)-ATPase). Some of their properties have been compared: their affinity for Ca2+ ions, dependence on exogenous calmodulin (CaM) and sensitivity to the anti-CaM drug calmidazolium (R24571). The properties of Ca2+-CaM-dependent brain phosphodiesterase (PDE) have also been examined. R24571 appeared to be the most potent inhibitor from brain PDE. For the three enzymes studied, exogenously added CaM was able to antagonize the R24571 inhibition, although the efficiency to counteract was rather low in the case of the SL Ca2+/Mg2+-ATPase. R24571 decreased the affinity of the Ca2+/Mg2+-ATPase for Ca2+ ions (KCa 0.35 versus 0.75 microM) and exerted an inhibition non-competitive with Ca2+ ions on the other CaM-dependent enzymes. Membrane-bound CaM, which is involved in controlling the Ca2+/Mg2+-ATPase, appeared to be present in a stoichiometry varying from 1:1 to 1:4 compared to the 32P-intermediate of the ATPase. R24571 treatment of SL vesicles selectively solubilized a number of proteins in the molecular range of 13-20 kD, which may include CaM. The results suggest that different mechanisms are involved in the CaM control of the two SL enzymes studied.
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PMID:Inhibition of Ca2+-dependent protein kinase and Ca2+/Mg2+-ATPase in cardiac sarcolemma by the anti-calmodulin drug calmidazolium. 613 71

Plasma membranes of vertebrate lens fiber cells contain large numbers of gap junctions that may provide pathways for metabolic cooperation. Characterization of fiber cell gap junctions is thus necessary to understand this function. In this study, plasma membrane fractions were isolated from bovine lens according to established techniques, but without urea, detergents, or proteolytic enzymes. Electron microscopy indicated that isolated plasma membranes with gap junctions form double-membrane vesicles, and gap junctions comprised approximately 35% of the total membrane area in the crude fraction. These vesicles were impermeable to cationized ferritin, suggesting that they were sealed, and may be useful for permeability studies. Treatment of the crude fraction with 2.5% beta-mercaptoethanol or dithiothreitol caused reversible separation of junctional membranes, suggesting that disulfide bonds may be important in maintaining gap junction structure. Fractions with varying proportions of gap junctions were isolated using linear sucrose density gradient centrifugation. The proportional area of gap junction membrane versus total membrane in the various fractions ranged from 10% to at least 51%. The following plasma membrane enzymes were assayed in all fractions: Mg++-ATPase, Ca++-ATPase, alkaline phosphatase, phosphodiesterase, 5'-nucleotidase, and Na+, K+-ATPase. There was no correlation between enzyme activity and gap junction enrichment. This suggests that these enzymes are not associated with fiber cell gap junctions.
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PMID:Biochemical and structural characterization of membrane fractions from bovine lens. 613 51

Incubation of human erythrocyte ghosts with an equal volume of 0.2 mM EDTA in isotonic KCl decreased both the activity and Ca2+ sensitivity of the (Ca2+ + Mg2+)-ATPase remaining associated with the membrane. Readdition of the EDTA-extract activated the (Ca2+ + Mg2+)-ATPase activity. The activator activity was trypsin sensitive, heat stable and retained by a phenothiazine affinity column, consistent with properties expected of calmodulin. However, unlike calmodulin, the activity was not retained by DEAE Sephadex A-50 and it eluted from Sephacryl S-200 as heterogeneous peaks of activator activity of apparent molecular weight between 107,000 and 178,000. Nevertheless, the activator in the EDTA extract both before and after gel filtration contained calmodulin, as determined by radioimmunoassay and by its activation of calmodulin - deficient phosphodiesterase. SDS-gel electrophoresis of the activator isolated by gel filtration showed a protein of Mr 56,000 in addition to a low molecular weight protein corresponding to calmodulin. It is suggested that the red cell membrane contains a calmodulin binding protein which tightly binds calmodulin as a polymeric complex in a Ca2+-independent manner.
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PMID:Characterization of a (Ca2+ + Mg2+)-ATPase activator bound to human erythrocyte membranes. 614 20


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