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Hydrolyse ATP (hydrolyse + atp)
Selected AbstractsChanges in expression and activity levels of ecto-5,-nucleotidase/CD73 along the mouse female estrous cycleACTA PHYSIOLOGICA, Issue 2 2010E. Aliagas Abstract Aim:, Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those requiring contraction, hormone synthesis and maintenance of fluid composition. Moreover, adenosine is a key molecule for sperm capacitation. Extracellular nucleotide and nucleoside levels are affected by cell surface ectonucleotidases, amongst which the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family is the most abundant and effective to hydrolyse ATP and ADP to AMP. In the female reproductive tract three members of this family have been recently identified: NTPDase1, NTPDase2 and NTPDase3 (Histochem. Cell Biol.131, 2009, 615). The purpose of the present study was to characterize in this system the expression profile of ecto-5,-nucleotidase (CD73), the enzyme generating adenosine from AMP. Methods:, Immunological techniques and in situ enzymatic assays were used to characterize the ecto-5,-nucleotidase expression in the mouse female reproductive tract along the four stages of the estrous cycle, that were determined by vaginal smear examination. Results:, Ecto-5,-nucleotidase was abundantly detected in the corpora lutea of the ovaries, as well as in several epithelia, such as that of oviducts, uterus and endometrial glands. Marked changes in endometrial ecto-5,-nucleotidase expression and activity along the estrous cycle are described, these being maximum at estrus phase, coinciding with optimal female sexual receptivity. Conclusion:, The adenosine generated thereby, besides other functions, might contribute to sperm capacitation, thus significantly influencing fertility. [source] The presence of phosphate at a catalytic site suppresses the formation of the MgADP-inhibited form of F1 -ATPaseFEBS JOURNAL, Issue 1 2002Noriyo Mitome F1 -ATPase is inactivated by entrapment of MgADP in catalytic sites and reactivated by MgATP or Pi. Here, using a mutant ,3,3, complex of thermophilic F1 -ATPase (,W463F/,Y341W) and monitoring nucleotide binding by fluorescence quenching of an introduced tryptophan, we found that Pi interfered with the binding of MgATP to F1 -ATPase, but binding of MgADP was interfered with to a lesser extent. Hydrolysis of MgATP by F1 -ATPase during the experiments did not obscure the interpretation because another mutant, which was able to bind nucleotide but not hydrolyse ATP (,W463F/,E190Q/,Y341W), also gave the same results. The half-maximal concentrations of Pi that suppressed the MgADP-inhibited form and interfered with MgATP binding were both ,,20 mm. It is likely that the presence of Pi at a catalytic site shifts the equilibrium from the MgADP-inhibited form to the enzyme,MgADP,Pi complex, an active intermediate in the catalytic cycle. [source] Presence of a Na+ -stimulated P-type ATPase in the plasma membrane of the alkaliphilic halotolerant cyanobacterium Aphanothece halophyticaFEMS MICROBIOLOGY LETTERS, Issue 1 2007Kanjana Wiangnon Abstract Aphanothece cells could take up Na+ and this uptake was strongly inhibited by the protonophore, carbonyl cyanide m -chlorophenylhydrazone (CCCP). Cells preloaded with Na+ exhibited Na+ extrusion ability upon energizing with glucose. Na+ was also taken up by the plasma membranes supplied with ATP and the uptake was abolished by gramicidin D, monensin or Na+ -ionophore. Orthovanadate and CCCP strongly inhibited Na+ uptake, whereas N, N, -dicyclohexylcarbodiimide (DCCD) slightly inhibited the uptake. Plasma membranes could hydrolyse ATP in the presence of Na+ but not with K+, Ca2+ and Li+. The Km values for ATP and Na+ were 1.66±0.12 and 25.0±1.8 mM, respectively, whereas the Vmax value was 0.66±0.05 ,mol min,1 mg,1. Mg2+ was required for ATPase activity whose optimal pH was 7.5. The ATPase was insensitive to N -ethylmaleimide, nitrate, thiocyanate, azide and ouabain, but was substantially inhibited by orthovanadate and DCCD. Amiloride, a Na+/H+ antiporter inhibitor, and CCCP showed little or no effect. Gramicidin D and monensin stimulated ATPase activity. All these results suggest the existence of a P-type Na+ -stimulated ATPase in Aphanothece halophytica. Plasma membranes from cells grown under salt stress condition showed higher ATPase activity than those from cells grown under nonstress condition. [source] Reversal of cancer multidrug resistance by green tea polyphenolsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 10 2004Yuying Mei The aim of this study was to examine the effect and mechanism of green tea polyphenols (TP) on reversal of multidrug resistance (MDR) in a carcinoma cell line. Using the MTT assay, TP was examined for its modulating effects on the drug-resistant KB-A-1 cells and drug-sensitive KB-3,1 cells. When 10 ,g mL,1 (-)-epigallocatechin gallate (EGCG) or 40 ,g mL,1 TP were present simultaneously with doxorubicin (DOX), the IC50 of DOX on KB-A-1 cells decreased from 10.3 ± 0.9 ,g mL,1 to 4.2 ± 0.2 and 2.0 ± 0.1 ,g mL,1, respectively. TP and EGCG enhanced the DOX cytotoxicity on KB-A-1 cells by 5.2-and 2.5-times, respectively, but did not show a modulating effect on KB-3,1 cells. This indicated that both TP and EGCG had reversal effects on the MDR phenotype in-vitro. A KB-A-1 cell xenograft model was established, and the effect of TP on reversing MDR in-vivo was determined. Mechanistic experiments were conducted to examine the uptake, efflux and accumulation of DOX. Cloning and expression of the nucleotide binding domain of the human MDR1 gene in Escherichia coli was established, and by using colorimetry to examine the activity of ATPase to hydrolyse ATP, the ATPase activity of target nucleotide binding domain protein was determined. TP exerted its reversal effects through the inhibition of ATPase activity, influencing the function of P-glycoprotein, and causing a decreased extrusion of anticancer drug and an increased accumulation of anticancer drug in drug resistant cells. Using reverse transcription-polymerase chain reaction, the inhibitory effect of TP on MDR1 gene expression was investigated. Down-regulation of MDR1 gene expression was the main effect, which resulted in the reversal effect of TP on the MDR phenotype. TP is a potent MDR modulator with potential in the treatment of P-glycoprotein mediated MDR cancers. [source] Phosphate metabolite concentrations and ATP hydrolysis potential in normal and ischaemic heartsTHE JOURNAL OF PHYSIOLOGY, Issue 17 2008Fan Wu To understand how cardiac ATP and CrP remain stable with changes in work rate , a phenomenon that has eluded mechanistic explanation for decades , data from 31phosphate-magnetic resonance spectroscopy (31P-MRS) are analysed to estimate cytoplasmic and mitochondrial phosphate metabolite concentrations in the normal state, during high cardiac workstates, during acute ischaemia and reactive hyperaemic recovery. Analysis is based on simulating distributed heterogeneous oxygen transport in the myocardium integrated with a detailed model of cardiac energy metabolism. The model predicts that baseline myocardial free inorganic phosphate (Pi) concentration in the canine myocyte cytoplasm , a variable not accessible to direct non-invasive measurement , is approximately 0.29 mm and increases to 2.3 mm near maximal cardiac oxygen consumption. During acute ischaemia (from ligation of the left anterior descending artery) Pi increases to approximately 3.1 mm and ATP consumption in the ischaemic tissue is reduced quickly to less than half its baseline value before the creatine phosphate (CrP) pool is 18% depleted. It is determined from these experiments that the maximal rate of oxygen consumption of the heart is an emergent property and is limited not simply by the maximal rate of ATP synthesis, but by the maximal rate at which ATP can be synthesized at a potential at which it can be utilized. The critical free energy of ATP hydrolysis for cardiac contraction that is consistent with these findings is approximately ,63.5 kJ mol,1. Based on theoretical findings, we hypothesize that inorganic phosphate is both the primary feedback signal for stimulating oxidative phosphorylation in vivo and also the most significant product of ATP hydrolysis in limiting the capacity of the heart to hydrolyse ATP in vivo. Due to the lack of precise quantification of Piin vivo, these hypotheses and associated model predictions remain to be carefully tested experimentally. [source] | ||||||||||