The plasma membrane Ca pump catalyzes the hydrolysis of ATP at low rate in the absence of Ca

The plasma membrane Ca²⁺ ATPase catalyzed the hydrolysis of ATP in the presence of millimolar concentrations of EGTA and no added Ca²⁺ at a rate near 1.5% of that attained at saturating concentrations of Ca²⁺. Like the Ca-dependent ATPase, the Ca-independent activity was lower when the enzyme was autoinhibited, and increased when the enzyme was activated by acidic lipids or partial proteolysis. The ATP concentration dependence of the Ca²⁺-independent ATPase was consistent with ATP binding to the low affinity modulatory site. In this condition a small amount of hydroxylamine-sensitive phosphoenzyme was formed and rapidly decayed when chased with cold ATP. We propose that the Ca²⁺-independent ATP hydrolysis reflects the well known phosphatase activity which is maximal in the absence of Ca²⁺ and is catalyzed by E₂-like forms of the enzyme. In agreement with this idea pNPP, a classic phosphatase substrate was a very effective inhibitor of the ATP hydrolysis.     

Entamoeba histolytica: ouabain-insensitive Na(+)-ATPase activity

Our aim was to determine the presence of sodium pumps in Entamoeba histolytica. It is shown through the measurement of ouabain-sensitive ATPase activity and immunoblotting that E. histolytica does not express (Na(+)+K(+))ATPase. On the other hand, we observed a Na(+)-ATPase with the following characteristics: (1) stimulated by Na(+) or K(+), but these effects are not addictive; (2) the apparent affinity is similar for Na(+) and K(+) (K(0.5) = 13.3 +/- 3.7 and 15.4 +/- 3.1mM, respectively), as well as the V(max) (24.9 +/- 1.5 or 27.5 +/- 1.6 nmol Pi mg(-1)min(-1), respectively); (3) insensitive up to 2mM ouabain; and (4) inhibited by furosemide with an IC(50) of 0.12 +/- 0.004 mM. Furthermore, this enzyme forms a Na(+)- or K(+)-stimulated, furosemide- and hydroxylamine-sensitive ATP-driven acylphosphate phosphorylated intermediate.     

Leishmania amazonensis: characterization of an ouabain-insensitive Na+-ATPase activity

We characterized ouabain-insensitive Na+-ATPase activity present in the plasma membrane of Leishmania amazonensis and investigated its possible role in the growth of the parasite. An increase in Na+ concentration in the presence of 1mM ouabain, increased the ATPase activity with a V(max) of 154.1+/-13.5nmol Pi x h(-1) x mg(-1) and a K0.5 of 28.9+/-7.7mM. Furosemide and sodium orthovanadate inhibited the Na+-stimulated ATPase activity with an IC(50) of 270microM and 0.10microM, respectively. Furosemide inhibited the growth of L. amazonensis after 48h incubation, with maximal effect after 96h. The IC50 for furosemide was 840. On the other hand, ouabain (1mM) did not change the growth of the parasite. Taken together, these results show that L. amazonensis expresses a P-type, ouabain-insensitive Na+-ATPase that could be involved with the growth of the parasite.     

Characterization and partial isolation of ouabain-insensitive Na(+) -ATPase in MDCK I cells

We show that MDCK I cells express, besides the classical (Na(+)+K(+))ATPase, a Na(+)-stimulated ATPase activity with the following characteristics: (1) K(0.5) for Na(+) 7.5+/-1.5 mM and V(max) 23.12+/-1.1 nmol Pi/mg per min; (2) insensitive to 1 mM ouabain and 30 mM KCl; and (3) inhibited by furosemide and vanadate (IC(50) 42.1+/-8.0 and 4.3+/-0.3 microM, respectively). This enzyme forms a Na(+)-stimulated, furosemide- and hydroxylamine-sensitive ATP-driven acylphosphate phosphorylated intermediate with molecular weight of 100 kDa. Immunoprecipitation of the (Na(+)+K(+))ATPase with monoclonal anti-alpha(1) antibody reduced its activity in the supernatant by 90%; the Na(+)-ATPase activity was completely maintained. In addition, the formation of the Na(+)-stimulated, furosemide- and hydroxylamine-sensitive ATP-driven acylphosphate intermediate occurred at the same magnitude as that observed before immunoprecipitation. These data suggest that Na(+)-ATPase and (Na(+)+K(+))ATPase activities are independent, with Na(+)-ATPase belonging to a different enzyme entity.     

植物金属硫蛋白Ⅱ型与谷胱甘肽在烟草中的表达和合成特性

利用从印度芥菜(Brassica juncea)中获得的金属硫蛋白2型（BjMT2）基因通过农杆菌介导法转入模式植物种烟草中。转基因烟草在经过继代组织培养和抗生素筛选以及PCR和Western blot检测后,确定目的基因BjMT-2被整合到烟草基因组中并具备表达功能。与对照野生型相比,转入金属硫蛋白2型的烟草在植物形态和根部发育方面都发生了显著的性状变化。在正常条件下,转基因植物叶片组织中谷胱甘肽的含量与野生型没有明显差别,但经过100 mmol·L^-1 Cu²⁺胁迫后,谷胱甘肽的含量明显高于野生型。在转基因植物细胞膜系统中V-型ATP酶的活性低于野生型,而P-型ATP酶的活性则表现出明显的升高趋势。