Solubilization, purification, and reconstitution of α2β1 isozyme of Na+/K+-ATPase from caveolae of pulmonary smooth muscle plasma membrane: comparative studies with DHPC, C12E8, and Triton X-100

We identified α₂, α₁, and β₁ isoforms of Na⁺/K⁺-ATPase in caveolae vesicles of bovine pulmonary smooth muscle plasma membrane. The biochemical and biophysical characteristics of the α₂β₁ isozyme of Na⁺/K⁺-ATPase from caveolae vesicles were studied during solubilization and purification using the detergents 1,2-heptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C₁₂E₈), and Triton X-100, and reconstitution with the phospholipid dioleoyl-phosphatidylcholine (DOPC). DHPC was determined to be superior to C₁₂E₈, whereas C₁₂E₈ was better than Triton X-100 in the active enzyme yields and specific activity. Fluorescence studies with DHPC-purified α₂β₁ isozyme of Na⁺/K⁺-ATPase elicited higher E1Na?E2 K transition compared with that of the C₁₂E₈- and Triton X-100-purified enzyme. The rate of Na⁺ efflux in DHPC–DOPC-reconstituted isozyme was higher compared to the C₁₂E₈–DOPC- and Triton X100–DOPC-reconstituted enzyme. Circular dichroism analysis suggests that the DHPC-purified α₂β₁ isozyme of Na⁺/K⁺-ATPase possessed more organized secondary structure compared to the C₁₂E₈- and Triton X-100-purified isozyme.     

Effects of aspirin and paracetamol on ATPases of human fetal brain: an in vitro study

In vitro effects of aspirin and paracetamol at the doses 200, 400, 600, 800 nmole/mg protein on ATPases activity were studied in the cerebrum and cerebellum of human fetus covering the age range from 10 weeks to 32 weeks of gestation. Both aspirin and paracetamol inhibit Na+K+ ATPase and Mg2+ ATPase in a dose dependent manner. The inhibition of Na+K+ ATPase and Mg2+ ATPase activity which may affect the release and uptake of biogenic amines in CNS, hinders the maturation of human fetal brain.     

Role of phospholemman and the 70 kDa inhibitor protein in regulating Na/K ATPase activity in pulmonary artery smooth muscle cells under U46619 stimulation

Treatment of bovine pulmonary smooth muscle cells with U46619 inhibited the Na⁺/K⁺ ATPase activity in two parallel pathways: one of which is mediated via glutathionylation of the pump and the other by augmenting the inhibitory activity of the 70 kDa inhibitor protein of Na⁺/K⁺ ATPase. Although phospholemman deglutathionylates the pump leading to its activation, the inhibitor is responsible for irreversible inhibition of Na⁺/K⁺ ATPase in an isoform specific manner during treatment of the cells with U46619.     

Involvement of a serine esterase in oxidant-mediated activation of phospholipase A2 in pulmonary endothelium

Exposure of bovine pulmonary arterial endothelial cells to 1 mM H2O2 stimulated associated TAME-esterase and PLA2 activities. Pretreatment with the serine esterase inhibitors: PMSF (1 mM), DFP (1 mM), and alpha 1-PI (1 mg/ml) inhibited H2O2-induced stimulation of TAME-esterase and PLA2 activities. The TAME-esterase and PLA2 activities under H2O2 exposure were determined to be linearly correlated. Affinity labelling of the endothelial cell membrane with [3H]DFP demonstrated that the serine esterase resides in a protein having molecular weight of 29,000 daltons (29 kDa) which is similar to that of elastase. Treatment of the endothelial cell homogenate with trypsin (1 microgram/ml) also stimulated PLA2 activity.