Persistent stimulation of lens fiber cell Na,K-ATPase by sodium thiocyanate.
The Na,K-ATPase partially purified from porcine lens fiber cells (Sen and Pfeiffer, 1982) is stimulated fourfold (specific activity) by treatment with sodium thiocyanate. The optimum conditions are 1.5 M NaSCN, 2 mg protein ml-1 reaction mixture, pH 7.0, with incubation continued for 30 min at 23 degrees C. Sodium docecyl sulphate-gel electrophoresis and [3H]ouabain binding studies indicate that the extent of purity is not increased significantly by the procedure. The high-activity preparation has elevated phospholipid:protein and phosphatidylethanolamine:sphingomyelin ratios compared with the deoxycholate-extracted starting material. The cholesterol:phospholipid ratio and phospholipid acyl group composition are not significantly altered by SCN- treatment. Measurements of 1,6-diphenyl-1,3,5-hexatriene fluorescence polarization show that SNC- treatment produces approximately a 5 degrees C decrease in a membrane phase transition temperature. The phase transition also affects the activation energy of the Na,K-ATPase reaction and probably reflects the onset of the gel to liquid crystalline transition rather than the midpoint location of the transition per se. p-Nitrophenylphosphatase activity and Na,K-ATPase activity in the gel state membrane are also increased by SCN- treatment. Increased specific activity may result, in part, from a membrane fluidity-dependent enzyme activation but is also due, in part, to the expression of latent enzyme activity. Using ouabain-binding data and the specific activity of the activated preparation, it can be shown that the turnover number of the fiber cell enzyme is approximately 1% of that observed in most other tissues.
D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D007908:Lens, Crystalline / Q000032:analysis / Q000201:enzymology*; D008055:Lipids / Q000032:analysis; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification / Q000378:metabolism*; D013268:Stimulation, Chemical; D013552:Swine; D013696:Temperature; D013861:Thiocyanates / Q000494:pharmacology*
D008055:Lipids; D013861:Thiocyanates; C024553:sodium thiocyanate; D000254:Sodium-Potassium-Exchanging ATPase