Persistent stimulation of lens fiber cell Na,K-ATPase by sodium thiocyanate

• Published in: Experimental eye research Vol. 43; no. 3; pp. 315 - 327
• Main Authors: Sen, Parimal C., Krebsbach, Randy J., Pfeiffer, Douglas R.
• Format: Journal Article
• Language: English
• Published: London Elsevier Ltd 01.09.1986; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; cataract; Enzyme Activation - drug effects; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases; Lens, Crystalline - analysis; Lens, Crystalline - enzymology; Lipids - analysis; membrane properties; Na,K-ATPase; porcine lens; sodium thiocyanate stimulation; Sodium-Potassium-Exchanging ATPase - isolation & purification; Sodium-Potassium-Exchanging ATPase - metabolism; Stimulation, Chemical; Swine; Temperature; Thiocyanates - pharmacology; membrane properties; porcine lens; cataract; sodium thiocyanate stimulation; Na,K-ATPase; Eye; Activation; Lens; Pore; Enzyme
• ISSN: 0014-4835; 1096-0007
• DOI: 10.1016/S0014-4835(86)80069-0

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°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°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.