Partial purification and characterization of (Na++K+)-ATPase from garfish olfactory nerve axon plasma membrane
- 48 Downloads
The (Na++K+)-ATPase of garfish olfactory nerve axon plasma membrane was purified about sixfold by treatment of the membrane with sodium dodecyl sulfate followed by sucrose density gradient centrifugation. The estimated molecular weights of the two major polypeptide components of the enzyme preparation on sodium dodecyl sulfate gels were 110,000 and 42,000 daltons, which were different from those of the corresponding peptides of rabbit kidney (Na++K+)-ATPase. No carbohydrate was detected in the 42,000-dalton component either by the periodic acid-Schiff reagent or by the more sensitive concanavalin A-peroxidase staining procedure. The molecular properties of the garfish (Na++K+)-ATPase, such as theK m for ATP, pH optimum, energies of activation, Na and K ion dependence and vanadium inhibition, were, however, similar to those of the kidney enzyme.
The partially purified garfish (Na++K+)-ATPase was reconstituted into phospholipid vesicles by a freeze-thaw-sonication procedure. The reconstituted enzyme was found to catalyze a time and ATP dependent22Na+ transport. The ratio of22Na+ pumped to ATP hydrolyzed was about 1; under the same reconstitution and assay conditions, eel electroplax (Na++K+)-ATPase, however, gave a22Na+ pumped to ATP hydrolyzed ratio of nearly 3.
Key wordsAxon membrane (Na++K+)-ATPase polypeptide components enzyme reconstitution garfish olfactory nerve
Unable to display preview. Download preview PDF.
- 2.Beauge, L. A., Glynn, I. M. 1978. Commerical ATP containing traces of vanadate alters the response of (Na++K+) ATPase to external potassium.Nature (London) 272:551–552Google Scholar
- 9.Fiske, C. H., Subbarow, Y. 1925. Colorimetric determination of phosphorus.J. Biol. Chem. 66:375–400Google Scholar
- 16.Hokin, L. E., Dahl, J. L., Deupree, J. D., Dixon, J. F., Hackney, J. F., Perdue, J. F. 1973. Studies on the characterization of the sodium-potassium transport adenosine triphosphatase: Purification of the enzyme from the rectal gland ofSqualus acanthias.J. Biol. Chem. 248:2593–2605PubMedGoogle Scholar
- 17.Hokin, L. E., Dixon, J. F. 1979. Parameters of reconstituted Na+ and K+ transport in liposomes in which purified Na,K-ATPase is incorporated by “freeze-thaw-sonication.”In: Na,K-ATPase, Structure and Kinetics. J. C. Skou and J. G. Nørby, editors. pp. 47–67. Academic Press, LondonGoogle Scholar
- 21.Jorgensen, P. L. 1974. Techniques for the study of steroid effects on membraneous (Na++K+)-ATPase.Methods Enzymol. 36:434–439Google Scholar
- 22.Kagawa, Y., Racker, E. 1971. Partial resolution of the enzymes catalyzing oxidative phosphorylation.J. Biol. Chem. 246:5477–5487Google Scholar
- 28.Lillie, R. D. 1965. Histopathological Techniques and Practical Histochemistry. (3rd Ed.) pp. 196 and 270. MacGraw Hill, New YorkGoogle Scholar
- 32.Perrone, J. R., Hackney, J. F., Dixon, J. F., Hokin, L. E. 1975. Molecular properties of purified (sodium+potassium)-activated adenosine triphosphatases and their subunits from the rectal gland ofSqualus acanthias and the electric organ ofElectrophorus electricus.J. Biol. Chem. 250:4178–4184PubMedGoogle Scholar
- 35.Rouser, G., Siakotos, A. N., Fleisher, S. 1966. Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots.Lipids 1:85–86Google Scholar