PLANT PHYSIOLOGY , Vol 108, Issue 4 1615-1621, Copyright © 1995 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Partial Purification of a Na+ -ATPase from the Plasma Membrane of the Marine Alga Heterosigma akashiwo
M. Shono, M. Wada and T. Fujii
Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan
A Na+ -ATPase was partially purified from plasma membranes of the marine
alga Heterosigma akashiwo. The plasma membranes of H. akashiwo cells were
collected by differential centrifugation with subsequent discontinuous
gradient centrifugation. Na+ -ATPase activity was associated with the
resultant plasma membrane fraction and was stimulated to the greatest
extent in the presence of 100 to 200 mM Na+, 10 mM K+, and 5 mM Mg2+ ions,
pH 8.0. The Km value for Na+ ions was 12.2 mM. An apparent Km value for ATP
was 880 [mu]M. A 140-kD phosphorylated intermediate was also detected in
the same fraction in the presence of both Mg2+ and Na+ ions, and this
protein was dephosphorylated upon the addition of K+ ions. We could
partially purify the 140-kD protein after solubilization by Suc monolaurate
and fractionation by sequential column chromatography on Sephacryl S-300,
DEAE-Sepharose CL-6B, and Mono-Q columns. The purified 140-kD polypeptide
could also be phosphorylated and be detected after acid sodium dodecyl
sulfate-polyacryl-amide gel electrophoresis in the presence of Na+ and Mg2+
ions.
