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J. Biochem, 1982, Vol. 91, No. 1 97-105
© 1982 Japanese Biochemical Society

research-article

Duality of {alpha}-Subunit of Canine Kidney Sodium- and Potassium- Activated Adenosinetriphosphatase in Electrophoresis1

Toshiko OHTA*, Masaru KAWAMURA*, Tsunemi HASEGAWA**, Hisayuki ISHIKURA** and Kei NAGANO*

*Department of Biology Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi 329–04
**Department of Chemistry, Jichi Medical School Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi 329–04

Sodium- and potassium-activated adenosinetriphosphatase (Na+,K+-ATPase) purified from dog kidney outer medulla was examined by polyacrylamide gel electrophoresis and by photoaffinity labeling with N-(ouabain)-N'-(2-nitro-4-azidophenyl)-ethylenediamine (NAP-ouabain).

The large subunit band ({alpha}-band) split into two bands on the gel after the enzyme was heat-treated in the presence of 1% sodium dodecylsulfate (SDS). Of the two bands ({alpha}I and {alpha}II), {alpha}I had the same electrophoretic mobility as the original band, while {alpha}II moved slightly faster. Total conversion into {alpha}II was not observed, about half of the original remaining as {alpha}I. Below 60°C, heat treatment did not produce {alpha}II. Phenylmethylsulfonyl fluoride did not prevent the appearance of {alpha}II.

Both {alpha}I and {alpha}II were labeled with [3H]NAP-ouabain. Nonspecific incorporation of [3H]NAP-ouabain also occurred irrespective of illumination, but it was removed either by diffusion during staining and destaining of the gel or by treatment of the enzyme with trichioroacetic acid.

It is tentatively concluded that the splitting of the band reflects some intrinsic differences in situ of the {alpha}-subunit of dog kidney membrane Na+,K+-ATPase.

1 This work was supported in part by a grant for scientific research from the Ministry of Education, Science and Culture of Japan. A part of this work was presented at the 53rd annual meeting of the Japanese Biochemical Society held in Tokyo in October, 1980, and at the 6th annual meeting of the Japan Bioenergetics Group held in Nagoya in December, 1980.


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