Toshiaki Imagawa, Mariko Shida, Kaori Matsuzawa, Shunji Kaya and Kazuya
Taniguchi
Biochemistry, Division of Chemistry, Graduate School of Science, Hokkaido
University, Kita-ku, Sapporo 060-0810, Japan
Abstract: To ascertain whether ouabain binding to human alpha1-subunit
influences coexpression of rat alpha1-subunit, the ouabain-sensitive profiles
of Na+,K+-ATPase activity and 86Rb+
uptake activity and ouabain binding capacity were measured in HeLa cells
stably expressing rat alpha1-subunit. The ouabain-sensitive profile of ATPase
and 86Rb+ uptake activity seemed to be the sum of
two components, one with high and one with low apparent affinity to ouabain,
which were similar to that observed in HeLa and NRK-52E cells derived from
human and rat, respectively. The ATPase activity with low sensitivity to
ouabain increased in simple proportion to the amount of the rat alpha1 mRNA
derived from transfected cDNA, which was determined by the reverse transcription-polymerase
chain reaction method. The turnover number of the human Na+,K+-ATPase
activity obtained from the ratio of the Na+,K+-ATPase
activity to the ouabain binding capacity is about 150/sec. The expression
of the rat alpha1-subunit had no effect on the turnover numbers of the Na+,K+-ATPase
activity with high affinity to ouabain estimated from the ouabain binding
capacity as the active site concentration. These results suggested that
the ouabain bound to human alpha1-subunit did not inhibit the ATPase activity
of the coexpressing rat alpha1 in these cells.
Keywords: HeLa cell, Na+,K+-ATPase, Ouabain binding,
Phosphorylation, Rb+ uptake