Kinetic properties of the alpha 2 beta 1 and alpha 2 beta 2 isozymes of the Na,K-ATPase.
The presence of multiple isoforms of the alpha and beta subunits of the Na,K-ATPase in most mammalian tissues has hindered the understanding of the roles of the individual isoforms in directing Na,K-ATPase function. Expression of the Na,K-ATPase subunits in insect cells using recombinant baculoviruses has proven to be a useful system for the study of Na,K-ATPase function. Using this system, we have expressed the rat Na,K-ATPase alpha 2 beta 1 and alpha 2 beta 2 isoforms in Sf-9 insect cells, a cell line derived from the ovary of the fall armyworm, Spodoptera frugiperda. Both beta 1 and beta 2 isoforms can efficiently assemble with the alpha 2 subunit to produce catalytically competent Na,K-ATPase molecules. The analysis of the kinetic properties of both isozymes showed that alpha 2 beta 1 and alpha 2 beta 2 have equivalent sensitivities to ouabain, and similar turnover numbers and apparent affinities for K+ and ATP. The dependence on Na+, however, differs between the isozymes, with alpha 2 beta 2 displaying a slightly higher apparent affinity for the cation than alpha 2 beta 1. In addition, the even greater kinetic differences between Na,K-ATPase isozymes varying in alpha isoforms may be important in further differentiating the enzyme. Thus, when compared to the rat alpha 1 beta 1 Na,K-ATPase expressed in Sf-9 cells, the alpha 2 beta 1 and alpha 2 beta 2 isozymes have a lower apparent affinity for K+ and a higher affinity for Na+ and ATP.(ABSTRACT TRUNCATED AT 250 WORDS)
Blanco
G
G
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Koster
J C
JC
Sánchez
G
G
Mercer
R W
RW
D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D015151:Immunoblotting; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D011233:Precipitin Tests; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D018411:Spodoptera
D007527:Isoenzymes; D011994:Recombinant Proteins; D000254:Sodium-Potassium-Exchanging ATPase