Functional characterization of a testes-specific α-subunit isoform of the sodium/potassium adenosinetriphosphatase

Different isoforms of the sodium/potassium adenosinetriphosphatase (Na,K-ATPase) alpha and beta subunits have been identified in mammals. The association of the various alpha and beta polypeptides results in distinct Na,K-ATPase isozymes with unique enzymatic properties. We studied the function of the Na,K-ATPase alpha 4 isoform in Sf-9 cells using recombinant baculoviruses. When alpha 4 and the Na pump beta 1 subunit are coexpressed in the cells, Na,K-ATPase activity is induced. This activity is reflected by a ouabain-sensitive hydrolysis of ATP, by a Na+-dependent, K+-sensitive, and ouabain-inhibitable phosphorylation from ATP, and by the ouabain-inhibitable transport of K+. Furthermore, the activity of alpha 4 is inhibited by the P-type ATPase blocker vanadate but not by compounds that inhibit the sarcoplasmic reticulum Ca-ATPase or the gastric H,K-ATPase. The Na,K-ATPase alpha 4 isoform is specifically expressed in the testis of the rat. The gonad also expresses the beta 1 and beta 3 subunits. In insect cells, the alpha 4 polypeptide is able to form active complexes with either of these subunits. Characterization of the enzymatic properties of the alpha 4 beta 1 and alpha 4 beta 3 isozymes indicates that both Na,K-ATPases have similar kinetics to Na+, K+, ATP, and ouabain. The enzymatic properties of alpha 4 beta 1 and alpha 4 beta 3 are, however, distinct from the other Na pump isozymes. A Na,K-ATPase activity with similar properties as the alpha 4-containing enzymes was found in rat testis. This Na,K-ATPase activity represents approximately 55% of the total enzyme of the gonad. These results show that the alpha 4 polypeptide is a functional isoform of the Na,K-ATPase both in vitro and in the native tissue.