title: Functional characterization of a testes-specific alpha-subunit isoform of the sodium/potassium adenosinetriphosphatase.
abstract: 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 alpha4 isoform in Sf-9 cells using recombinant baculoviruses. When alpha4 and the Na pump beta1 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 alpha4 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 alpha4 isoform is specifically expressed in the testis of the rat. The gonad also expresses the beta1 and beta3 subunits. In insect cells, the alpha4 polypeptide is able to form active complexes with either of these subunits. Characterization of the enzymatic properties of the alpha4beta1 and alpha4beta3 isozymes indicates that both Na,K-ATPases have similar kinetics to Na(+), K(+), ATP, and ouabain. The enzymatic properties of alpha4beta1 and alpha4beta3 are, however, distinct from the other Na pump isozymes. A Na, K-ATPase activity with similar properties as the alpha4-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 alpha4 polypeptide is a functional isoform of the Na,K-ATPase both in vitro and in the native tissue.
authors.affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. gblanco@cellbio.wustl.edu
authors.forename: G
authors.initials: G
authors.lastname: Blanco
authors.forename: R J
authors.initials: RJ
authors.lastname: Melton
authors.forename: G
authors.initials: G
authors.lastname: Sánchez
authors.forename: R W
authors.initials: RW
authors.lastname: Mercer
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D001692:Biological Transport / Q000235:genetics; D002384:Catalysis; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010042:Ouabain / Q000378:metabolism; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera / Q000201:enzymology / Q000235:genetics; D013737:Testis / Q000201:enzymology*
chemical_list: D007527:Isoenzymes; D010042:Ouabain; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium