The alpha-subunit of the Na,K-ATPase has catalytic activity independent of the beta-subunit.
All catalytic activities of the Na,K-ATPase have been ascribed to the alpha-subunit; however, normal activity requires the presence of the beta-subunit. Using recombinant baculoviruses to infect insect cells, we demonstrate that the alpha-subunit, without the beta-subunit, has catalytic activity. During the normal catalytic cycle of the Na,K-ATPase, the alpha-subunit is transiently phosphorylated by ATP at an aspartate residue. This phosphorylation requires Na+, in the presence of K+ the enzyme undergoes rapid dephosphorylation. In contrast, phosphorylation of the independent alpha-subunit by ATP occurs in the presence of Mg2+, does not require Na+ or K+, and is not affected by ouabain. The phosphorylation is, however, inhibited by EGTA and increasing ionic strength. Chemical properties of the alpha-subunit phosphointermediate are consistent with phosphorylation at the normal aspartyl residue. Membranes from cells infected with the recombinant alpha baculovirus exhibit an EGTA-sensitive Mg(2+)-ATPase activity that is not present in the uninfected cells. The Mg(2+)-ATPase of the alpha-infected cells is reduced under conditions of high ionic strength and completely inhibited by EGTA. Thus the phosphorylation of the unassociated alpha-subunit is representative of the ATPase activity of the enzyme. These results suggest that the alpha-subunit of the Na,K-ATPase can catalyze an activity not normally associated with the enzyme and demonstrate that the bea-subunit plays an important role in conferring normal activity to the enzyme complex.
Blanco
G
G
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
DeTomaso
A W
AW
Koster
J
J
Xie
Z J
ZJ
Mercer
R W
RW
D000818:Animals; D016367:Baculoviridae; D002384:Catalysis; D004285:Dogs; D004533:Egtazic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D009036:Moths; D011994:Recombinant Proteins; D012377:Rodentia; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D006898:Hydroxylamines; D011994:Recombinant Proteins; D019811:Hydroxylamine; D014638:Vanadates; D004533:Egtazic Acid; D000254:Sodium-Potassium-Exchanging ATPase