TY - JOUR
T1 - The α-subunit of the Na,K-ATPase has catalytic activity independent of the β-subunit
AU - Blanco, Gustavo
AU - DeTomaso, Anthony W.
AU - Koster, Joseph
AU - Xie, Zi Jian
AU - Mercer, Robert W.
PY - 1994/9/23
Y1 - 1994/9/23
N2 - All catalytic activities of the Na,K-ATPase have been ascribed to the α- subunit; however, normal activity requires the presence of the β-subunit. Using recombinant baculoviruses to infect insect cells, we demonstrate that the α-subunit, without the β-subunit, has catalytic activity. During the normal catalytic cycle of the Na,K-ATPase, the α-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 α-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 α-subunit phosphointermediate are consistent with phosphorylation at the normal aspartyl residue. Membranes from cells infected with the recombinant α baculovirus exhibit an EGTA- sensitive Mg2+-ATPase activity that is not present in the uninfected cells. The Mg2+-ATPase of the α-infected cells is reduced under conditions of high ionic strength and completely inhibited by EGTA. Thus the phosphorylation of the unassociated α-subunit is representative of the ATPase activity of the enzyme. These results suggest that the α-subunit of the Na,K-ATPase can catalyze an activity not normally associated with the enzyme and demonstrate that the β-subunit plays an important role in conferring normal activity to the enzyme complex.
AB - All catalytic activities of the Na,K-ATPase have been ascribed to the α- subunit; however, normal activity requires the presence of the β-subunit. Using recombinant baculoviruses to infect insect cells, we demonstrate that the α-subunit, without the β-subunit, has catalytic activity. During the normal catalytic cycle of the Na,K-ATPase, the α-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 α-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 α-subunit phosphointermediate are consistent with phosphorylation at the normal aspartyl residue. Membranes from cells infected with the recombinant α baculovirus exhibit an EGTA- sensitive Mg2+-ATPase activity that is not present in the uninfected cells. The Mg2+-ATPase of the α-infected cells is reduced under conditions of high ionic strength and completely inhibited by EGTA. Thus the phosphorylation of the unassociated α-subunit is representative of the ATPase activity of the enzyme. These results suggest that the α-subunit of the Na,K-ATPase can catalyze an activity not normally associated with the enzyme and demonstrate that the β-subunit plays an important role in conferring normal activity to the enzyme complex.
UR - http://www.scopus.com/inward/record.url?scp=0028168363&partnerID=8YFLogxK
M3 - Article
C2 - 8089106
AN - SCOPUS:0028168363
VL - 269
SP - 23420
EP - 23425
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 38
ER -