Expression of an active Na,K-ATPase with an alpha-subunit lacking all twenty-three native cysteine residues.
We have constructed a mutant Na,K-ATPase alpha1-subunit with all native cysteine residues replaced. Using the baculovirus system, this cysteine-less alpha1-subunit and wild-type beta1-subunit were expressed in High Five cells. After 3 days of infection, cells were fractionated, and endoplasmic reticulum, Golgi apparatus, and plasma membranes were isolated. The molecular activity of the cysteine-less mutant in the plasma membranes was close to the wild-type protein (8223 min(-)(1) versus 6655 min(-)(1)). Cation and ATP activation of Na,K-ATPase activities revealed that replacing all 23 cysteines resulted in only a 50% reduction of K(m) for Na(+), a 2-fold increase in K(m) for K(+), and no changes in K(m) for ATP. The distribution of alpha-subunits among the membranes showed a high percentage of cysteine-less protein in the endoplasmic reticulum and Golgi apparatus compared with the wild-type protein. Furthermore, the cellular stability of the alphabeta assembly appeared reduced in the cysteine-less mutant. Cells harvested after more than 3 days of infection showed extensive degradation of the cysteine-less alpha-subunit, which is not observed with the wild-type enzyme. Thus the Na,K-ATPase contains no cysteine residues that are critical for function, but the folding and/or assembly pathway of this enzyme is affected by total cysteine substitution.
Hu
Y K
YK
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201-3098, USA.
Eisses
J F
JF
Kaplan
J H
JH
D000255:Adenosine Triphosphate / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology; D008958:Models, Molecular; D009154:Mutation; D010042:Ouabain / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D012756:Sheep; D012965:Sodium Chloride / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera / Q000166:cytology; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000817:Anilino Naphthalenesulfonates; D004791:Enzyme Inhibitors; D013439:Sulfhydryl Reagents; D012965:Sodium Chloride; D010042:Ouabain; D011189:Potassium Chloride; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D003545:Cysteine