A model for the stepwise radiation inactivation of the alpha 2-dimer of Na,K-ATPase.
This study is a direct continuation of Jensen, J., and Nørby, J. G., (1988) J. Biol. Chem. 263, 18063-18070. A new model in which we propose that the in situ organization of the Na,K-ATPase alpha-subunit is an alpha 2-dimer and which describes the stepwise degradation by radiation inactivation of this assembly is presented on the basis of the following findings. Radiation inactivation size for alpha-peptide integrity, normal nucleotide, vanadate and ouabain binding, and K-pNPPase activity is close to m(alpha) = 112 kDa; for Na-ATPase activity it is 135 kDa and for Na,K-ATPase activity it increases from 140 to about 195 kDa with increasing assay ATP concentration (equal to increasing average turnover). Normal Tl+ occlusion had the same radiation inactivation size as Vmax for Na,K-ATPase, i.e. about 195 kDa. The binding experiments disclosed radiation-produced molecules with active binding sites but with a lower than normal affinity. Radiation inactivation size for the total binding capacity of ADP and ouabain was therefore smaller than the size of an alpha-peptide, namely about 70 kDa, and for total Tl+ occlusion it was down to 40 kDa. We can explain all these observations by using a new approach to target size analysis and by assuming a dimeric organization of the alpha-subunit. Each alpha-peptide is degraded stepwise by first destruction of either a 42- or a 70-kDa domain, and the partly damaged peptide may retain biochemical activity. We conclude that there is no role for the beta-subunit in catalysis and that the alpha-peptide is organized as an alpha 2-dimer in the membrane with each alpha-subunit being able to perform complete catalytic cycles (and probably also active transport), provided that it is stabilized by an adjacent alpha-peptide or a sufficiently large fragment thereof.
Nørby
J G
JG
Institute of Biophysics, University of Aarhus, Denmark.
Jensen
J
J
D000818:Animals; D004307:Dose-Response Relationship, Radiation; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008958:Models, Molecular; D008962:Models, Theoretical; D010042:Ouabain / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000528:radiation effects*; D013552:Swine
D046911:Macromolecular Substances; D010042:Ouabain; D005954:Glucosephosphate Dehydrogenase; D000254:Sodium-Potassium-Exchanging ATPase