Membrane-bound Na,K-ATPase: target size and radiation inactivation size of some of its enzymatic reactions.
Frozen samples of membrane-bound pig kidney Na,K-ATPase were subjected to target size analysis by radiation inactivation with 10-MeV electrons at -15 degrees C. The various properties investigated decreased monoexponentially with radiation dose, and the decay constants, gamma, were independent of the presence of other proteins and of sucrose concentrations above 0.25 M. The temperature factor was the same as described by others. Irradiation of four proteins of known molecular mass, m, showed that gamma for protein integrity was proportional to m with a proportionality factor about 20% higher than that conventionally used. By this standard curve, glucose-6-phosphate dehydrogenase activity used as internal standard gave a radiation inactivation size of 110 +/- 5 kDa, very close to m = 104-108 kDa for the dimer, as expected. For Na+/K+-transporting ATPase the following target sizes and radiation inactivation size values were very close to m = 112 kDa for the alpha-peptide: peptide integrity of alpha, 115 kDa; unmodified binding sites for ATP and vanadate, 108 kDa; K+-activated p-nitrophenylphosphatase activity, 106 kDa. There was thus no sign of dimerization of the alpha-peptide or involvement of the beta-peptide. In contrast, optimal Na+/K+-transporting ATPase activity had a radiation inactivation size = 189 +/- 7 kDa, and total nucleotide binding capacity corresponded to 72 +/- 3 kDa. These latter results will be extended and discussed in a forthcoming paper.
Jensen
J
J
Institute of Physiology, University of Aarhus, Denmark.
Nørby
J G
JG
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D004307:Dose-Response Relationship, Radiation; D005954:Glucosephosphate Dehydrogenase / Q000037:antagonists & inhibitors / Q000528:radiation effects; D007679:Kidney Medulla / Q000201:enzymology*; D007700:Kinetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000528:radiation effects*; D013552:Swine
D005954:Glucosephosphate Dehydrogenase; D000254:Sodium-Potassium-Exchanging ATPase