Stoichiometry and molecular weight of the minimum asymmetric unit of canine renal sodium and potassium ion-activated adenosine triphosphatase.
Sodium and potassium ion-activated adenosine triphosphatase is known to be composed of at least two different polypeptides, alpha and beta. When a detergent-treated supernatant preparation of the enzyme is reacted with the cross-linking reagent, cupric phenanthroline, a single, covalent heterodimer is formed. This product is formed from one of each of the two polypeptides. The remaining, unreacted alpha and beta chains maintain a constant ratio to each other throughout the reaction. The same heterodimer is formed in membrane-bound enzyme when reacted with several other cross-linking reagents. The protein mass ratio between the chains in the native enzyme, determined by two methods, is 2.15 +/- 0.16. Using this value and a value of 121,000 +/- 6,000 for the molecular weight of the larger polypeptide, a molecular weight of 56,000 +/- 7,000 can be calculated for the protein portion of the smaller polypeptide. Upon removal of a substantial portion of the carbohydrate from the smaller polypeptide, a change in its electrophoretic mobility is observed, while that of the larger polypeptide remains unaffected. The apparent length of this unglycosylated small chain is 450 residues, corresponding to a molecular weight of 51,000. Taken together, these results demonstrated that the two polypeptides of the (Na+ + K+)-ATPase exist in an equimolar, noncovalent association in the native enzyme, and that the protein molecular weight of the minimum asymmetric unit is 177,000 +/- 13,000, Previous results which address the question of the quaternary structure of the ATPase are re-examined in light of these determinations.
D000818:Animals; D003432:Cross-Linking Reagents; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis; D010618:Phenanthrolines; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis*; D013296:Streptococcus pneumoniae / Q000201:enzymology
D003432:Cross-Linking Reagents; D010455:Peptides; D010618:Phenanthrolines; D006026:Glycoside Hydrolases; D000254:Sodium-Potassium-Exchanging ATPase