Abstract
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.
Highlights
Information concerning the molecular weightsof each chain and their stoichiometry in the native enzyme is pertinent to proposals which have been presented for the mechanism of active transport
It can be seen from the scans of these gels that the amounts of both the a polypeptide and the p polypeptide decreaseas a new component of lower electrophoretic mobility appearTs.he new component is the only significantproduct of the cross-linking
The two polypeptides, initially observed in purified canine renal (Na' + K')-ATPase (l),have always been found in the purified preparations from a variety of species [2,3,4,5,6,7]
Summary
Information concerning the molecular weightsof each chain and their stoichiometry in the native enzyme is pertinent to proposals which have been presented for the mechanism of active transport. Values reported for the molecular weight of the a chain have ranged from 85,000 to 140,000 [6, 7, 10, 12,13,14,15] and those for the p chain from 35,000 to 67,000. The apparent length of this unglycosylated small chain that themolar stoichiometry of the a and /3 polypeptides in a is 450 residues, corresponding to a molecular weight of mammalian (Na++ K+)-ATPase is1:l.The molecular weight. Previous results which address the question of the quaternary structure of the ATPase are re-examined in light of these determinations
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