Chemical Characterization and Pronase Susceptibility of the Na:K Pump-Associated Phosphoprotein of Human Red Blood Cells

The phosphoproteins formed by incubation of red cell ghosts with [γ-³²P]ATP in the presence of Mg and Na + Mg have been characterized by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The ³²P-labeled phosphoprotein was seen as a single peak confined to the region of the diffuse 90,000 dalton polypeptide band; labeling with Na + Mg considerably increased the quantity of ³²P-phosphoprotein contained in this band relative to labeling with Mg alone. Treatment of intact cells with Pronase known to partially hydrolyze the glycoproteins and the 90,000 daltons polypeptide did not change either the amount or the position of the ³²P-phosphoprotein present in the gels. The molecular weight of the ³²P-phosphoprotein is estimated to be 103,000. Pronase treatment of intact cells also did not significantly alter any of the transport parameters of the membrane such as the K pump flux, ouabain binding, or Na,K-ATPase. In contrast, treatment of ghosts with Pronase not only resulted in drastic alteration of the transport parameters but also inhibited the formation of the phosphoprotein under all conditions. Thus, while the Na:K pump is not intrinsically resistant to Pronase, those elements of the pump which are susceptible are not accessible from the outside of the cell. Further, SDS-polyacrylamide gel electrophoresis after Pronase treatment of intact cells results in a substantial increase in the purification of the phosphoprotein relative to that which was previously possible in ghosts.