Previous results suggest that Ehrlich ascites tumor plasma membrane-bound NaK-ATPase cation translocation is regulated by phosphorylation of the enzyme's glycoprotein beta subunit. It is planned to study this putative control mechanism by investigating the effects of beta subunit phosphorylation and dephosphorylation on reconstituted enzyme ATP-dependent Na+ uptake; on substrate and inhibitor binding; and on enzyme conformation as detected by trapping conformational states by chemically cross-linking NaK-ATPase subunits, preparing limit digests of the cross-linked enzymes, and then comparing two-dimensional peptide maps of the resulting peptide fragments. The putative mechanism regulating NaK-ATPase cation pumping efficiency will be further examined by investigating quercetin- and normal brain NaK-ATPase beta subunit 29,000 dalton polypeptide fragment-dependent repair of inefficient ascites NaK-ATPase cation pumping. It is also planned to investigate the possibility that differences in the post-translational processing of normal mouse brain and Ehrlich ascites tumor NaK-ATPase beta subunit processing of normal mouse brain and Ehrlich ascites tumor NaK-ATPase beta subunit oligosaccharides account for the fact that tumor, but not normal, enzyme is phosphorylated in vivo. Specifically stained peptide maps of normal mouse brain and Ehrlich ascites NaK-ATPase beta subunits will be analyzed in order to determine if differences in oligosaccharide structure exist between the two. Systematic deglycosylations will be performed in order to study the importance of oligosaccharide to NaK-ATPase function and susceptibility to beta subunit phosphorylation and dephosphorylation. In addition, NaK-ATPase oligosaccharide structural differences, if present, will be examined in order to learn the identities of metabolic lesions in Ehrlich ascites tumor which effect the post-translational processing of olgiosaccharides.