An essential role for the extracellular domain of the Na,K-ATPase beta-subunit in cation occlusion.

The role of the Na,K-ATPase beta-subunit in stabilization of ion-binding sites has been investigated. Treatment of the purified renal Na,K-ATPase with 0.25 M DTT at 40 degrees C for 1 h resulted in 50% loss of Rb occlusion, which correlates with partial reduction of S-S bridges in the extracellular portion of the beta-subunit; both of these effects were prevented by the presence of 20 mM RbCl. To clarify the role of the extracellular portion of the beta-subunit, "19-kDa membranes" (Na,K-ATPase posttryptic residues, which have been shown to possess many of the cation-binding properties) were used. Incubation of the "19-kDa membranes" with 0.2 M DTT for 1 h at 37 degrees C abolished 70-80% of the 86Rb occlusion capacity. This was accompanied by accumulation of 16- and 17-kDa peptides (in SDS-PAGE of the membranes) and release of a 45-kDa band derived from the Na,K-ATPase beta-subunit to the supernatant. The appearance of the 45-kDa fragment of the beta-subunit in the supernatant confirms the existence of only one transmembrane fragment in this subunit. N-Terminal sequence analysis of the 16- and 17-kDa bands revealed the same structure, A-K-E-E-G-, which corresponds to the beta-subunit sequence beginning at Ala5. The simultaneous presence of 25 mM RbCl (but not 25 mM choline chloride) during DTT treatment prevents almost all (85%) of the loss of Rb occlusion, the appearance of 16- and 17-kDa bands, and reduction and release of the 45-kDa fragment.(ABSTRACT TRUNCATED AT 250 WORDS)