The role of the Na, K-ATPase β-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 °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 β-subunit; both of these effects were prevented by the presence of 20 mM RbCl. To clarify the role of the extracellular portion of the β-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 °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 β-subunit to the supernatant. The appearance of the 45-kDa fragment of the β-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 β-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. Fluorescent labeling with CPM [3-(4-maleimidophenyl)-4-methyl-7-(diethylamino)coumarin] of “19-kDa membranes” following DTT treatment revealed no changes either in the “19-kDa peptide” or in any peptides smaller than 11 kDa. Similar procedures were used on Pronase-digested “19-kDa membranes”. DTT treatment and CPM reaction following Pronase digestion revealed changes in both the mobility and fluorescence intensity of a 45-kDa peptide derived from the β-subunit concomitant with loss in the Rb occluding capacity. We conclude that the β-subunit of the Na,K-ATPase participates in stabilizing the occluded cation intermediate and that the extracellular domain structure of the β-subunit which is maintained by S–S bridges plays an important role in the K-binding function.
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