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)
Department of Physiology, University of Pennsylvania, Philadelphia 19104-6085.
S
S
Lutsenko
J H
JH
Kaplan
D000818:Animals; D001665:Binding Sites; D002412:Cations / Q000378:metabolism; D003374:Coumarins; D004229:Dithiothreitol / Q000494:pharmacology; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D008301:Maleimides; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D012413:Rubidium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D002412:Cations; D003374:Coumarins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D008301:Maleimides; D008565:Membrane Proteins; D010446:Peptide Fragments; C030860:N-(4-(7-diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D004229:Dithiothreitol