Solubilization of a complex of tryptic fragments of Na,K-ATPase containing occluded Rb ions and bound ouabain.
The nonionic detergent C12E10 (polyoxyethylene 10-laurylether) has been used to solubilize a complex of tryptic fragments of Na, K-ATPase containing occluded Rb ions and bound ouabain. The aim was to define which fragments are required to maintain Rb occlusion. The experiments utilize "19 kDa membranes" consisting of a 19 kDa and several smaller tryptic fragments (8-11.7 kDa) of the alpha subunit, which include trans-membrane segments M7/M10 and the pairs M1/M2, M3/M4, and M5/M6 [Capasso, J. M., et al (1992) J. Biol. Chem. 267, 1150-1158]. The beta subunit is partially split into a 16 kDa fragment and a glycosylated approximately 50 kDa fragment. Cation occlusion and ouabain binding are intact. After preincubation of "19 kDa membranes" with Rb (5 mM) and then ouabain (10 mM), 90-100% of occluded Rb was solubilized by C12E10 at 0 degrees C. All fragments of the alpha and beta subunits, and also the gamma subunit, were cosolubilized by C12E10, and were observed to sediment together on a sucrose density gradient as a complex containing occluded Rb ions. The soluble complex consists of a monomer containing one copy of each fragment, as indicated by size-exclusion HPLC, as well as estimates of specific Rb occlusion (20.0 +/- 1.2 nmol/mg of protein). In the absence of Rb ions and ouabain, the complex was unstable. Whereas the 19 kDa fragment (M7-M10) and beta subunit remained associated, the smaller fragments, containing M5/M6 and M3/M4 and M1/M2, and the subunit dissociated. Observations on the thermal inactivation of Rb occlusion, and effect of pH and ionic strength, suggest that the soluble complex is stabilized by multiple interactions, both within the lipid bilayer and in hydrophilic domains (e.g., salt bridges).
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Biochemistry Department, Weizmann Institute of Science, Rehovot, Israel.
Goldshleger
E D
ED
Tal
D M
DM
Karlish
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D010042:Ouabain / Q000032:analysis* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D012413:Rubidium / Q000032:analysis* / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013552:Swine; D014357:Trypsin / Q000378:metabolism*
D010446:Peptide Fragments; D010042:Ouabain; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium