title: The Na/Ca-K exchanger of rod photoreceptor exists as dimer in the plasma membrane.
abstract: The oligomeric state of the Na/Ca-K exchanger in the plasma membrane of bovine photoreceptors was investigated using chemical cross-linking techniques. In the natural membrane, virtually all Na/Ca-K exchanger could be cross-linked mainly to a complex having an apparent molecular mass of 490 kDa by cupric phenanthroline catalyzed disulfide bonding as evidenced by Western blotting. Stable cross-links of the exchanger were also obtained with the thiol-specific reagent N,N'-p-phenylidenedimaleimide. Neuraminidase treatment reduced the apparent molecular mass of the highly glycosylated Na/Ca-K exchanger and of the 490 kDa cross-link product by 50 and 85 kDa, respectively. DL-1,4-Bismaleimido-2,3-butanediol (BMBD), a novel cleavable dimaleimide, was synthesized in order to produce cross-links that were stable to reductive conditions. Purification of the BMBD cross-linked exchanger followed by two-dimensional SDS polyacrylamide electrophoresis identified the cross-linked homodimers of the exchanger. There was no indication of higher oligomers, suggesting that the exchanger exists as a dimer in the plasma membrane. Hydrodynamic properties of the detergent-solubilized exchanger were determined by velocity sedimentation and gel filtration chromatography. The Triton X-100-solubilized exchanger ran as a single species having a Stokes radius of 10.0 nm, a sedimentation coefficient of 5.4 S, and a partial specific volume of 0.74 mL/g in Triton X-100. Similar results were obtained for the CHAPS-solubilized exchanger. A molecular mass of 236 and 205 kDa was calculated for the exchanger-detergent complex and the detergent-free protein, respectively. Neuraminidase treatment further reduced the molecular mass of the exchanger indicating that glycosylation contributes significantly to the mass of the exchanger. Cross-links of the exchanger were not detected if cross-linking was attempted after solubilization in 10 mM CHAPS. However, after reconstitution of the purified exchanger into soybean phosphatidylcholine vesicles, chemical cross-linking yielded again dimers. On the basis of these cross-linking and hydrodynamic studies, we conclude that the exchanger exists as a homodimer in the rod outer segment plasma membrane but dissociates into a monomer when solubilized in detergent.
authors.affiliation: Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Schwarzer
authors.forename: T S
authors.initials: TS
authors.lastname: Kim
authors.forename: V
authors.initials: V
authors.lastname: Hagen
authors.forename: R S
authors.initials: RS
authors.lastname: Molday
authors.forename: P J
authors.initials: PJ
authors.lastname: Bauer
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D019281:Dimerization; D008565:Membrane Proteins / Q000737:chemistry*; D008970:Molecular Weight; D009439:Neuraminidase; D017830:Octoxynol; D012374:Rod Cell Outer Segment / Q000737:chemistry*; D019831:Sodium-Calcium Exchanger; D012995:Solubility; D013438:Sulfhydryl Compounds
chemical_list: D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D019831:Sodium-Calcium Exchanger; D013438:Sulfhydryl Compounds; C074530:potassium-dependent sodium-calcium exchanger; D017830:Octoxynol; D009439:Neuraminidase