Cross-linking of the erythrocyte (Na+,K+)-ATPase. Chemical cross-linkers induce alpha-subunit-band 3 heterodimers and do not induce alpha-subunit homodimers.

D.W Martin,J.R Sachs

doi:10.1016/s0021-9258(18)35925-8

Abstract

Earlier studies (Periyasamy, S. M., Huang, W.-H., and Askari, A. (1983) J. Biol. Chem. 258, 9878-9885) suggested that Cu2+ and o-phenanthroline induced the formation of cross-linked homodimers between alpha-subunits of the erythrocyte (Na+,K+)-ATPase. This was interpreted as indicating that alpha-subunits existed in close proximity in native erythrocyte membranes. The alpha-subunit and band 3 monomers have similar molecular weights (M(r) approximately 100,000) and exist in the membrane in molar ratios of approximately 1:3000 alpha-subunit:band 3. We explored the possibility that alpha-subunit and band 3 could be induced to form heterodimeric structures in the presence of cross-linking reagents. Using methods similar to those employed in the above-cited reference we demonstrated that cross-linked dimers containing phosphorylated alpha-subunits had proteolytic sensitivity that was inconsistent with the formation of alpha-subunit homodimers and fully consistent with heterodimer formation between alpha-subunit and band 3. The data also indicated that alpha-subunit-band 3 heterodimer formation is dependent on the conformational state of the (Na+,K+)-ATPase. Using the appropriate reagents we obtained cross-linked products which were consistent with heterodimer formation between alpha- and beta-subunits of the (Na+,K+)-ATPase. Our data argue against a close association between pairs of (Na+,K+)-ATPase alpha-subunits in the human red cell membrane.

Highlights

Chem. 258,9878-9885) suggested that Cu2+ ando-phenanthroline induced the formation of cross-linked homodimers between a-subunits of the erythrocyte (Na+,K+)-ATPase.This was interpreted as indicating that a-subunits existedin close proximity in native erythrocyte membranes
The(Na+,K+)-ATPaserepresentsthe predomiphorylated a-subunits had proteolytic sensitivity that nant protein species in purified kidney preparations, and the was inconsistent with the formation of a-subunit hom- subunit associations could bedirectly assayed by densitometodimers and fully consistent with heterodimer forma- ric scans of Coomassie Blue-stained polyacrylamide gels
The data are consiwstiethnt he phosphorylated species of the high molecular weight band originating with the a-subunit and do not suggest that Cu2+and o-phenanthroline (CUP) induced a uniquephosphorylation of some other red cell membrane protein

Summary

RESULTS

The data are consiwstiethnt he phosphorylated species of the high molecular weight band originating with the a-subunit and do not suggest that CUP induced a uniquephosphorylation of some other red cell membrane protein. The above data clearly show that while Pronase treatment did not alter theM , of the a-subunit itdid reduce the M,of the high molecular weight cross-linked species.

10 Chymotrypstn Treated RBC x 0 Trypsln Treated RBC x Untreated RBC

Findings

DISCUSSION