Abstract
Since ATP and 2,3-diphosphoglycerate cause an increase in the lateral mobility of integral membrane proteins in the erythrocyte (Schindler, M., Koppel, D., and Sheetz, M. P. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 1457-1461), we have studied their effects on the membrane skeletal complex or shell (composed of spectrin, actin, and bands 4.1 (78,000 daltons) and 4.9 (50,000 daltons)) and its interaction with the erythrocyte membrane. Both phosphate compounds dissociated the delipidated shell complex, with half-maximal dissociation at 2.5 mM 2,3-diphosphoglycerate and 8 mM ATP, whereas equivalent concentrations of EDTA did not. Concomitant with complex dissociation, spectrin was solubilized but band 4.1 and actin remained in a complexed or polymeric form. When proteins which were involved in linking spectrin to the membrane were present on the shell, higher concentrations of the phosphate compounds still dissociated the complex but less spectrin was solubilized. Treatment of erythrocyte membranes with the same phosphate compounds caused membrane vesiculation but no proteins were solubilized. We suggest that ATP and 2,3-diphosphoglycerate, at concentrations which are normally present in erythrocytes, can weaken associations in the shell but will not dissociate the complex from membrane attachment sites.
Highlights
We suggest that ATP and 2,3-diphospho- 2,3-P2 glycerate (Tris salt), glutathione, and diisopropyl fluorophosglycerate, at concentrations which are normally pres- phate were obtained from Sigma
Whenerythrocytemembranes were incubatedwith low concentrations (t10 mMo)f 2,3-P2 glycerateorATP,no morphological change or solubilization of membrane protein was found
Control membranes incubated for the sameperiod of time under similar condition(sFig. l a ) did not vesiculate
Summary
When the effect of25 lll~ATP was analyzed after 15 min at 37°C (cytoskeletons were still visible), there was a release of 26% of cytoskeletal spectrin whereas only 3%of the spectrin was released in control samples.Analysis of supernatant andpellet fractions by SDS polyacrylamide gels (Fig. 2, c and d) after extensive dissociation by 40 mM A T P showed that all of the major proteins were inthesupernatant fraction. The effect of2,3-P2 glycerate on the shells was dramatic (Fig. 3) with maximal dissociation of 60% of the pelletable A m material at 4 mM 2,3-P2glycerate (half-maximal at 2.5 mM).SDS polyacrylamide gel analysis indicated that 60 f 10%of the shell spectrin was released into the supernatant along with 40 f 5%of actin and band 4.1 (Fig. 4).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
