Preliminary Identification of ATP Compartment Associated Proteins On Erythrocyte Membrane.

Abstract 3019

Poster Board II-995

A compartment/pool of ATP that is generated by glycolytic enzymes has been reported to exist on the erythrocyte membrane where it channels ATP directly to the Na⁺/K⁺-ATPase (Proverbio F and Hoffman JF. J. Gen. Physiol. 1977, 69:605-632. Mercer RW and Dunham PB. J. Gen. Physiol. 1981, 78:547-567). In order to identify the protein components enclosing this compartment of ATP, a photoactivatable probe, 8-azido-[αa-³²P]ATP, was loaded into porous erythrocytes under conditions that fill the compartment with ATP, and the radiolabeled ATP was induced to label proximal proteins by illumination with UV light. Analysis of radiolabeled bands reveals that spectrin, adducin, protein 4.1, and actin constitute major components of the compartment. To further verify the involvement of these proteins in the ATP compartment, antibodies against the aforementioned proteins were pre-incubated with porous erythrocytes before attempting to load the compartment with ATP. Analysis of the efficiency of ATP loading in the presence of these blocking antibodies reveals that antibody binding prevents normal filling of the ATP pool. These findings confirm Hoffman's earlier hypothesis that a membrane-bound compartment of ATP exists. The data also suggest that the location of the compartment might reside at the junctional complex, and that the complex of enzymes that fill the ATP pool is different from the complex organized around band 3.