IDENTIFICATION OF TRANSMEMBRANE DOMAINS OF THE RED-CELL CALCIUM-PUMP WITH A NEW PHOTOACTIVATABLE PHOSPHOLIPIDIC PROBE

The membrane-associated regions of the human erythrocyte Ca2+ pump were investigated by hydrophobic photolabeling. Purified Ca2+ pump was reconstituted in asolectin vesicles loaded with [H-3]DIPETPD, a photochemical probe designed to label deeply into the hydrophobic core of the lipid bilayer (Delfino et al. J. Am. Chem. Sec. 115, 3458-3474, 1993). After photolysis and SDS-PAGE analysis, a significant light-dependent labeling of the Ca2+ pump was found. Controlled proteolysis of the photoadduct with trypsin or protease Vg followed by SDS-PAGE and immunoblotting yielded individual labeled fragments. The labeling pattern indicated the existence of three sequential clusters of transmembrane regions, consistent with the current model for the topography of this enzyme. (C) 1994 Academic Press, Inc.