title: Identification of transmembrane domains of the red cell calcium pump with a new photoactivatable phospholipidic probe.
abstract: 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 [3H]DIPETPD, a photochemical probe designed to label deeply into the hydrophobic core of the lipid bilayer (Delfino et al. J. Am. Chem. Soc. 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 V8 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.
authors.affiliation: IQUIFIB, Instituto de Química y Fisicoquímica Biológicas (UBA-CONICET), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.
authors.forename: P R
authors.initials: PR
authors.lastname: Castello
authors.forename: A J
authors.initials: AJ
authors.lastname: Caride
authors.forename: F L
authors.initials: FL
authors.lastname: González Flecha
authors.forename: H N
authors.initials: HN
authors.lastname: Fernández
authors.forename: J P
authors.initials: JP
authors.lastname: Rossi
authors.forename: J M
authors.initials: JM
authors.lastname: Delfino
mesh_terms: D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000648:ultrastructure*; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000648:ultrastructure; D010743:Phospholipids / Q000737:chemistry; D012697:Serine Endopeptidases / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000494:pharmacology
chemical_list: D008562:Membrane Glycoproteins; D010743:Phospholipids; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D000252:Calcium-Transporting ATPases