Premium
Caloxin 1c2: Experimental Evidence That It Binds Plasma Membrane Calcium Pump
Author(s) -
Pande Jyoti,
Szewczyk Magdalena M,
Kuner Rajneet,
Ghosh Paromita,
Grover Ashok K
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1157.3
Subject(s) - biotinylation , streptavidin , chemistry , extracellular , biotin , membrane , biochemistry , biophysics , microbiology and biotechnology , biology
We engineered caloxin 1c2, a novel high affinity peptide (TAWSEVLDLLRRGGGSK‐amide) inhibitor of the Plasma Membrane Ca 2+ pump (PMCA). To show that caloxin 1c2 acts by binding to the PMCA, we synthesized a photoreactive derivative 3Bpa‐caloxin 1c2 (TA(Bpa)SEVLDLLRRGGGSK‐biotin), containing the photoreactive residue Bpa (benzoylphenylalanine) and biotin. 3Bpa‐caloxin 1c2 inhibited the Ca 2+ ‐Mg 2+ ‐ATPase activity of PMCA. The human erythrocyte membranes were photolabeled with 3Bpa‐caloxin 1c2 and the proteins were detected in the Western blots using HRP‐streptavidin to identify biotin or PMCA antibodies to detect PMCA. The photolabeled erythrocyte membranes showed a 250–270 kDa doublet with HRP‐streptavidin. The degree of biotinylation of the erythrocyte membranes depended on the length of the cross‐linking time, and concentrations of 3Bpa‐caloxin 1c2 and the membrane protein. Immunoprecipitates from the photolabeled erythrocyte membranes using a PMCA antibody showed a 250–270 kDa doublet with HRP‐streptavidin. Biotinylated proteins isolated from the photolabeled erythrocyte membranes using captavidin also showed a 250–270 kDa doublet with HRP‐streptavidin and PMCA antibodies. Caloxin 1c2 had been selected for binding to the extracellular domain 1 (amino acids 115–131) of PMCA isoform 4. Caloxin 1c2 and the extracellular domain 1 peptide of PMCA 4 competed with 3Bpa‐caloxin 1c2 for photolabeling the erythrocyte membranes. Pieces of de‐endothelialized pig coronary artery cross‐linked to 3Bpa‐caloxin 1c2 and treated with Alexa‐streptavidin were examined by confocal microscopy, using the Na + ‐pump inhibitor Bodipy‐ouabain as a positive control. Both the probes bound to the cell surface. Thus, the photoreactive derivative of caloxin 1c2 binds to PMCA at the cell surface. Supported by Heart & Stroke Foundation of Ontario.