Interaction of ruthenium red with Ca2(+)-binding proteins.
The interaction of ruthenium red, [(NH3)5Ru-O-Ru(NH3)4-O-Ru(NH3)5]Cl6.4H2O, with various Ca2(+)-binding proteins was studied. Ruthenium red inhibited Ca2+ binding to the sarcoplasmic reticulum protein, calsequestrin, immobilized on Sepharose 4B. Furthermore, ruthenium red bound to calsequestrin with high affinity (Kd = 0.7 microM; Bmax = 218 nmol/mg protein). The dye stained calsequestrin in sodium dodecyl sulfate-polyacrylamide gels or on nitrocellulose paper and was displaced by Ca2+ (Ki = 1.4 mM). The specificity of ruthenium red staining of several Ca2(+)-binding proteins was investigated by comparison with two other detection methods, 45Ca2+ autoradiography and the Stains-all reaction. Ruthenium red bound to the same proteins detected by the 45Ca2+ overlay technique. Ruthenium red stained both the erythrocyte Band 3 anion transporter and the Ca2(+)-ATPase of skeletal muscle sarcoplasmic reticulum. Ruthenium red also stained the EF hand conformation Ca2(+)-binding proteins, calmodulin, troponin C, and S-100. This inorganic dye provides a simple, rapid method for detecting various types of Ca2(+)-binding proteins following electrophoresis.
MRC Group in Membrane Biology, Department of Medicine, University of Toronto, Ontario, Canada.
J H
JH
Charuk
C A
CA
Pirraglia
R A
RA
Reithmeier
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002132:Calcium Radioisotopes; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002155:Calsequestrin / Q000378:metabolism; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D009132:Muscles / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011817:Rabbits; D012430:Ruthenium Red / Q000494:pharmacology*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013194:Staining and Labeling
D002132:Calcium Radioisotopes; D002135:Calcium-Binding Proteins; D002155:Calsequestrin; D012430:Ruthenium Red; D002118:Calcium