Detection of bis(sulfosuccinimidyl) suberate binding in electrophoresis: determination of membrane sidedness of proteins.
The applicability of the membrane-impermeant protein cross-linker bis(sulfosuccinimidyl) suberate (BS(3)) to the determination of membrane sidedness of proteins was tested in 3T3-L1 cells and in erythrocytes. Binding of BS(3) to proteins was apparent in electrophoresis. In three proteins of 3T3-L1 cells, protein kinase-Cepsilon, protein kinase-Czeta, and glyceraldehyde-3-phosphate dehydrogenase, BS(3) action was detectable in SDS-PAGE with immunoblotting. This enabled confirmation of the well-known intracellular localization of these proteins. In cathepsin E of erythrocytes, a mobility increase in nondenaturing PAGE was the most prominent effect of BS(3) treatment. A mechanism for the increase in mobility due to BS(3) binding is suggested. Cathepsin E was found to be located at the intracellular side of the membrane, in accordance with existing evidence.
Till
Olaf
O
Institute of Anatomy I, Hospital of the Friedrich Schiller University, Jena, Germany. olaf.till@uni-jena.de
Schmidt
Martin
M
Linss
Werner
W
Baumann
Eckehard
E
D041721:3T3-L1 Cells; D000818:Animals; D020059:Cathepsin E / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000737:chemistry; D006801:Humans; D051379:Mice; D011493:Protein Kinase C / Q000737:chemistry; D051744:Protein Kinase C-epsilon / Q000737:chemistry; D013388:Succinimides / Q000737:chemistry*; D000099221:Protein Kinase C zeta
D020059:Cathepsin E; D003432:Cross-Linking Reagents; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D013388:Succinimides; D000099221:Protein Kinase C zeta; C035760:bis(sulfosuccinimidyl)suberate