Purification and oligomeric state of the major lens fiber cell membrane proteins.
Purification of the lens fiber cell membrane proteins MP20 and MP26, and the partial co-purification of the lens connexin-related proteins MP70 and connexin 46 has been achieved using anion- and cation-exchange chromatography of lens fiber cell membrane proteins solubilized in n-octyl-beta-D-glucopyranoside (octyl glucoside). The apparent molecular weights of the solubilized protein-detergent complexes were significantly greater than that expected for the monomeric proteins. The purified proteins retained their ability to be phosphorylated by cAMP-dependent protein kinase, and to bind calmodulin in a calcium and magnesium dependent manner. The heterobifunctional covalent chemical crosslinking agent N-5-azido-2-nitro-benzoyloxysuccinimide (ANB-NOS), and the thiol oxidant cupric phenanthroline were used to identify the oligomeric states of these proteins. Crosslinking of either the purified proteins or native lens membranes generated a ladder of crosslinked MP20 or MP26 homo-oligomers. The largest detectable crosslinked homo-oligomer of MP20 was at least a hexamer, while for MP26 the largest crosslinked homo-oligomer was at least a tetramer. The possible oligomeric states of MP70 and connexin 46 could not be determined with the crosslinking reagents used in this study. The procedure described here for the purification of detergent-solubilized major lens proteins should provide a valuable approach in future studies aimed at clarifying the roles of these different lens membrane proteins.
Department of Biochemistry, University of Minnesota, St Paul 55108, USA.
L J
LJ
Jarvis
C F
CF
Louis
D000345:Affinity Labels; D000818:Animals; D020346:Aquaporins; D015153:Blotting, Western; D002147:Calmodulin / Q000737:chemistry; D002417:Cattle; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000302:isolation & purification*; D007908:Lens, Crystalline / Q000737:chemistry*; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000302:isolation & purification*; D010766:Phosphorylation
D000345:Affinity Labels; D020346:Aquaporins; D002147:Calmodulin; D003432:Cross-Linking Reagents; D005136:Eye Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C030328:aquaporin 0