Delipidization of membrane glycoproteins solubilized in acidified chloroform/methanol. Preparation of N-retinylidene opsin in a water soluble form without-detergent.
Bovine retina membrane proteins and glycoproteins were insoluble in chloroform/methanol (2:1, v/v) unless the membrane suspension was precipitated with trichloroacetic acid and the organic solvent mixture added to the precipitated membranes. The presence of millimolar amount of trichloroacetic acid in the organic solvent led to the total solubilization of membranes. The glycoproteins precipitated at the interphase after partition of the acidified chloroform/methanol solution with water and were resolubilized from the interphase with chloroform/methanol/water (1:1:0.3, by vol). The solubility properties of the membrane glycoproteins in the acidified organic solvent mixtures allow to remove the bulk of membrane lipids and to recover from the chloroform/methanol/water solution the glycoprotein of rod outer segment membranes, rhodopsin, as protonated N-retinylidene opsin in a water soluble form.
Curtino
J A
JA
Chiodi
J C
JC
D000818:Animals; D002417:Cattle; D002725:Chloroform; D003902:Detergents; D005136:Eye Proteins / Q000302:isolation & purification*; D006023:Glycoproteins / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008563:Membrane Lipids / Q000302:isolation & purification*; D008565:Membrane Proteins / Q000302:isolation & purification*; D000432:Methanol; D012168:Retinal Pigments / Q000302:isolation & purification; D017299:Rod Opsins; D012995:Solubility; D013053:Spectrophotometry
D003902:Detergents; D005136:Eye Proteins; D006023:Glycoproteins; D008563:Membrane Lipids; D008565:Membrane Proteins; D012168:Retinal Pigments; D017299:Rod Opsins; D002725:Chloroform; D000432:Methanol