The procedures for purification and reconstitution of rat brain microsomal membrane protein that causes fusion of liposomes at acidic pH are described. A 1,860-fold purification was achieved, starting from the detergent-solubilized microsomal membranes. The fusion process was assayed spectrofluorimetrically by monitoring the formation of terbium-dipicolinic acid complex (Wilschut, J. et al. 1980. Biochemistry 19:6011-6021) evoked by the protein after mixing of two populations of liposomes. The fusogenic activity of the protein inserted into the membrane of Tb3+- containing vesicles was found to be strongly dependent on phospholipid composition and was higher in vesicles enriched with exogenous phosphatidylserine, phosphatidylglycerol and phosphatidylethanolamine than in those prepared with an excess of phosphatidylcholine. The vesicles enriched in negatively charged phospholipids were bound to Concanavalin A coupled to Sepharose-4B and could be released from this column only in the presence of a high concentration of o-methylmannopyranoside and detergent, indicating a glycoprotein nature of the fusogenic protein. Furthermore, these data show that protein inserted into membrane has its oligosaccharide chains exposed to the environment.
A fusogenic protein from rat brain microsomal membranes: partial purification and reconstitution into liposomes
CORAZZI, Lanfranco
1994
Abstract
The procedures for purification and reconstitution of rat brain microsomal membrane protein that causes fusion of liposomes at acidic pH are described. A 1,860-fold purification was achieved, starting from the detergent-solubilized microsomal membranes. The fusion process was assayed spectrofluorimetrically by monitoring the formation of terbium-dipicolinic acid complex (Wilschut, J. et al. 1980. Biochemistry 19:6011-6021) evoked by the protein after mixing of two populations of liposomes. The fusogenic activity of the protein inserted into the membrane of Tb3+- containing vesicles was found to be strongly dependent on phospholipid composition and was higher in vesicles enriched with exogenous phosphatidylserine, phosphatidylglycerol and phosphatidylethanolamine than in those prepared with an excess of phosphatidylcholine. The vesicles enriched in negatively charged phospholipids were bound to Concanavalin A coupled to Sepharose-4B and could be released from this column only in the presence of a high concentration of o-methylmannopyranoside and detergent, indicating a glycoprotein nature of the fusogenic protein. Furthermore, these data show that protein inserted into membrane has its oligosaccharide chains exposed to the environment.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
