Solubilization and reconstitution of the sodium-and-chloride-coupled glycine transporter from rat spinal cord.
Synaptic membranes from rat spinal cord were solubilized in the presence of 2% sodium cholate, phospholipids and 15% ammonium sulphate. The soluble extract was incorporated into liposomes consisting of asolectin and crude rat brain lipids. Reconstitution of the functional transporter protein was achieved by removal of detergent by gel filtration. Several parameters proved to be important for optimal reconstitution efficiency: (a) the lipid composition of the liposomes, (b) the type of detergent, and (c) the phospholipid/protein and detergent/protein ratio during reconstitution. In the reconstituted system, the transport of glycine showed a specific activity about twice that of native vesicles. The ionic dependence of the transport, the inhibitory effect of nigericin in the presence of external sodium and the stimulatory effect of valinomycin in the presence of internal potassium on glycine transport were preserved and more clearly observed in the reconstituted system. These results indicate that, in this preparation, the glycine transporter protein retains the same features displayed in the synaptic plasma membrane vesicles, namely dependence on sodium and chloride, electrogenicity and inhibitor sensitivity.
Lopez-Corcuera
B
B
Departamento de Biología Molecular, Facultad de Ciencias, Universidad Autónoma, Madrid, Spain.
Aragon
C
C
D026921:Amino Acid Transport Systems, Neutral; D000818:Animals; D001921:Brain / Q000502:physiology; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002712:Chlorides / Q000494:pharmacology*; D005998:Glycine / Q000378:metabolism*; D050487:Glycine Plasma Membrane Transport Proteins; D007700:Kinetics; D008055:Lipids / Q000502:physiology; D008081:Liposomes; D008297:Male; D010713:Phosphatidylcholines; D010743:Phospholipids; D051381:Rats; D011919:Rats, Inbred Strains; D012964:Sodium / Q000494:pharmacology*; D012995:Solubility; D013116:Spinal Cord / Q000378:metabolism*; D013570:Synaptic Membranes / Q000378:metabolism*
D026921:Amino Acid Transport Systems, Neutral; D002352:Carrier Proteins; D002712:Chlorides; D050487:Glycine Plasma Membrane Transport Proteins; D008055:Lipids; D008081:Liposomes; D010713:Phosphatidylcholines; D010743:Phospholipids; C024990:asolectin; D012964:Sodium; D005998:Glycine