Solubilization and reconstitution characteristics of hepatic system A-mediated amino acid transport.
In the liver, System A-mediated uptake of neutral amino acids may play a key role in metabolic control. Knowing the properties of the solubilized and reconstituted System A activity is important for future studies on the purification of the carrier protein. Solubilization of System A activity by the combination of 2.5% cholate and 4 M urea resulted in greater than 85% extraction of the activity. Previous removal of easily extracted plasma membrane proteins with 1% cholate alone followed by solubilization of the transporter with cholate/urea resulted in a 2-fold enrichment in transport activity. Based on the observation that the carrier protein aggregates in the presence of low detergent concentrations, a selective polyethylene glycol precipitation procedure was developed resulting in recovery of more than 70% of the initial transport activity and less than 10% of the total protein. A concomitant 10-fold enrichment in carrier activity was achieved. The precipitated carrier could be resuspended in buffer containing Triton X-100, asolectin, and glycerol. Transporter activity in this buffer was stable for up to 5 days when maintained at -20 degrees C or for 2 days at 4 degrees C. The general applicability of the devised reconstitution is illustrated by the presence of Systems N and Gly in the reconstituted proteoliposomes at specific activities greater than those in the native vesicles.
Fafournoux
P
P
Laboratoire des Maladies Metaboliques, INRA Theix, Ceyrat, France.
Dudenhausen
E E
EE
Kilberg
M S
MS
D026905:Amino Acid Transport Systems; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002021:Buffers; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D003902:Detergents; D004355:Drug Stability; D008099:Liver / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D051381:Rats; D012995:Solubility
D026905:Amino Acid Transport Systems; D000596:Amino Acids; D002021:Buffers; D002352:Carrier Proteins; D003902:Detergents; D008565:Membrane Proteins