Solubilization and reconstitution of proline carrier in Escherichia coli; quantitative analysis and optimal conditions.
Proline carrier of Escherichia coli was extracted from the carrier-overproducing membranes with dodecylmaltoside in the presence of phospholipid. The solubilized carrier showed the same proline binding activity as that in normal membranes. As judged from determinations of the binding activity in the micellar state as a marker of active carrier and the radioactivity of N-[ethyl-2-3H]ethylmaleimide-labeled carrier as a marker of carrier polypeptide, 80% of the carrier molecules in the membranes were extracted. Optimal conditions for reconstitution of the solubilized carrier were established. By a combination of freeze-thawing, sonication and dilution procedures, 70% of the solubilized carrier molecules were incorporated into proteoliposomes and the restored active transport of proline showed an apparent Kt of 1 microM and turnover number of 0.6 s-1. The transport of proline was driven by a membrane potential in a Na+ (or Li+)-dependent manner.
Hanada
K
K
Department of Biology, Faculty of Science, University of Tokyo, Japan.
Yamato
I
I
Anraku
Y
Y
D026921:Amino Acid Transport Systems, Neutral; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000032:analysis; D004926:Escherichia coli / Q000032:analysis*; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide; D005615:Freezing; D005960:Glucosides / Q000494:pharmacology; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D010743:Phospholipids / Q000494:pharmacology; D011392:Proline / Q000378:metabolism; D012995:Solubility; D013010:Sonication; D027981:Symporters
D026921:Amino Acid Transport Systems, Neutral; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D005960:Glucosides; D008081:Liposomes; D010743:Phospholipids; D027981:Symporters; C041832:proline transport protein; C060609:PutP protein, E coli; C018619:octyl-beta-D-glucoside; C040358:dodecyl maltoside; D011392:Proline; D005033:Ethylmaleimide