Frontal affinity chromatographic analysis of membrane protein reconstitution
1997 (English)In: Materials science & engineering. C, biomimetic materials, sensors and systems, ISSN 0928-4931, Vol. 4, no 4, 221-226 p.Article in journal (Refereed) Published
The human red cell glucose transporter Glut1 was solubilized with octaoxyethylene n-dodecyl ether (low critical micelle concentration (CMC)), purified, mixed with egg phospholipids and cholate, and reconstituted by gel filtration on Superdex 75. Free protepliposomes showed relatively high D-glucose transport activity. Frontal affinity chromatographic analysis with the proteoliposomes sterically immobilized in Superdex 200 gel beads revealed that the number of operative cytochalasin B (CB) binding sites increased during the first days of chromatographic runs to become the same as with 1-O-n-octyl β-d-glucopyranoside (high CMC) as solubilizer and Sephadex G-50 as gel filtration medium. The average number of sites per Glut1 monomer was 0.32 ± 0.02. The average Kd for CB was 66 ± 3 nM at 150 mM NaCl, similarly as for Glut1 in membrane vesicles, whereas the affinity of d-glucose for reconstituted Glut1 was lower (Kd = 44 ± 3 mM) than for membranous Glut1 (Kd = 15 ± 5 mM). Two theoretical treatments of affinity chromatographic data gave the same values in agreement with competitive and monovalent interactions.
Place, publisher, year, edition, pages
1997. Vol. 4, no 4, 221-226 p.
Medical and Health Sciences
IdentifiersURN: urn:nbn:se:uu:diva-90670DOI: 10.1016/S0928-4931(97)80004-5OAI: oai:DiVA.org:uu-90670DiVA: diva2:163114