Active and monomeric human red cell glucose transporter after high performance molecular-sieve chromatography in the presence of octyl glucoside and phosphatidylserine or phosphatidylcholine.
The human red cell glucose transporter (Glut 1) was purified by ion-exchange chromatography in the presence of octyl glucoside. The state of association of the protein was studied, and the transport activity was determined after exchange of copurified membrane lipids for phosphatidylserine (PS) or phosphatidylcholine (PC). The purpose was to analyze the Glut 1 preparation for homogeneity and activity prior to attempts at crystallization. Analyses by high performance molecular-sieve chromatography showed that the Glut 1 was monomeric immediately after the ion-exchange purification: the Mr of the Glut 1 polypeptide was estimated to be 49,000 +/- 6000 by TSKgel G3000SW chromatography monitored by low-angle laser light-scattering photometry, differential refractometry and UV photometry. This required determination of the absorption coefficient of the Glut 1, which was measured to be 1.13 +/- 0.03 ml mg-1 cm-1 at 280 nm, referring to the polypeptide concentration. The Mr value is consistent with the cDNA-deduced Mr 54,117 of the very similar HepG2 glucose transporter polypeptide. At 2 degrees C, pH 7 and an ionic strength of 0.06 M, the Glut 1 associated gradually during three days to form oligomers. These formed much more rapidly at room temperature or at high ionic strength. Freshly prepared Glut 1 retained high activity after separation from membrane lipids on a TSKgel G3000SW column in the presence of 40 mM octyl glucoside and 1 mM PS or PC. In contrast, most of the activity was lost when the membrane lipids were separated from the protein in the absence of eluent lipids. The presence of a phospholipid was thus essential for retention of high activity of the Glut 1 in octyl glucoside and PC was nearly as effective as PS.
Lundahl
P
P
Department of Biochemistry, Uppsala University, Sweden.
Mascher
E
E
Andersson
L
L
Englund
A K
AK
Greijer
E
E
Kameyama
K
K
Takagi
T
T
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004912:Erythrocytes / Q000378:metabolism*; D005960:Glucosides / Q000737:chemistry*; D006801:Humans; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000737:chemistry*; D010718:Phosphatidylserines / Q000737:chemistry*; D012031:Refractometry; D013056:Spectrophotometry, Ultraviolet
D005960:Glucosides; D009004:Monosaccharide Transport Proteins; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; C018619:octyl-beta-D-glucoside