An assay for the alpha-tocopherol binding protein mediated transfer of vitamin E between membranes.
A model system consisting of donor membrane (egg lecithin liposomes) and acceptor membrane (human erythrocyte ghosts or rat liver mitochondria) were used to investigate the alpha-tocopherol binding protein (alpha TBP) mediated transfer of alpha-tocopherol. Liposomes containing RRR-[alpha-3H]tocopherol ([alpha-3H]T) were incubated with acceptor membrane at 37 degrees C for 0-45 min in the presence or absence of rat liver cytosol or a dialyzed 30-60% saturated ammonium sulfate precipitated fraction of rat liver cytosol (Fraction B). Erythrocyte ghosts and liver mitochondria were compared and found to behave similarly in the presence of Fraction B. alpha-Tocopherol transfer activity (alpha TTA) typically varied 0- to 27-fold greater than buffer blanks, depending upon type and concentration of protein preparation. Gel filtration of Fraction B yielded one alpha TTA peak (liver mitochondria as acceptor) with an estimated Mr of 39,000. [alpha-3H]T recovered from erythrocyte ghosts pellets by HPLC suggest that the [alpha-3H]T was transferred intact. alpha TTA of Fraction B in the presence of varying concentrations of erythrocyte ghosts and liposomal [alpha-3H]T followed saturation kinetics. Optimal concentrations gave alpha TTA responses directly proportional to rat liver cytosol concentration. alpha TTA was inhibited only 5% in the presence of a 32-fold excess of cold liposomal alpha-tocopheryl acetate suggesting that the free hydroxyl group on the chromanol ring of alpha-tocopherol is needed for transfer. Coefficient of variation of repeated measures of alpha TTA in rat liver cytosol was 2.9%. Thus, the intermembrane transfer phenomenon of alpha-tocopherol can be studied quantitatively and can be used to compare liver protein preparations exhibiting transfer activity.
Verdon
C P
CP
USDA Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts 02111.
Blumberg
J B
JB
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008081:Liposomes / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism; D010758:Phosphorus / Q000032:analysis; D051381:Rats; D014810:Vitamin E / Q000378:metabolism*
D002352:Carrier Proteins; D008081:Liposomes; C017763:alpha-tocopherol transfer protein; D014810:Vitamin E; D010758:Phosphorus