Cytochrome b5 and a recombinant protein containing the cytochrome b5 hydrophobic domain spontaneously associate with the plasma membranes of cells.
Both cytochrome b5, isolated from rabbit liver microsomes, and LacZ:HP, a recombinant protein consisting of enzymatically active Escherichia coli beta-galactosidase coupled to the C-terminal membrane-anchoring hydrophobic domain of cytochrome b5, were shown to spontaneously associate with the plasma membranes of erythrocytes and 3T3 cells. Association was promoted by low pH values, but proceeded satisfactorily over several hours at physiological pH and temperature. About 150,000 cytochrome b5 molecules or 100,000 LacZ:HP molecules could be associated per erythrocyte. These proteins were not removed from the membrane by extensive washing, even at high ionic strength. After incubation with fluorescently labeled cytochrome b5 or LacZ:HP, cells displayed fluorescent membranes. The lateral mobility of fluorescently labeled cytochrome b5 and LacZ:HP was measured by photo-bleaching techniques. In the plasma membrane of erythrocytes and 3T3 cells, the apparent lateral diffusion coefficient D ranged from 1.0.10(-9) to 8.10(-9) cm2 s-1 with a mobile fraction M between 0.4 and 0.6. The lateral mobility of these proteins closely resembled that reported for lipid-anchored proteins and was much higher than that reported for Band 3, an erythrocyte membrane-spanning protein with a large cytoplasmic domain. These results suggest that the hydrophobic domain of cytochrome b5 could be employed as a universal, laterally mobile membrane anchor to associate a variety of diagnostically and therapeutically useful recombinant proteins with cells.
George
S K
SK
Department of Medical Biochemistry and Genetics, Texas A&M College of Medicine, College Station.
Xu
Y H
YH
Benson
L A
LA
Pratsch
L
L
Peters
R
R
Ihler
G M
GM
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D015786:Cytochromes b5 / Q000737:chemistry / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D046911:Macromolecular Substances; D008560:Membrane Fluidity / Q000502:physiology; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013696:Temperature; D013861:Thiocyanates; D014966:Xanthenes / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
D005452:Fluoresceins; D005456:Fluorescent Dyes; D008081:Liposomes; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D013861:Thiocyanates; D014966:Xanthenes; C034657:Texas red; D015786:Cytochromes b5; D001616:beta-Galactosidase; D016650:Fluorescein-5-isothiocyanate