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Yeungnam Univ J Med > Volume 3(1); 1986 > Article
Yeungnam University Journal of Medicine 1986;3(1):41-48.
DOI: https://doi.org/10.12701/yujm.1986.3.1.41    Published online December 31, 1986.
Purification of Band 3 from the Human Erythrocyte Membrane and its Incorporation into Liposome.
Jae Ryong Kim, Jung Hye Kim, Ki Yung Lee
Abstract
Band 3, the predominant 95,000 dalton anion transport protein, is the major intrinsic glycoprotein of the human erythrocyte membrane. This anion carrier exists as a dimer and binds the cytoskeletons such as spectrin, ankyrin and actin. And the liposomes are vesicular structures which form spontaneously upon hydration of phospholipids. These artificial lipid vesicles have been investigated as model of the biological membranes and as a mean of improving the delivery of nucleic acids, drugs, proteins and biological substances to specific target tissues and cells. In this study, we were purified Band 3 from the human erythrocyte membrane (ghost) was prepared by hemolysis of intact human erythrocyte with weak alkali-hypotonic solution. Band 6 was removed from ghost by extracting with solution of an ionic strength of 0.15. Band 3 and Band 4 were solubilized selectively by extracting Band 6-depleted ghosts with Triton X-100 under nondenaturing conditions. Band 3 was then purified from Triton X-100 extract treated with p-chloromercuribenzoate by sucrose density gradient ultracentrifugation. This purified Band 3 was incorporated into liposomes prepared by reverse-phase evaporation. Phosphatidyl L-serine and cholesterol (1:1 molar ratio) were dissolved in chloroform and the chloroform was removed by rotator evaporation under reduced pressure. Band 3 solution without Triton X-100 was introduced into a mixture of lipids and diethylether. Diethylether was subsequently removed by evaporation. This purified Band 3 and its incorporation into liposomes were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
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