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(Received for publication, February 27, 1996, and in revised form, June 17, 1996)
From the Insitut de Biologie et Chimie des Protéines, UPR 412 CNRS, 7 Passage du Vercors, F-69367 Lyon Cedex 07, France
Plasma membrane P-glycoprotein is known as an
ATP-dependent drug efflux pump that confers multidrug
resistance to tumor cells. None of the reported purification
procedures worked properly for our P-glycoprotein-overproducing cell
lines, i.e. murine lymphoid leukemia P388/ADR25, rat
hepatoma AS30-D/COL10, and human lymphoblastic leukemia CEM/VLB5 cells.
We have thus developed a general procedure for efficient purification
of P-glycoprotein by combining solubilization with sodium dodecyl
sulfate and chromatography on ceramic hydroxyapatite. This procedure
was successful for the three cell lines and yielded 70% of the
P-glycoprotein present in the starting plasma membranes with more than
99% purity. After exchanging sodium dodecyl sulfate into dodecyl
maltoside and reconstitution into liposomes, purified P-glycoprotein
exhibited a specific ATPase activity of about 200 nmol/min/mg, which
was very similar to that obtained for P-glycoprotein solubilized and
purified with
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
This ATPase activity was sensitive to orthovanadate inhibition and
stimulated by verapamil and other drugs. More importantly, drug
transport properties of the reconstituted P-glycoprotein were
comparable with those of P-glycoprotein embedded in plasma membranes.
Since it is virtually devoid of lipids, this preparation is suitable
for both functional and structural investigations.
Volume 271, Number 46,
Issue of November 15, 1996
pp. 28875-28883
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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