Abstract
To study the molecular function of the multidrug-resistance gene product P-glycoprotein, we purified and reconstituted it into liposomes. Twelve detergents were examined in an attempt to solubilize and reconstitute the transport activity of K562/ADM membrane proteins containing P-glycoprotein. We found that transport activity was effectively reconstituted after solubilization with cholate, glycocholate and taurocholate. Other detergents, such as CHAPS, Triton X-100 and deoxycholate, diminished the transport activity. The K562/ADM membrane was solubilized by 1% glycocholate, and P-glycoprotein was purified by MRK-16 immunoaffinity column chromatography to a homogeneous single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The purified P-glycoprotein was reconstituted by detergent dialysis into liposomes composed of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. The reconstituted P-glycoprotein specifically bound [3H]azidopine and had an ATPase activity that was slightlystimulated when vincristine was added. Furthermore, though its activity was reduced, the reconstituted P-glycoprotein was shown to be an ATP-dependent transporter of vincristine.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Al-Shawi MK, Senior AE (1993) Characterization of the adenosine triphosphatase activity of Chinese hamster P-glycoprotein. J Biol Chem 268:4197–4206
Ambudkar SV, Lelong IH, Zhang J, Cardarelli CO, Gottesman MM, Pastan I (1992) Partial purification and reconstitution of the human multidrug-resistance pump: characterization of the drug-stimulatable ATP hydrolysis. Proc Natl Acad Sci USA 89:8472–8476
Boscoboinik D, Debanne MT, Atafford AR, Jung CY, Gupta RS, Epand RM (1990) Dimerization of the P-glycoprotein in membranes. Biochim Biophys Acta 1027:225–228
Chen CJ, Chin JE, Ueda K, Clark DP, Pastan I, Gottesman MM Roninson IB (1986) Internal duplication and homology with bacterial transport in the mdr1 (P-glycoprotein) gene from multidrug-resistant human cells. Cell 47:381–389
Endicott JA, Ling V (1989) The biochemistry of P-glycoproteinmediated multidrug resistance. Annu Rev Biochem 58:137–171
Gottesman MM, Pastan I (1993) Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu Rev Biochem 62:385–427
Gros P, Croop J, Housman D (1986) Mammalian multidrug resistance gene: complete cDNA sequence indicates strong homology to bacterial transport proteins. Cell 47:371–380
Hamada H, Tsuruo T (1986) Functional role for the 170- to 180-kDa glycoprotein specific to drug-resistant tumor cells as revealed by monoclonal antibodies. Proc Natl Acad Sci USA 83:7785–7789
Hamada H, Tsuruo T (1988) Purification of the 170- to 180-kilodalton membrane glycoprotein associated with multidrug resistance. J Biol Chem 263:1454–1458
Hamada H, Tsuruo T (1989) Characterization of the ATPase activity of theM r 170000 to 180000 membrane glycoprotein (P-glycoprotein) associated with multidrug resistance in K562/ADM cells. Cancer Res 48:4926–4932
Hinkle PC, Kim JJ, Racker E (1971) Ion transport and respiratory control in vesicles formed from cytochrome oxidase and phospholipids. J Biol Chem 247:1338–1339
Loo TW, Clarke DM (1994) Reconstitution of drug-stimulated ATPase activity following co-expression of each half of human P-glycoprotein as separate polypeptides. J Biol Chem 269:7750–7755
Naito M, Tsuruo T (1989) Competitive inhibition by verapamil of ATP-dependent high affinity vincristine binding to the plasma membrane of multidrug-resistant K562 cells without calcium ion involvement. Cancer Res 49:1452–1455
Naito M, Tsuruo T (1992) Functionally active homodimer of P-glycoprotein in multidrug-resistant tumor cells. Biochem Biophys Res Commun 185:284–290
Naito M, Hamada H, Tsuruo T (1988) ATP/Mg2+-dependent binding of vincristine to the plasma membrane of multidrug-resistant K562 cells. J Biol Chem 263:11887–11891
Naito M, Oh-hara T, Yamazaki A, Danki T, Tsuruo T (1992) Reversal of multidrug resistance by an immunosuppressive agent FK-506. Cancer Chemother Pharmacol 29:195–200
Saeki T, Shimabuku AM, Ueda K, Komano T (1992) Specific drug binding by purified lipid-reconstituted P-glycoprotein: dependence on the lipid composition. Biochim Biophys Acta 1107:105–110
Sarkadi B, Price EM, Boucher RC, Germann UA, Scarborough GA (1992) Expression of the human multidrug resistance cDNA in insect cells generates a high activity drug-stimulated membrane ATPase. J Biol Chem 267:4854–4858
Sehested M, Simpson D, Skovsgaard T, Buhl-Jensen P (1989) Freezefracture study of plasma membranes in wild-type and daunorubicin-resistant Ehrlich ascites tumor and P388 leukemia cells. Virchows Arch [B] 56:327–335
Shapiro AB, Ling V (1994) ATPase activity of purified and reconstituted P-glycoprotein from Chinese hamster ovary cells. J Biol Chem 269:3745–3754
Sharom FJ, Yu X, Doige CA (1993) Functional reconstitution of drug transport and ATPase activity in proteoliposomes containing partially purified P-glycoprotein. J Biol Chem 268:24197–24202
Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bisinchoninic acid. Anal Biochem 150:76–85
Tsuruo T (1988) Mechanism of multidrug resistance and implications for therapy. Jpn J Cancer Res 79:285–296
Tsuruo T, Iida-Saito H, Kawabata H, Oh-hara T, Hamada H, Utakoji T (1986) Characteristics of resistance to Adriamycin in human myelogenous leukemia K562 resistant to Adriamycin and in isolated clones. Jpn J Cancer Res 77:682–692
Author information
Authors and Affiliations
Additional information
Work dedicated to Dr. Haruo Sugano on the occasion of his 70th birthday
Rights and permissions
About this article
Cite this article
Naito, M., Tsuruo, T. Reconstitution of purified P-glycoprotein into liposomes. J Cancer Res Clin Oncol 121, 582–586 (1995). https://doi.org/10.1007/BF01197774
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01197774