Partial purification and reconstitution of the human multidrug-resistance pump: characterization of the drug-stimulatable ATP hydrolysis. (original) (raw)

• Journal List
• Proc Natl Acad Sci U S A
• v.89(18); 1992 Sep 15
• PMC49942

Proc Natl Acad Sci U S A. 1992 Sep 15; 89(18): 8472–8476.

Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205.

Abstract

Multidrug-resistant human tumor cells overexpress the MDR1 gene product P-glycoprotein, which is believed to function as an ATP-dependent efflux pump. In this study we demonstrate that the partially purified P-glycoprotein, when reconstituted in an artificial membrane, catalyzes drug-stimulated ATP hydrolysis. Plasma membrane proteins of a human multidrug-resistant cell line, KB-V1, were solubilized with 1.4% (wt/vol) octyl beta-D-glucopyranoside in the presence of 0.4% phospholipid and 20% (vol/vol) glycerol, and the crude detergent extract was chromatographed on DEAE-Sepharose CL-6B. The 0.1 M NaCl fraction, enriched in P-glycoprotein but devoid of Na,K-ATPase, was reconstituted by the detergent-dilution method. P-glycoprotein constituted 25-30% of the reconstituted protein in proteoliposomes. ATP hydrolysis by proteoliposomes was stimulated 3.5-fold by the addition of vinblastine but was unaffected by the hydrophobic antitumor agent camptothecin, which is not transported by P-glycoprotein. The stimulatory effect of vinblastine was observed only if the protein was reconstituted in proteoliposomes, suggesting that either the substrate binding site(s) was masked by detergent or that the conformation of the soluble P-glycoprotein might not be suitable for substrate-induced activation. Several other drugs that are known to be transported by P-glycoprotein enhanced the ATPase activity in a dose-dependent manner with relative potencies as follows: doxorubicin = vinblastine greater than daunomycin greater than actinomycin D greater than verapamil greater than colchicine. The basal and vinblastine-stimulated ATPase activities were inhibited by vanadate (50% inhibition observed at 7-10 microM) but were not affected by agents that inhibit other ATPases and phosphatases. These data indicate that the P-glycoprotein, similar to other ion-transporting ATPases, exhibits a high level of ATP hydrolysis (5-12 mumol per min per mg of protein).

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• Gottesman MM, Pastan I. The multidrug transporter, a double-edged sword. J Biol Chem. 1988 Sep 5;263(25):12163–12166. [PubMed] [Google Scholar]
• Endicott JA, Ling V. The biochemistry of P-glycoprotein-mediated multidrug resistance. Annu Rev Biochem. 1989;58:137–171. [PubMed] [Google Scholar]
• Pastan I, Gottesman MM. Multidrug resistance. Annu Rev Med. 1991;42:277–286. [PubMed] [Google Scholar]
• Chen CJ, Chin JE, Ueda K, Clark DP, Pastan I, Gottesman MM, Roninson IB. Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycoprotein) gene from multidrug-resistant human cells. Cell. 1986 Nov 7;47(3):381–389. [PubMed] [Google Scholar]
• Ueda K, Clark DP, Chen CJ, Roninson IB, Gottesman MM, Pastan I. The human multidrug resistance (mdr1) gene. cDNA cloning and transcription initiation. J Biol Chem. 1987 Jan 15;262(2):505–508. [PubMed] [Google Scholar]
• Hyde SC, Emsley P, Hartshorn MJ, Mimmack MM, Gileadi U, Pearce SR, Gallagher MP, Gill DR, Hubbard RE, Higgins CF. Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport. Nature. 1990 Jul 26;346(6282):362–365. [PubMed] [Google Scholar]
• Monaco JJ, Cho S, Attaya M. Transport protein genes in the murine MHC: possible implications for antigen processing. Science. 1990 Dec 21;250(4988):1723–1726. [PubMed] [Google Scholar]
• Cornwell MM, Tsuruo T, Gottesman MM, Pastan I. ATP-binding properties of P glycoprotein from multidrug-resistant KB cells. FASEB J. 1987 Jul;1(1):51–54. [PubMed] [Google Scholar]
• Hamada H, Tsuruo T. Purification of the 170- to 180-kilodalton membrane glycoprotein associated with multidrug resistance. 170- to 180-kilodalton membrane glycoprotein is an ATPase. J Biol Chem. 1988 Jan 25;263(3):1454–1458. [PubMed] [Google Scholar]
• Azzaria M, Schurr E, Gros P. Discrete mutations introduced in the predicted nucleotide-binding sites of the mdr1 gene abolish its ability to confer multidrug resistance. Mol Cell Biol. 1989 Dec;9(12):5289–5297. [PMC free article] [PubMed] [Google Scholar]
• Rothenberg M, Ling V. Multidrug resistance: molecular biology and clinical relevance. J Natl Cancer Inst. 1989 Jun 21;81(12):907–910. [PubMed] [Google Scholar]
• Horio M, Gottesman MM, Pastan I. ATP-dependent transport of vinblastine in vesicles from human multidrug-resistant cells. Proc Natl Acad Sci U S A. 1988 May;85(10):3580–3584. [PMC free article] [PubMed] [Google Scholar]
• Shen DW, Cardarelli C, Hwang J, Cornwell M, Richert N, Ishii S, Pastan I, Gottesman MM. Multiple drug-resistant human KB carcinoma cells independently selected for high-level resistance to colchicine, adriamycin, or vinblastine show changes in expression of specific proteins. J Biol Chem. 1986 Jun 15;261(17):7762–7770. [PubMed] [Google Scholar]
• Ambudkar SV, Maloney PC. Bacterial anion exchange. Use of osmolytes during solubilization and reconstitution of phosphate-linked antiport from Streptococcus lactis. J Biol Chem. 1986 Aug 5;261(22):10079–10086. [PubMed] [Google Scholar]
• Ambudkar SV, Maloney PC. Anion exchange in bacteria: reconstitution of phosphate: hexose 6-phosphate antiport from Streptococcus lactis. Methods Enzymol. 1986;125:558–563. [PubMed] [Google Scholar]
• Maloney PC, Ambudkar SV. Functional reconstitution of prokaryote and eukaryote membrane proteins. Arch Biochem Biophys. 1989 Feb 15;269(1):1–10. [PubMed] [Google Scholar]
• D'Souza MP, Ambudkar SV, August JT, Maloney PC. Reconstitution of the lysosomal proton pump. Proc Natl Acad Sci U S A. 1987 Oct;84(20):6980–6984. [PMC free article] [PubMed] [Google Scholar]
• Schaffner W, Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. [PubMed] [Google Scholar]
• Wessel D, Flügge UI. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem. 1984 Apr;138(1):141–143. [PubMed] [Google Scholar]
• Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed] [Google Scholar]
• Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. [PMC free article] [PubMed] [Google Scholar]
• Tanaka S, Currier SJ, Bruggemann EP, Ueda K, Germann UA, Pastan I, Gottesman MM. Use of recombinant P-glycoprotein fragments to produce antibodies to the multidrug transporter. Biochem Biophys Res Commun. 1990 Jan 15;166(1):180–186. [PubMed] [Google Scholar]
• Takeyasu K, Tamkun MM, Renaud KJ, Fambrough DM. Ouabain-sensitive (Na+ + K+)-ATPase activity expressed in mouse L cells by transfection with DNA encoding the alpha-subunit of an avian sodium pump. J Biol Chem. 1988 Mar 25;263(9):4347–4354. [PubMed] [Google Scholar]
• Chen CC, Wilson TH. The phospholipid requirement for activity of the lactose carrier of Escherichia coli. J Biol Chem. 1984 Aug 25;259(16):10150–10158. [PubMed] [Google Scholar]
• Bishop L, Agbayani R, Jr, Ambudkar SV, Maloney PC, Ames GF. Reconstitution of a bacterial periplasmic permease in proteoliposomes and demonstration of ATP hydrolysis concomitant with transport. Proc Natl Acad Sci U S A. 1989 Sep;86(18):6953–6957. [PMC free article] [PubMed] [Google Scholar]
• Hamada H, Tsuruo T. Characterization of the ATPase activity of the Mr 170,000 to 180,000 membrane glycoprotein (P-glycoprotein) associated with multidrug resistance in K562/ADM cells. Cancer Res. 1988 Sep 1;48(17):4926–4932. [PubMed] [Google Scholar]
• Davidson AL, Shuman HA, Nikaido H. Mechanism of maltose transport in Escherichia coli: transmembrane signaling by periplasmic binding proteins. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2360–2364. [PMC free article] [PubMed] [Google Scholar]
• Chen AY, Yu C, Potmesil M, Wall ME, Wani MC, Liu LF. Camptothecin overcomes MDR1-mediated resistance in human KB carcinoma cells. Cancer Res. 1991 Nov 15;51(22):6039–6044. [PubMed] [Google Scholar]
• Sarkadi B, Price EM, Boucher RC, Germann UA, Scarborough GA. Expression of the human multidrug resistance cDNA in insect cells generates a high activity drug-stimulated membrane ATPase. J Biol Chem. 1992 Mar 5;267(7):4854–4858. [PubMed] [Google Scholar]
• Lyon RC, Cohen JS, Faustino PJ, Megnin F, Myers CE. Glucose metabolism in drug-sensitive and drug-resistant human breast cancer cells monitored by magnetic resonance spectroscopy. Cancer Res. 1988 Feb 15;48(4):870–877. [PubMed] [Google Scholar]
• Kaplan O, Jaroszewski JW, Clarke R, Fairchild CR, Schoenlein P, Goldenberg S, Gottesman MM, Cohen JS. The multidrug resistance phenotype: 31P nuclear magnetic resonance characterization and 2-deoxyglucose toxicity. Cancer Res. 1991 Mar 15;51(6):1638–1644. [PubMed] [Google Scholar]
• Van Belle H. Kinetics and inhibition of alkaline phosphatases from canine tissues. Biochim Biophys Acta. 1972 Nov 10;289(1):158–168. [PubMed] [Google Scholar]
• Kamimoto Y, Gatmaitan Z, Hsu J, Arias IM. The function of Gp170, the multidrug resistance gene product, in rat liver canalicular membrane vesicles. J Biol Chem. 1989 Jul 15;264(20):11693–11698. [PubMed] [Google Scholar]
• Ames GF, Nikaido K, Groarke J, Petithory J. Reconstitution of periplasmic transport in inside-out membrane vesicles. Energization by ATP. J Biol Chem. 1989 Mar 5;264(7):3998–4002. [PubMed] [Google Scholar]
• Rosen BP, Weigel U, Karkaria C, Gangola P. Molecular characterization of an anion pump. The arsA gene product is an arsenite(antimonate)-stimulated ATPase. J Biol Chem. 1988 Mar 5;263(7):3067–3070. [PubMed] [Google Scholar]

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