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Beljanski V , Soulika A , Tucker JM , Townsend DM , Davis Jr W , Tew KD
Characterization of the ATPase activity of human ATP-binding cassette transporter-2 (ABCA2)
In Vivo. 2005 ;19(4) :657-660
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Abstract
Background: ABCA2 is a member of the ATP binding cassette transporter family with functional roles in cholesterol homeostasis and drug resistance. Materials and methods: In order to characterize its ATPase activity, we transfected HEK293 cells with an ABCA2 mammalian expression system and isolated ABCA2-enriched membranes. Results: We found no measurable ATPase activity of ABCA2 in isolated membranes, except in the presence of the methyl-?-cyclodextrin. However, competitive binding of a pseudo-substrate, 8-azido-[?-32P]-ATP, was demonstrated. CHO cells transfected with ABCA2 did not have a higher rate of endogenous ATP hydrolysis when compared to the mock-transfected cells. Conclusion: Overall, we conclude that, while ABCA2 may have low levels of ATPase activity that can be substrate-stimulated, it is more likely to have a regulatory role in cell physiology.
Notes
0258851X (ISSN) Cited By: 1; Export Date: 25 May 2006; Source: Scopus CODEN: IVIVE Language of Original Document: English Correspondence Address: Tew, K.D.; Medical University of South Carolina; Department of Cell and Molecular Pharmacology and Experimental Therapeutics; 173 Ashley Avenue Charleston, SC 29425, United States; email: tewk@musc.edu Chemicals/CAS: adenosine triphosphatase, 37289-25-1, 9000-83-3 References: Ambudkar, S.V., Kimchi-Sarfaty, C., Sauna, Z.E., P-glycoprotein: From genomics to mechanism (2003) Oncogene, 22, pp. 7468-7485; Bakos, E., Evers, R., Calenda, G., Characterization of the amino-terminal regions in the human multidrug resistance protein (MRP1) (2000) J. Cell Sci., 113 (PART 24), pp. 4451-4461; Bakos, E., Evers, R., Szakacs, G., Functional multidrug resistance protein (MRP1) lacking the N-terminal transmembrane domain (1998) J. Biol. Chem., 273, pp. 32167-32175; Boonstra, R., Timmer-Bosscha, H., van Echten-Arends, J., Mitoxantrone resistance in a small cell lung cancer cell line is associated with ABCA2 upregulation (2004) Br. J. Cancer, 90, pp. 2411-2417; Borst, P., Multidrug resistance: A solvable problem? (1999) Ann. Oncol., 10 (SUPPL. 4), pp. 162-164; Borst, P., Elferink, R.O., Mammalian ABC transporters in health and disease (2002) Ann. Rev. Biochem., 71, pp. 537-592; Breuninger, L.M., Paul, S., Gaughan, K., Expression of multidrug resistance-associated protein in NIH/3T3 cells confers multidrug resistance associated with increased drug efflux and altered intracellular drug distribution (1995) Cancer Res., 55, pp. 5342-5347; Chen, W., Sun, Y., Welch, C., Preferential ATP-binding cassette transporter A1-mediated cholesterol efflux from late endosomes/lysosomes (2001) J. Biol. Chem., 276, pp. 43564-43569; Cornwell, M.M., Gottesman, M.M., Pastan, I.H., Increased vinblastine binding to membrane vesicles from multidrug-resistant KB cells (1986) J. Biol. Chem., 261, pp. 7921-7928; Davis Jr., W., Boyd, J.T., Ile, K.E., Human ATP-binding cassette transporter-2 (ABCA2) positively regulates low-density lipoprotein receptor expression and negatively regulates cholesterol esterification in Chinese hamster ovary cells (2004) Biochim. Biophys. Acta, 1683, pp. 89-100; Garewal, H.S., Ahmann, F.R., Schifman, R.B., ATP assay: Ability to distinguish cytostatic from cytocidal anticancer drug effects (1986) J. Natl. Cancer Inst., 77, pp. 1039-1045; Fliggins, C.F., Linton, K.J., The ATP switch model for ABC transporters (2004) Nat. Struct. Mol. Biol., 11, pp. 918-926; Hoffmann, U., Kroemer, H.K., The ABC transporters MDR1 and MRP2: Multiple functions in disposition of xenobiotics and drug resistance (2004) Drug Metab. Rev., 36, pp. 669-701; Holland, I.B., Blight, M.A., ABC-ATPases, adaptable energy generators fuelling transmembrane movement of a variety of molecules in organisms from bacteria to humans (1999) J. Mol. Biol., 293, pp. 381-399; He, K.E., Davis Jr., W., Boyd, J.T., Identification of a novel first exon of the human ABCA2 transporter gene encoding a unique N-terminus (2004) Biochim. Biophys. Acta, 1678, pp. 22-32; Kilsdonk, E.P., Yancey, P.G., Stoudt, G.W., Cellular cholesterol efflux mediated by cyclodextrins (1995) J. Biol. Chem., 270, pp. 17250-17256; Klein, U., Gimpl, G., Fahrenholz, F., Alteration of the myometrial plasma membrane cholesterol content with beta-cyclodextrin modulates the binding affinity of the oxytocin receptor (1995) Biochemistry, 34, pp. 13784-13793; Laing, N.M., Belinsky, M.G., Kruh, G.D., Amplification of the ATP-binding cassette 2 transporter gene is functionally linked with enhanced efflux of estramustine in ovarian carcinoma cells (1998) Cancer Res., 58, pp. 1332-1337; Leonard, G.D., Fojo, T., Bates, S.E., The role of ABC transporters in clinical practice (2003) Oncologist, 8, pp. 411-424; Luciani, F., Spada, M., De Milito, A., Effect of proton pump inhibitor pretreatment on resistance of solid tumors to cytotoxic drugs (2004) J. Natl. Cancer Inst., 96, pp. 1702-1713; Luckie, D.B., Wilterding, J.H., Krha, M., CFTR and MDR: ABC transporters with homologous structure but divergent function (2003) Curr. Genomics, 4, pp. 109-121; Ramachandra, M., Ambudkar, S.V., Chen, D., Human P-glycoprotein exhibits reduced affinity for substrates during a catalytic transition state (1998) Biochemistry, 37, pp. 5010-5019; Sauna, Z.E., Smith, M.M., Muller, M., Functionally similar vanadate-induced 8-azidoadenosine 5?-[alpha-(32)P]diphosphate-trapped transition state intermediates of human P-glycoprotin are generated in the absence and presence of ATP hydrolysis (2001) J. Biol. Chem., 276, pp. 21199-21208; Szakacs, G., Langmann, T., Ozvegy, C., Characterization of the ATPase cycle of human ABCA1: Implications for its function as a regulator rather than an active transporter (2001) Biochem. Biophys. Res. Commun., 288, pp. 1258-1264; Vulevic, B., Chen, Z., Boyd, J.T., Cloning and characterization of human adenosine 5?-triphosphate-binding cassette, sub-family A, transporter 2 (ABCA2) (2001) Cancer Res., 61, pp. 3339-3347.