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J. Biol. Chem., Vol. 279, Issue 47, 48855-48864, November 19, 2004

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Multidrug Resistance Protein 4 (ABCC4)-mediated ATP Hydrolysis

EFFECT OF TRANSPORT SUBSTRATES AND CHARACTERIZATION OF THE POST-HYDROLYSIS TRANSITION STATE^*

Zuben E. Sauna, Krishnamachary Nandigama, and Suresh V. Ambudkar

From the Laboratory of Cell Biology, Center for Cancer Research, NCI, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892-4256

Multidrug resistance protein 4 (MRP4/ABCC4), transports cyclic nucleoside monophosphates, nucleoside analog drugs, chemotherapeutic agents, and prostaglandins. In this study we characterize ATP hydrolysis by human MRP4 expressed in insect cells. MRP4 hydrolyzes ATP (K_m, 0.62 mM), which is inhibited by orthovanadate and beryllium fluoride. However, unlike ATPase activity of P-glycoprotein, which is equally sensitive to both inhibitors, MRP4-ATPase is more sensitive to beryllium fluoride than to orthovanadate. 8-Azido[-³²P]ATP binds to MRP4 (concentration for half-maximal binding 3 µM) and is displaced by ATP or by its non-hydrolyzable analog AMPPNP (concentrations for half-maximal inhibition of 13.3 and 308 µM). MRP4 substrates, the prostaglandins E1 and E2, stimulate ATP hydrolysis 2- to 3-fold but do not affect the K_m for ATP. Several other substrates, azidothymidine, 9-(2-phosphonylmethoxyethyl)adenine, and methotrexate do not stimulate ATP hydrolysis but inhibit prostaglandin E2-stimulated ATP hydrolysis. Although both post-hydrolysis transition states MRP4·8-azido[-³²P]ADP·Vi and MRP4·8-azido[-³²P]ADP·beryllium fluoride can be generated, nucleotide trapping is 4-fold higher with beryllium fluoride. The divalent cations Mg²⁺ and Mn²⁺ support comparable levels of nucleotide binding, hydrolysis, and trapping. However, Co²⁺ increases 8-azido[-³²P]ATP binding and beryllium fluoride-induced 8-azido[-³²P]ADP trapping but does not support steady-state ATP hydrolysis. ADP inhibits basal and prostaglandin E2-stimulated ATP hydrolysis (concentrations for half-maximal inhibition 0.19 and 0.25 mM, respectively) and beryllium fluoride-induced 8-azido[-³²P]ADP trapping, whereas P_i has no effect up to 20 mM. In aggregate, our results demonstrate that MRP4 exhibits substrate-stimulated ATP hydrolysis, and we propose a kinetic scheme suggesting that ADP release from the post-hydrolysis transition state may be the rate-limiting step during the catalytic cycle.

Received for publication, August 3, 2004 , and in revised form, August 30, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Laboratory of Cell Biology, Center for Cancer Research, NCI, National Institutes of Health, Dept. of Health and Human Services, Bethesda, MD 20892-4256. Tel.: 301-402-4178; Fax: 301-435-8188; E-mail: ambudkar{at}helix.nih.gov.

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