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J Biol Chem, Vol. 273, Issue 27, 16631-16634, July 3, 1998

COMMUNICATION
Mechanism of Action of Human P-glycoprotein ATPase Activity
PHOTOCHEMICAL CLEAVAGE DURING A CATALYTIC TRANSITION STATE USING ORTHOVANADATE REVEALS CROSS-TALK BETWEEN THE TWO ATP SITES

Christine A. Hrycyna, Muralidhara Ramachandra^¶, Suresh V. Ambudkar, Young Hee Ko^**, Peter L. Pedersen^**, Ira Pastan^¶, and Michael M. Gottesman

From the  Laboratory of Cell Biology and ^¶ Laboratory of Molecular Biology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 and ^** Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Human P-glycoprotein (P-gp), an ATP-dependent efflux pump responsible for cross-resistance of human cancers to a variety of lipophilic compounds, is composed of two homologous halves, each containing six transmembrane domains and an ATP-binding/utilization domain. To determine whether each site can hydrolyze ATP simultaneously, we used an orthovanadate (Vi)-induced ADP-trapping technique (P-gp·MgADP·Vi). In analogy with other ATPases, a photochemical peptide bond cleavage reaction occurs within the Walker A nucleotide binding domain consensus sequence (GX₄GK(T/S)) when the molecule is trapped with Vi in an inhibited catalytic transition state (P-gp·MgADP·Vi) and incubated in the presence of ultraviolet light. Upon reconstitution into proteoliposomes, histidine-tagged purified P-gp from baculovirus-infected insect cells had drug-stimulated ATPase activity. Reconstituted P-gp was incubated with either ATP or 8-azido-ATP in the presence or absence of Vi under ultraviolet (365 nm) light on ice for 60 min. The resultant products were separated by SDS-polyacrylamide gel electrophoresis and subjected to immunoblotting with seven different human P-gp-specific antibodies covering the entire length of the molecule. Little to no degradation of P-gp was observed in the absence of Vi. In the presence of Vi, products of approximately 28, 47, 94, and 110 kDa were obtained, consistent with predicted molecular weights from cleavage at either of the ATP sites but not both sites. An additional Vi-dependent cleavage site was detected at or near the trypsin site in the linker region of P-gp. These results suggest that both the amino- and carboxyl-terminal ATP sites can hydrolyze ATP. However, there is no evidence that ATP can be hydrolyzed simultaneously by both sites.

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Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

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