Characterization of the adenosine triphosphatase activity of Chinese hamster P-glycoprotein.

M.K Al-Shawi,A.E Senior

doi:10.1016/s0021-9258(18)53597-3

Abstract

A multidrug-resistant Chinese hamster ovary cell line (CR1R12) was obtained which constitutively expresses P-glycoprotein, up to 32% by weight of plasma membrane protein. CR1R12 plasma membranes had high, drug-activated ATPase activity referable to P-glycoprotein. The specific ATPase activity in the presence of verapamil was calculated to be approximately 9 mumol/min/mg (identical to 21 s-1) at 37 degrees C, pH 7.4. KM ATP was 1.4 mM, and ADP and 5'-adenylyl imidodiphosphate were competitive inhibitors with Ki values 0.35 and 0.44 mM, respectively. 2'-dATP was a good substrate, GTP and ITP were real but poor substrates, and ADP and AMP were not hydrolyzed. Optimal pH for ATP hydrolysis was 7.3. MgATP was the preferred substrate, and CaATP was hydrolyzed very weakly. 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) covalently labeled the P-glycoprotein, and incorporation of 1.1 mol of NBD-Cl/mol of P-glycoprotein gave 100% inactivation. ATP protected against NBD-Cl inactivation. N-Ethylmaleimide was a potent inhibitor in the absence of ATP, and in its presence significant protection from inhibition could be achieved. Vanadate and fluoroaluminate were also strong inhibitors. The plasma membranes from CR1R12 cells should provide material for purification and reconstitution of P-glycoprotein and for screening of potential "multidrug-reversal" reagents by enzymic assay.

Highlights

IntroductionK M ATP was 1.4 mM, glycoprotein (Azzariaet al., 1989)
Summarizing this section, we find a large ATPase activity in plasma membranes of multidrug-resistant cell lines that is not due to Ca-ATPase, Na,K-ATPase, ecto-ATPase, or mitochondrial ATPase and is correlated with the amounts of Pglycoprotein present in the membranes
Tein in amounts up to 32% by weight of total plasma membrane protein. This cell line grows well in suspension culture, providing a good source of plasma membranes enriched in Pglycoprotein for enzymatic and mechanistic studies, and potentially for purification and reconstitution studies

Summary

Introduction

K M ATP was 1.4 mM, glycoprotein (Azzariaet al., 1989). All of these resultsstrongly and ADP and5”adenylyl imidodiphosphatewere competitive inhibitors with K i values 0.35 and 0.44 mM, respectively. MgATP was thepreferredsubstrate,and suggest that P-glycoprotein is an ATP-dependent drug-exporting transport protein. The plasma ilarly, immunoprecipitated human P-glycoproteinlfl-galactomembranes from CRlR12 cells should provide mate- sidase fusion protein showed a relatively low specific activity rial for purification and reconstitution of P-glycopro-(turnover = 0.77 s-’) (Shimabuku et al, 1992).Recently, tein and for screening of potential “multidrug-rever- Sarkadi et al (1992) expressed human P-glycoprotein in Sf9 sal” reagents by enzymic assay. P-glycoprotein was calculated to constitute about 3% by weight of the total plasma membrane proteins, andby extrapolation it was cal-

Methods

Results

Conclusion