©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
P-glycoprotein Is Stably Inhibited by Vanadate-induced Trapping of Nucleotide at a Single Catalytic Site (*)

(Received for publication, April 19, 1995; and in revised form, June 16, 1995)

Ina L. Urbatsch Banumathi Sankaran Joachim Weber Alan E. Senior

From the Department of Biochemistry, University of Rochester Medical Center, Rochester, New York 14642

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

P-glycoprotein (Pgp or multidrug-resistance protein) shows drug-stimulated ATPase activity. The catalytic sites are known to be of low affinity and specificity for nucleotides. From the sequence, two nucleotide sites are predicted per Pgp molecule. Using plasma membranes from a multidrug-resistant Chinese hamster ovary cell line, which are highly enriched in Pgp, we show that vanadate-induced trapping of nucleotide at a single catalytic site produces stably inhibited Pgp, with t for reactivation of ATPase activity of 84 min at 37 °C and >30 h at 4 °C. Reactivation of ATPase correlated with release of trapped nucleotide. Concentrations of MgATP and MgADP required to produce 50% inhibition were 9 and 15 µM, respectively, thus the apparent affinity for nucleotide is greatly increased by vanadate-trapping. The trapped nucleotide species was ADP. Divalent cation was required, with magnesium, manganese, and cobalt all effective; cobalt yielded a very stable inhibited species, t at 37 °C = 18 h. No photocleavage of Pgp was observed after vanadate trapping with MgATP, nor was UV-induced photolabeling of Pgp by trapped adenine nucleotide observed. Vanadate-trapping with 8-azido-ATP followed by UV irradiation caused permanent inactivation and specific labeling of Pgp. Vanadate-induced inhibition was also shown with pure, reconstituted Pgp, with similar characteristics to those in plasma membranes. Vanadate trapping overcomes technical difficulties posed by lack of high affinity nucleotide-binding site(s) or a covalent enzyme-phosphate catalytic intermediate in Pgp. The finding that vanadate trapping of nucleotide at just one site/Pgp is sufficient to give full inhibition of ATPase activity shows that the two predicted nucleotide sites can not function independently as catalytic sites.


INTRODUCTION

P-glycoprotein (Pgp) (^1)is a plasma membrane protein which endows a multidrug resistance phenotype on cells. It acts by hydrolyzing ATP at catalytic sites, which project from the cytoplasmic side of the membrane, and coupling the hydrolysis of ATP to transport of drugs or other hydrophobic molecules across the membrane to the outside of the cell (Ruetz and Gros, 1994; Sharom et al., 1993). Pgp has attracted considerable interest because of its possible role in the resistance of human cancers to chemotherapy (for recent reviews, see Endicott and Ling, 1989; Gottesman and Pastan, 1993; Gros and Buschman, 1993).

Our laboratory has been working to characterize the catalytic sites of Pgp, and in recent work we obtained a highly Pgp-enriched plasma membrane preparation from multidrug-resistant Chinese hamster ovary cells that showed substantial ATPase activity referable to Pgp (Al-Shawi and Senior, 1993). We purified the Pgp to >95% homogeneity and reconstituted it in proteoliposomes, with retention of ATPase activity (Urbatsch et al., 1994). The ATPase activity is activated and/or inhibited by a range of drugs, and it is also sensitive to the membrane lipid environment (Urbatsch and Senior, 1995) and subject to covalent inhibition by N-ethylmaleimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, 8-azido-ATP, and 2-azido-ATP (Al-Shawi et al., 1994). Several other laboratories have also demonstrated and characterized ATPase activity in plasma membrane-located Pgp (Sarkardi et al., 1992), partially purified and reconstituted Pgp (Ambudkar et al., 1992; Sharom et al., 1993), or purified, reconstituted Pgp (Shapiro and Ling, 1994).

It has been established that Pgp-ATPase activity follows simple Michaelis-Menten kinetics with a single K(MgATP) around 1 mM. Free ATP is not hydrolyzed. Catalysis is relatively nonspecific in that a wide range of magnesium nucleotides are hydrolyzed, all with relatively high K values. MgADP and MgAMPPNP show competitive inhibition with high K values, around 0.4 mM (Al-Shawi et al., 1994; Urbatsch et al., 1994). Therefore, there is no evidence from kinetic studies for a high affinity site involved in catalysis. We have applied other techniques to try to detect a tight nucleotide site(s). (^2)Direct analyses of bound ATP or ADP using the luciferin-luciferase method in Pgp-enriched plasma membranes failed to detect any bound nucleotide, either before or after incubation with 5 mM MgATP. ``Unisite''-type ATP hydrolysis assays, using concentrations of [-P]ATP stoichiometric with Pgp (conduced as described by Penefsky(1986)) showed there was little binding or hydrolysis of the ATP under these conditions. Direct binding experiments with [alpha-P]ATP or [^3H]ADP, using centrifuge columns, gel filtration, or vacuum-assisted filtration methods failed to detect significant specific binding of nucleotide to Pgp at concentrations of added nucleotide up to 100 µM. Above this concentration, nonspecific binding of nucleotide became problematic, in both plasma membrane and purified reconstituted Pgp, probably due to the particulate nature of these preparations. Finally, in an exhaustive, and we believe conclusive, series of experiments aimed at detecting a covalent enzyme-phosphate catalytic intermediate, we obtained negative results with both plasma membrane and purified reconstituted Pgp.

As we point out elsewhere, the lack of a high-affinity nucleotide-binding site or covalent enzyme-phosphate intermediate in Pgp has important mechanistic implications (Senior et al., 1995). From a technical standpoint, it also renders investigation of the enzyme mechanism difficult, e.g. by impeding direct determination of number of nucleotide-binding sites by equilibrium binding techniques, by reducing stoichiometry and specificity of labeling in (photo)affinity labeling studies, by limiting applicability of fluorescent nucleotides as reporter probes, and so forth. In several ATPase enzymes, of which myosin is a pre-eminent example (Goodno, 1979, 1982; Yount, et al., 1992), vanadate-induced trapping of nucleotides in catalytic sites, with resultant generation of a stably inhibited enzyme species, has proven extremely valuable. It provides a way of stoichiometrically and specifically binding nucleotides and nucleotide analogs in the sites, such that their dissociation rate is appreciably slowed (t values of days or even weeks have been reported). Both binding and (photo)labeling experiments are thereby facilitated (e.g. Cremo et al., 1989; Garabedian and Yount, 1991; Yount et al., 1992). Moreover, the vanadate-nucleotide-enzyme complex is thought to resemble the catalytic transition state.

The ATPase activity of both plasma membrane Pgp and purified reconstituted Pgp is inhibited when vanadate is included in the ATPase assay medium. Under these conditions, vanadate causes 50% inhibition of ATP hydrolysis at around 10 µM and complete inhibition at higher concentrations (Sarkadi et al., 1992; Ambudkar et al., 1992; Al-Shawi and Senior, 1993; Urbatsch et al., 1994). Here we carried out experiments to determine whether preincubation of Pgp with nucleotide and vanadate, followed by removal of unbound ligands, generated a species of Pgp showing long-lived inhibition of ATPase activity, as seen, for example, in myosin. We found that this was the case, and in this paper we have characterized the vanadate-induced inhibited Pgp species.


EXPERIMENTAL PROCEDURES

Preparation of Plasma Membranes

Plasma membranes were prepared from the multidrug-resistant Chinese hamster ovary cell-line (CR1R12) as described by Al-Shawi and Senior(1993). One modification was that the Parr N(2)-disruption apparatus was replaced by use of the BioNeb cell disruption system (Glas-Col, Terre Haute, IN) The cells were passed through the apparatus at 30 pounds/square inch of N(2), monitoring disruption in the microscope. Usually five passes were performed. Using this modification, yields of plasma membranes were significantly increased. The membranes contained from 15-24% (w/w) of Pgp as a fraction of total membrane protein.

Assay of Pgp-ATPase Activity

ATPase activity was measured utilizing an ATP-regenerating system according to Vogel and Steinhart (1976). Ten µg or less of membrane protein was added to 1 ml of assay medium at 37 °C containing 10 mM ATP, 12 mM MgSO(4), 3 mM phosphoenolpyruvate, 0.33 mM NADH, 10 units of lactate dehydrogenase, 10 units of pyruvate kinase, and 40 mM Tris-HCl, pH 7.4. ATP hydrolysis, recorded as absorbance decrease at 340 nm, was linear in the range between 0.33-0.01 mM NADH. Unless otherwise stated the ATPase activity was calculated from the absorbance decrease during the first 5 min which was linear in all cases. EGTA (0.1 mM) and ouabain (2 mM) were included to eliminate Ca-ATPase and Na,K-ATPase activity, respectively, and 10 µM verapamil was included to maximally stimulate the Pgp. The Pgp-ATPase activity was around 1.3 µmol ATP hydrolyzed/min/mg membrane protein. Under the assay conditions described, the plasma membranes (which are prepared in 0.25 M sucrose-containing buffer) are osmotically disrupted (Al-Shawi and Senior, 1993).

Vanadate-induced Inhibition of Pgp

Orthovanadate solutions (100 mM) were prepared from Na(3)VO(4) (Fisher Scientific) at pH 10 as described (Goodno, 1982) and boiled for 2 min before each use to break down polymeric species. At concentration leq200 µM, monomeric vanadate is the predominant species in solution (Ringel et al., 1990). Plasma membranes (10 µg) were incubated with 200 µM vanadate, 1 mM nucleotide, 3 mM MgSO(4), 10 µM verapamil, 2 mM ouabain, 0.1 mM EGTA, and 40 mM Tris-HCl, pH 7.4, in a total volume of 100 µl for 20 min at 37 °C. (Variations of the conditions are detailed in the tables and figures.) The incubations were started by addition of membranes and stopped by passage of the 100-µl samples through centrifuge columns consisting of 1 ml of Sephadex G-50 (fine) topped with a 10-mm layer of Dowex AG1-X8 (Bio-Rad) (Penefsky, 1977; Wolodko et al., 1983) equilibrated with 0.1 mM EGTA, 40 mM Tris-HCl, pH 7.4. Unless otherwise stated the centrifuge column step was carried out at 23 °C. Control experiments using [alpha-P]ATP showed that <0.002% of the applied nucleotide eluted from the columns in the absence of membranes.

Determination of Stoichiometry of Vanadate-trapped Nucleotide in Pgp

Stoichiometry of vanadate-trapped nucleotide in Pgp was calculated as the amount of radioactive nucleotide in the centrifuge column eluates after vanadate-induced inhibition divided by the amount of Pgp in the plasma membranes in the same eluates. The amount of Pgp in the membranes was measured as described by Al-Shawi et al.(1994). Briefly, the eluates were run on SDS-gels, stained with Coomassie Blue, and subjected to laser densitometry. We have established previously that this method is reliable as a means of determining the Pgp concentration in membrane samples (Al-Shawi et al., 1994). For the experiments described (Table 3, Fig. 8), the Pgp content of membranes was 24% (w/w total membrane protein). The molecular size of Pgp was taken as 141 kDa.




Figure 8: Reactivation of Pgp-ATPase activity and release of trapped [alpha-P]nucleotide. Plasma membranes were preincubated with 130 µM [alpha-P]ATP in the presence of 200 µM vanadate and 3 mM MgSO(4), with 200 mM sodium phosphate present. All other conditions were as under ``Experimental Procedures.'' Unbound ligands were removed by passage through centrifuge columns (time 0), and the eluates were incubated at 37 °C. At time intervals aliquots were passed through second centrifuge columns, and ATPase activity and bound [alpha-P]nucleotide were determined. Panel A, , ATPase activity (relative to control without vanadate); ▾, [alpha-P]nucleotide bound. The lines are non-linear least-squares regression analysis fits to the data. Panel B, release of vanadate-trapped [alpha-P]nucleotide plotted against relative ATPase activity (circles). The solid line is that expected if 100% inactivation of ATPase corresponds to trapping of 1 mol nucleotide/mol Pgp.



Thin Layer Chromatography

Nucleotides were analyzed by thin layer chromatography using polyethyleneimine-cellulose plates (Brinkmann) and developing in 0.1 M potassium phosphate adjusted to pH 3.4 with phosphoric acid.

Routine Procedures

SDS-gel electrophoresis, immunoblotting, and protein assay by bicinchoninic acid method in the presence of 1% SDS were all performed as described previously (Al-Shawi and Senior, 1993).

Materials

[alpha-P]ATP, [alpha-P]UTP, and [^3H]ADP were from Amersham Corp. Tissue culture materials were from BRL Life Technology Inc. Lactate dehydrogenase (catalog no. 127868) and pyruvate kinase (catalog. no. 109053) (both supplied in 50% glycerol) were from Boehringer Mannheim. C219 anti-Pgp monoclonal antibody was from Signet Laboratories.


RESULTS

Long-lived Inhibition of Pgp-ATPase Activity by Vanadate and Conditions for Reactivation

We previously reported that vanadate is a potent inhibitor of ATPase activity in plasma membrane and purified reconstituted Pgp (Al-Shawi and Senior, 1993; Urbatsch et al., 1994). In those experiments vanadate was present during the assay, with 10 mM MgATP, and it was found that concentrations of vanadate required for 50% inhibition were 12 or 9 µM in plasma membranes or purified reconstituted Pgp, respectively. Essentially total inhibition was obtained in both cases at 100 µM vanadate.

Here we investigated whether vanadate induced a long-lived inhibited species of Pgp, as has been described for example in the case of myosin. We first preincubated plasma membranes from the multidrug-resistant Chinese hamster ovary cell line CR1R12, which are highly enriched in Pgp, with 200 µM vanadate in the presence of 1 mM MgATP, then removed unbound ligands by passage through centrifuge columns (see ``Experimental Procedures''). Membranes in the eluates were then added to ATPase assay medium, and the Pgp-ATPase activity was found to be inhibited by geq90%. This indicated that preincubation with vanadate and MgATP induced a stably inhibited Pgp species.

Conditions for reactivation of the inhibited Pgp species were examined in Fig. 1. The closed circles show inhibited membranes added directly to the ATPase assay medium at 37 °C. ATPase activity was followed over several hours and was seen to increase exponentially with an apparent rate constant (k) of 1.4 10 s (t = 84 min). Complete recovery of ATPase activity was eventually obtained. 10 mM MgATP, as present in the ATPase assay medium, was not required for reactivation; incubation of the eluate from the centrifuge column in 0.1 mM EGTA, 40 mM Tris-Cl, pH 7.4, at 37 °C followed by assay of ATPase at various times gave the same k for reactivation of 1.4 10 s (Fig. 1, open circles).


Figure 1: Reactivation of the ATPase activity of Pgp in plasma membranes after inducing inhibition with vanadate and MgATP. Inhibited Pgp was induced by preincubation of plasma membranes with 1 mM MgATP and 200 µM vanadate at 37 °C for 20 min followed by elution through centrifuge columns in 0.1 mM EGTA, 40 mM Tris-Cl at 23 °C as described under ``Experimental Procedures.'' ▪, uninhibited control with no vanadate in preincubation; bullet, eluate from centrifuge column added to ATPase assay medium and activity determined at indicated times; , , and ▾, eluates from centrifuge columns incubated at 37, 30, and 23 °C, respectively, and aliquots assayed at indicated times; , eluate from centrifuge column incubated at 4 °C and aliquots assayed at indicated times (in this case the centrifuge column elution was also at 4 °C). The lines are nonlinear least-squares regression fits to the data.



The rate of recovery of ATPase activity was temperature dependent, with k of 6.6 10 s (t = 175 min) and 3.7 10 s (t = 311 min) at 30 and 23 °C, respectively (Fig. 1). At 4 °C the extrapolated half-life for reactivation of the inhibited Pgp-ATPase was >30 h. This is similar to the situation with myosin ATPase, which is known to form a stable transition state-like complex with vanadate ions and MgADP that has a half-life of 3 days at 4 °C (Goodno, 1982).

In the studies reported above, the inhibited Pgp species was formed by preincubation of membranes with vanadate and MgATP at 37 °C for 20 min. Similar results were obtained if room temperature was used. For the studies reported below, we used 37 °C throughout. Additionally, verapamil was routinely present during the inhibition phase of the experiments (see ``Experimental Procedures''), but omission of it did not affect the degree or time course of the inhibition.

Effect of Drugs on Reactivation of ATPase Activity

The drugs verapamil and vinblastine which stimulate Pgp-ATPase, or cyclosporin A and mitomycin D which inhibit Pgp-ATPase, were added at various concentrations to the inhibited plasma membranes in eluates from the centrifuge columns. The samples were incubated at 23 °C, and it was noted that none of the drugs affected the rate of reactivation of Pgp-ATPase.

Factors Determining Formation of the Vanadate-induced Inhibited Pgp Species in Plasma Membranes

Table 1shows that formation of the inhibited Pgp species required preincubation with nucleotide, Mg, and vanadate, and that omission of any one of these resulted in zero inhibition. Neither PPi nor P(i) nor high ionic strength (200 mM Na(2)SO(4)) prevented the inhibition when 1 mM MgATP was present; however, we did note that at lower MgATP concentrations, 200 mM P(i) did reduce inhibition somewhat (see below). ADP substituted for ATP, whereas AMP did not. Pyrophosphate did not substitute for nucleotide.



Effect of Varying Vanadate, MgATP, and MgADP Concentrations on Formation of Inhibited Pgp

Plasma membranes were preincubated with varied concentrations of vanadate in the presence of ATP (1 mM) and MgSO(4) (3 mM) and the Pgp-ATPase activity determined after removal of unbound ligands (Fig. 2). The concentration of vanadate required for half-maximal inhibition of Pgp-ATPase was 4 µM. In the absence of nucleotide, vanadate in concentrations up to 3 mM had no effect.


Figure 2: Vanadate-induced inhibition of Pgp: dependence on vanadate concentration. Plasma membranes were incubated with varied concentrations of vanadate ± 1 mM MgATP. Pgp-ATPase activity was assayed after removal of unbound ligands (see ``Experimental Procedures'').



Fig. 3, A and B show the effects of varying the concentrations of MgATP and MgADP at saturating concentrations of vanadate (200 µM). The concentrations required for half-maximal inhibition were 9 µM MgATP and 15 µM MgADP. The K for MgATP hydrolysis is 1.4 mM, and the K for competitive inhibition by MgADP is 0.4 mM (Al-Shawi and Senior, 1993), thus vanadate strongly increased the apparent affinities of Pgp for both MgATP and MgADP. With both nucleotides the inhibition phase of the curves was steep (see Fig. 3legend). This aspect was not further investigated here but is of interest for future studies.


Figure 3: Vanadate-induced inhibition of Pgp: dependence on MgATP or MgADP concentration. Plasma membranes were incubated with 200 µM vanadate, 3 mM MgSO(4), and concentrations of ATP (A) or ADP (B) as indicated. Pgp-ATPase activity was assayed after removal of unbound ligands (see ``Experimental Procedures''). The dotted line is in each case the curve expected for simple non-cooperative binding (Michaelis-Menten equation).



Time Courses of Inhibition of Pgp by Vanadate with MgATP and MgADP and of Reactivation

Inhibition of ATPase activity was followed by varying the incubation times before applying the plasma membrane samples to centrifuge columns. The incubations contained 200 µM vanadate and 200 µM nucleotide. With MgATP, inhibition was rapid, reaching completion in <10 s (Fig. 4). However, with MgADP the apparent rate constant (k) for the inhibition was 2.4 10 s (t = 4.8 min), corresponding to an apparent second-order rate constant (k) of 12 M s. The rate of inhibition seen with ADP was therefore low in comparison with the rates of simple protein-ligand association reactions, and also in comparison with the observed k/K for Pgp-ATPase (1.5 10^4M s), suggesting that a slow protein isomerization step occurred after MgADP binding, to form the inhibited species.


Figure 4: Vanadate-induced inhibition of Pgp: time course of inhibition with MgATP and MgADP. Plasma membranes were incubated with 200 µM vanadate, 3 mM MgSO(4), and 200 µM ATP or ADP at 37 °C for varied times, then passed through centrifuge columns to remove unbound ligands, and the Pgp-ATPase activity assayed. The control (100%) was incubated without vanadate. , ATP; ▾, ADP.



The rate constants for the reactivation of ATPase activity were compared (Fig. 5) after Pgp was inhibited by incubation with either MgATP or MgADP in the presence of vanadate so as to achieve maximal inhibition. They were in fact very similar (k for MgATP-inhibited Pgp = 1.4 10 s and for MgADP-inhibited Pgp = 1.5 10 s). This indicated that the inhibited Pgp species likely contained bound MgADP and vanadate, whether inhibition was induced in presence of MgATP or MgADP.


Figure 5: Reactivation of inhibited Pgp after inhibition in presence of MgATP or MgADP. Vanadate-induced inhibition was effected in the presence of 200 µM MgATP or MgADP, followed by removal of unbound ligands (see ``Experimental Procedures''). The eluates from centrifuge columns were incubated at 37 °C and ATPase assays performed at intervals. The 100% value is that of control incubated with no vanadate. and solid line, ATP; ▾ and dashed line, ADP.



Formation of the Inhibited Pgp Species by Incubation with Vanadate and a Range of Different Nucleotides

As Table 2shows, a range of nucleoside triphosphates were able, in conjunction with vanadate, to induce the inhibition of Pgp. Many of these were shown previously to be substrates for Pgp (Urbatsch et al., 1994). TNP-ATP and lin-benzo-ATP (Table 2, lines 3 and 4) are fluorescent nucleotide analogs of ATP. The potential photoaffinity labeling analogs 8-azido-ATP and BzATP (Table 2, lines 5 and 6) were both effective (as also was 8-azido-ADP, not shown in Table 2). Furthermore, it was found that if, after induction of inhibition of Pgp by vanadate with 8-azido-ATP and removal of unbound ligands, the inhibited Pgp was irradiated with UV light, no reactivation of the ATPase activity could be achieved, whereas if the samples were not irradiated, complete reactivation of the ATPase activity was possible. These data indicated that vanadate had caused trapping of 8-azido-adenine nucleotide in the catalytic sites.



Fig. 6shows that vanadate-trapped 8-azido-[alpha-P]nucleotide was, on UV irradiation, selectively (>95%) cross-linked to Pgp in the plasma membranes. Immunoblotting experiments (not shown) demonstrated that the minor labeled bands seen in Fig. 6, lane 2, of molecular sizes 65 and 100 kDa, both reacted with C219 anti-Pgp monoclonal antibody, and correspond to proteolytic fragments of Pgp as shown by Georges et al.(1991). It is evident therefore that vanadate trapping offers an excellent approach to achieving specific (photo)labeling of Pgp catalytic sites.


Figure 6: Vanadate trapping of 8-azido-ATP and subsequent photolabeling of Pgp. Plasma membranes were preincubated with 200 µM 8-azido-[P]ATP, 3 mM MgSO(4), in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 200 µM vanadate. Unbound ligands were removed by passage through centrifuge columns. The degree of Pgp-ATPase inhibition was 80% (with vanadate) or zero (without vanadate). The eluates were placed on ice and irradiated for 5 min ( = 254 nm, 5.5 milliwatts/cm^2). Samples were subjected to SDS-polyacrylamide gel electrophoresis, then to autoradiography (lanes 1 and 2) or staining with Coomassie Blue (lanes 3 and 4).



Effects of Substituting Manganese or Cobalt for Magnesium on Vanadate-induced Inhibition

All of the experiments described so far used magnesium as the metal cofactor with the nucleotide. In other systems, again notably myosin, it has been found that the substitution of manganese or cobalt leads to a further increase in the apparent affinity for the vanadate-nucleotide complex, due to decrease in dissociation rate. We first established that both MnATP and CoATP were hydrolyzed by Pgp (V(max) values relative to MgATPase were 43% for MnATP and 10% for CoATP). Fig. 7A shows the time dependence of vanadate-induced inhibition in the presence of CoATP and MnATP. Inhibition with both divalent metal ions was fast and substantial, with >95% inhibition of the ATPase activity found after preincubation with CoATP. Reactivation of the inhibited Pgp species is shown in Fig. 7B, where it is seen that reactivation for Mn was faster than for Mg, with a half-life of only 24 min at 37 °C (k 4.8 10 s). In the presence of Co, in contrast, vanadate caused very stable inhibition of the Pgp, with a half-life for reactivation of 18 h at 37 °C (k 1.1 10 s).


Figure 7: Effects of manganese and cobalt on vanadate- induced inhibition and reactivation. A, vanadate-induced inhibition in the presence of 1 mM ATP was carried out as described under ``Experimental Procedures'' except that MnSO(4) or CoSO(4) replaced MgSO(4). The time course of inhibition was determined as in Fig. 4. B, reactivation of the inhibited Pgp samples at 37 °C was followed as in Fig. 5. , manganese; ▴, cobalt.



UV Irradiation of Vanadate-MgATP-inhibited Pgp

Samples which had been preincubated with vanadate and MgATP and passed through centrifuge columns were irradiated with light at 254 nm, then examined by SDS-gel electrophoresis. It was found that no photocleavage of the polypeptide backbone of Pgp occurred (data not shown). This is different from the situation in myosin and dynein, where UV irradiation of the inhibited species cleaves the protein within the nucleotide-binding site (Cremo et al., 1990; Gibbons et al., 1987). Experiments were also carried out using [alpha-P]ATP or [alpha-P]UTP with either magnesium or cobalt ions in the preincubation phase in order to trap radioactive nucleotide in the catalytic sites, then the samples eluting from centrifuge columns were irradiated at 254 nm in order to test whether direct photolabeling of the Pgp by nucleotide occurred. No labeling of the Pgp band on SDS-gels was detected by autoradiography. Again this was different to the experience with myosin, where direct photolabeling by vanadate-trapped adenine or uridine nucleotide occurred (Garabedian and Yount, 1990, 1991)

Demonstration That Vanadate-induced Inhibition Involves Trapping of Nucleotide

Plasma membranes were preincubated with [alpha-P]ATP in the presence or absence of vanadate and magnesium ions then unbound ligands were removed by centrifuge column elution (see ``Experimental Procedures'') and the radioactivity in the eluates was determined. In initial experiments a relatively high amount of nonspecific binding by the plasma membranes was noted, which could be reduced by inclusion of 200 mM P(i) in the preincubation and centrifuge column buffer. Table 3shows representative results obtained when membranes were preincubated at three different MgATP concentrations. As noted above, at this concentration of P(i) the degree of inhibition was somewhat decreased, but, for example, at 130 µM MgATP 81% inhibition was still achieved (see Table 3legend). The major conclusion to be drawn from the table is that vanadate-induced inhibition of Pgp was accompanied by trapping of nucleotide. This was shown by the fact that only under conditions where inhibition of Pgp occurred was trapping of nucleotide seen, and that the parent AUXB1 cell-line plasma membranes, which contain essentially zero Pgp, showed no vanadate-induced nucleotide trapping. The data in Table 3confirm that the trapped nucleotide was non-exchangeable with medium nucleotide, because after ``pre-loading'' with nonradioactive ATP in the presence of vanadate, followed by incubation with radioactive ATP, there was little incorporation of radioactive ATP. Other experiments (not shown) involved preincubation of plasma membranes with radioactive MgATP and vanadate, followed by exposure to 100-fold excess nonradioactive ATP in the presence of 10 mM EDTA, or to 100-fold excess of nonradioactive MgATP. These experiments confirmed that preincubation with MgATP and vanadate produces a stably trapped nucleotide complex because no release of radioactivity occurred. The experiments in Table 3indicated that full inhibition of Pgp-ATPase occurred with trapping of approximately 1 mol nucleotide/mol Pgp. For example, in Table 3, line 2, the total trapped [alpha-P]nucleotide in CR1R12 membranes (1.5 pmol/µg protein) corresponded to 1.09 mol/mol Pgp when extrapolated to 100% inhibition of ATPase activity. Correction for the amount of nucleotide bound in the absence of vanadate, or after preloading with nonradioactive ATP, or in the parent AUXB1 membranes (Table 3, lines 1-3) reduced this value to 0.80-0.94 mol nucleotide/mol Pgp. The experiments carried out with 50 or 25 µM [alpha-P]ATP in Table 3yielded similar values. Four other experiments, not shown in Table 3, using MgATP concentrations of 10, 20, 30, and 100 µM, gave a range of 0.71 to 1.11 mol of nucleotide trapped per mol of Pgp when extrapolated to full inhibition.

Demonstration That Recovery of ATPase Activity Is Accompanied by Release of Trapped Nucleotide and Calculation of Stoichiometry of Inhibitory Trapped Nucleotide

Plasma membranes were preincubated with [alpha-P]ATP in the presence of vanadate and magnesium in order to achieve inhibition of Pgp. After removal of unbound ligands, the eluates from centrifuge columns were incubated at 37 °C to reactivate the ATPase activity of Pgp. Samples were taken at intervals for estimation of ATPase and bound nucleotide (Fig. 8A). The half-time for nucleotide release was 87 min (k = 1.3 10 s), and this was very similar to the t for reactivation of ATPase (84 min; k = 1.4 10 s).

Fig. 8B emphasizes the excellent correlation between release of vanadate-trapped nucleotide and reactivation of ATPase activity. The solid line in Fig. 8B is the expected behavior if 100% inhibition of ATPase activity occurs with trapping of 1 mol nucleotide/mol Pgp. We conclude that vanadate trapping of nucleotide at one catalytic site/Pgp molecule is sufficient to give full inhibition of ATPase activity.

Analysis of Trapped Nucleotide by Thin Layer Chromatography

Plasma membranes were incubated with vanadate and [alpha-P]MgATP as in legend to Fig. 8, then passed through centrifuge columns in 40 mM Tris-Cl, 0.1 mM EGTA, pH 7.4, at 4 °C and the eluates collected directly in ice-cold 5% trichloroacetic acid. After brief centrifugation to remove protein precipitate, the radioactive nucleotides present in the supernatants were analyzed by thin layer chromatograpy and autoradiography. The major radioactive spot corresponded to ADP in mobility, and there was no radioactive spot at the position of ATP. Therefore, when ATP is used with vanadate to induce the inhibited Pgp species, the resultant trapped nucleotide is ADP.

Purified Reconstituted Pgp Shows Vanadate-induced Inhibition

Purified reconstituted Pgp in proteoliposomes (prepared as in Urbatsch et al., 1994) were preincubated with 200 µM MgATP and 200 µM vanadate at 37 °C for 20 min (as described for plasma membranes under ``Experimental Procedures''), followed by separation from unbound ligands by liquid chromatography on Sephadex G-50 in 0.1 mM EGTA, 40 mM Tris-Cl, pH 7.4, at 4 °C (the centrifuge column technique cannot be used with proteoliposomes). This resulted in an inhibited Pgp species. ATPase activity was inhibited by >90%, and complete reactivation could be achieved at 37 °C with a half-life of 80 min, the same as was seen for plasma membrane Pgp. A detailed study of vanadate-induced inhibition of pure Pgp will be presented elsewhere.


DISCUSSION

In this study we demonstrate long-lived inhibition of Pgp-ATPase activity by vanadate. Preincubation of plasma membrane Pgp with vanadate alone caused no inhibition, yet when ATP or ADP and Mg ions were included, a stable, enzymatically inactive complex formed which could be isolated free of unbound ligands. Inhibition of ATPase activity was fully reversible with a half-life of 84 min at 37 °C, but reactivation was very slow at 4 °C with a half-life of >30 h. These observations, reminiscent of previous studies of myosin and other ATPase enzymes, indicated that ADP, magnesium, and vanadate, together form a stable, ternary, noncovalent, catalytic site complex. Further evidence supporting this conclusion was that vanadate-trapped [alpha-P]ADP was demonstrated directly in the inhibited Pgp, that release of trapped, labeled nucleotide correlated well with reactivation of the ATPase activity, and that vanadate-trapped 8-azido-nucleotide caused permanent inhibition of Pgp-ATPase activity after UV irradiation.

These findings open up avenues of experimentation which had been precluded or rendered difficult because of the low affinity with which Pgp binds nucleotides and analogs. The main advantage is that vanadate greatly increases the apparent affinity for the nucleotide. This allows, for example, efficient trapping of photolabeling analogs at the catalytic sites with removal of unbound ligand, which should greatly improve the specificity of incorporated photoaffinity probes at catalytic sites and help identify residues in the catalytic sites by protein chemical approaches. Vanadate trapping allows stoichiometry of nucleotide binding to be analyzed with much greater accuracy, and may be valuable for application of fluorescent nucleotides, for example in fluorescence energy transfer experiments.

Vanadate-induced inhibition of Pgp-ATPase was seen in both plasma membranes from the multidrug-resistant Chinese hamster ovary cell-line CR1R12 and in purified reconstituted Pgp. In this paper we have characterized features of the vanadate-induced inhibition of Pgp using plasma membranes, which provide a good source of material for multiple experiments. We demonstrate that a variety of nucleotides which are substrates also support the inhibition, in concert with earlier findings that the catalytic sites are of low nucleotide specificity. We characterize the time course and affinity of inhibition with adenine nucleotide, the dependence of inhibition on vanadate concentration, and the effects of different divalent cations. Reactivation of the inhibited Pgp was also characterized and was seen to be temperature-dependent. At 4 °C the half-life for reactivation was >30 h. Inhibition in the presence of cobalt ions was seen to lead to formation of an inhibited Pgp species with an extremely long half-life of reactivation.

Vanadate-induced trapping of radioactive nucleotide was demonstrated directly in Table 3. It appeared from these experiments that full inhibition occurred with 1 mol of nucleotide trapped per mol of Pgp. On reactivation, release of radioactive nucleotide correlated well with recovery of Pgp-ATPase activity (Fig. 8A). Fig. 8B indicated that full relief of inhibition of Pgp-ATPase occurred with release of 1 mol of trapped nucleotide/mol of Pgp.

Based on the evidence that reactivation occurred at the same rate whether inhibition was induced with MgATP or MgADP (Fig. 5) it seemed likely that MgADP was the trapped nucleotide species in both cases. Using thin layer chromatography, it was confirmed that in experiments in which [alpha-P]ATP was used to induce inhibition the resultant trapped nucleotide was [alpha-P]ADP. In myosin, the vanadate-trapped nucleotide species is also the nucleoside diphosphate (Goodno, 1979, 1982). Considering the similarity in structure between vanadate and phosphate, the PgpbulletMgMgADPbulletV(i) complex may be a stable analog of the PgpbulletMgMgADPbulletP(i) complex formed by ATP hydrolysis in Pgp.

Finally, we note that vanadate-induced inhibition was demonstrated to occur in purified, reconstituted Pgp, with characteristics the same as those seen in plasma membrane Pgp. Therefore, the procedure of vanadate trapping of nucleotide may also be applied with advantage to studies of pure Pgp.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM50156 (to A. E. S.) and Deutsche Forschungsgemeinschaft Grant Ur 45/1-1 (to I. L. U.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: Pgp, P-glycoprotein; lin-benzo-ATP, 8-amino-3-(beta-D-ribofuranosyl)imidazo[4,5-g]quinazoline-triphosphate (contains formally a benzene ring inserted between pyrimidine and imidazole rings of adenosine, linearly extending the ring system); AMPPNP, adenyl-5`-yl-imidodiphosphate; TNP-ATP, 2`,3`-O-(2,4,6)-trinitrophenyl-ATP; BzATP, 3`-O-(4-benzoyl)benzoyl-ATP.

(^2)
I. L. Urbatsch, B. Sankaran, J. Weber, and A. E. Senior, unpublished results.


ACKNOWLEDGEMENTS

We thank Sumedha Bhagat for excellent technical assistance and Dr. E. Grell (Frankfurt, Germany) for a generous gift of lin-benzo-ATP.


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