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Characterization of the Adenosine Triphosphatase Activity of the Periplasmic Histidine Permease, a Traffic ATPase (ABC Transporter)

1997; Elsevier BV; Volume: 272; Issue: 35 Linguagem: Inglês

10.1074/jbc.272.35.21883

1083-351X

Cheng Eureka Liu, Peiqi Liu, Giovanna Ferro‐Luzzi Ames,

Advanced biosensing and bioanalysis techniques

The superfamily of traffic ATPases (ABC transporters) includes bacterial periplasmic transport systems (permeases) and eukaryotic transporters. The histidine permease ofSalmonella typhimurium is composed of a membrane-bound complex (HisQMP2) containing four subunits, and of a soluble receptor, the histidine-binding protein (HisJ). Transport is energized by ATP. In this article the ATPase activity of HisQMP2 has been characterized, using a novel assay that is independent of transport. The assay uses Mg2+ ions to permeabilize membrane vesicles or proteoliposomes, thus allowing access of ATP to both sides of the bilayer. HisQMP2 displays a low level of intrinsic ATPase activity in the absence of HisJ; unliganded HisJ stimulates the activity and liganded HisJ stimulates to an even higher level. All three levels of activity display positive cooperativity for ATP with a Hill coefficient of 2 and aK0.5 value of 0.6 mm. The activity has been characterized with respect to pH, salt, phospholipids, substrate, and inhibitor specificity. Free histidine has no effect. The activity is inhibited by orthovanadate, but not byN-ethylmaleimide, bafilomycin A1, or ouabain. Several nucleotide analogs, ADP, 5′-adenylyl-β,γ-imidodiphosphate, adenosine 5′-(β,γimino)triphosphate, and adenosine 5′-O-(3-thio)triphosphate, inhibit the activity. Unliganded HisJ does not compete with liganded HisJ for the stimulation of the ATPase activity of HisQMP2. The superfamily of traffic ATPases (ABC transporters) includes bacterial periplasmic transport systems (permeases) and eukaryotic transporters. The histidine permease ofSalmonella typhimurium is composed of a membrane-bound complex (HisQMP2) containing four subunits, and of a soluble receptor, the histidine-binding protein (HisJ). Transport is energized by ATP. In this article the ATPase activity of HisQMP2 has been characterized, using a novel assay that is independent of transport. The assay uses Mg2+ ions to permeabilize membrane vesicles or proteoliposomes, thus allowing access of ATP to both sides of the bilayer. HisQMP2 displays a low level of intrinsic ATPase activity in the absence of HisJ; unliganded HisJ stimulates the activity and liganded HisJ stimulates to an even higher level. All three levels of activity display positive cooperativity for ATP with a Hill coefficient of 2 and aK0.5 value of 0.6 mm. The activity has been characterized with respect to pH, salt, phospholipids, substrate, and inhibitor specificity. Free histidine has no effect. The activity is inhibited by orthovanadate, but not byN-ethylmaleimide, bafilomycin A1, or ouabain. Several nucleotide analogs, ADP, 5′-adenylyl-β,γ-imidodiphosphate, adenosine 5′-(β,γimino)triphosphate, and adenosine 5′-O-(3-thio)triphosphate, inhibit the activity. Unliganded HisJ does not compete with liganded HisJ for the stimulation of the ATPase activity of HisQMP2. The superfamily of traffic ATPases (or ABC transporters) comprises both prokaryotic and eukaryotic transport proteins which share a conserved nucleotide-binding domain (1Ames G.F.-L. Mimura C. Shyamala V. FEMS Microbiol. Rev. 1990; 75: 429-446Crossref Google Scholar, 2Hyde S.C. Emsley P. Hartshorn M.J. Mimmack M.M. Gileadi U. Pearce S.R. Gallagher M.P. Gill D.R. Hubbard R.E. Higgins C.F. Nature. 1990; 346: 362-365Crossref PubMed Scopus (949) Google Scholar). The superfamily includes bacterial periplasmic permeases, the yeast STE6 gene product, the mammalian P-glycoprotein (multidrug resistance protein or MDR), 1The abbreviations used are: MDR, multidrug resistance protein; HisQMP2, HisQ·HisM·HisP, membrane-bound complex of the histidine permease; PLS, proteoliposomes reconstituted from solubilized membrane vesicles, unless specified differently; CF, 5- (and 6)-carboxyfluorescein; PAGE, polyacrylamide gel electrophoresis; PE, phosphatidylethanolamine; AMP-PNP, 5′-adenylyl-β,γ-imidodiphosphate; AMP-PCP, adenosine 5′-(β,γ-imino)triphosphate; ATPγS, adenosine 5′-O-(3-thio)triphosphate; MOPS, 4-morpholinepropanesulfonic acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol; TNP-ATP, 2′-(3′)-D-(trinitrophenyl)adenosine 5′-triphosphate.1The abbreviations used are: MDR, multidrug resistance protein; HisQMP2, HisQ·HisM·HisP, membrane-bound complex of the histidine permease; PLS, proteoliposomes reconstituted from solubilized membrane vesicles, unless specified differently; CF, 5- (and 6)-carboxyfluorescein; PAGE, polyacrylamide gel electrophoresis; PE, phosphatidylethanolamine; AMP-PNP, 5′-adenylyl-β,γ-imidodiphosphate; AMP-PCP, adenosine 5′-(β,γ-imino)triphosphate; ATPγS, adenosine 5′-O-(3-thio)triphosphate; MOPS, 4-morpholinepropanesulfonic acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol; TNP-ATP, 2′-(3′)-D-(trinitrophenyl)adenosine 5′-triphosphate. the human cystic fibrosis transmembrane conductance regulator (CFTR), and the mammalian heterodimeric transporter (TAP1/TAP2) involved in antigen processing (3Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3323) Google Scholar, 4Doige C.A. Ames G.F.-L. Annu. Rev. Microbiol. 1993; 47: 291-319Crossref PubMed Scopus (264) Google Scholar). The periplasmic histidine permease in Salmonella typhimurium and the maltose permease in Escherichia coli have been extensively characterized; both are good model systems for this superfamily (5Shyamala V. Baichwal V. Beall E. Ames G.F.-L. J. Biol. Chem. 1991; 266: 18714-18719Abstract Full Text PDF PubMed Google Scholar, 6Liu C.E. Ames G.F.-L. J. Biol. Chem. 1997; 272: 859-866Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 7Nikaido H. FEBS Lett. 1994; 346: 55-58Crossref PubMed Scopus (64) Google Scholar). The histidine permease is composed of a soluble substrate-binding receptor, HisJ (the periplasmic histidine-binding protein), and a membrane-bound complex, HisQMP2 (8Kerppola R.E. Shyamala V.K. Klebba P. Ames G.F.-L. J. Biol. Chem. 1991; 266: 9857-9865Abstract Full Text PDF PubMed Google Scholar), which contains two integral membrane proteins, HisQ and HisM, and two copies of HisP which carries the ATP-binding motif (9Mimura C.S. Admon A. Hurt K.A. Ames G.F.-L. J. Biol. Chem. 1990; 265: 19535-19542Abstract Full Text PDF PubMed Google Scholar, 10Mimura C.S. Holbrook S.R. Ames G.F.-L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 84-88Crossref PubMed Scopus (139) Google Scholar). ATP hydrolysis provides the energy for the transport process (11Ames G.F.-L. Krulwich T.A. Bacterial Energetics. Academic Press, Inc., New York1990: 225-245Crossref Google Scholar). The histidine permease has been reconstituted into PLS that transport histidine when ATP is trapped internally and HisJ is added externally (6Liu C.E. Ames G.F.-L. J. Biol. Chem. 1997; 272: 859-866Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 12Bishop L. Agbayani R.J. Ambudkar S.V. Maloney P.C. Ames G.F.-L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6953-6957Crossref PubMed Scopus (125) Google Scholar). As usually assumed in current working models, the liganded receptor sends a signal that initiates ATP hydrolysis and results in subsequent ligand translocation (13Ames G.F.-L. Liu C.E. Joshi A.K. Nikaido K. J. Biol. Chem. 1996; 271: 14264-14270Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 14Davidson A.L. Shuman H.A. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2360-2364Crossref PubMed Scopus (231) Google Scholar). The characterization of the ATPase activity, which is likely to be common to all members of this superfamily of transporters because of the extensive sequence conservation, is crucial for understanding their mechanism of action. ATP hydrolysis has generally been ascribed to the nucleotide-binding domain. Therefore, several laboratories took the approach of purifying this domain (or component) for the purpose of studying its activity in vitro. MalK, following its solubilization with urea and purification from inclusion bodies, has ATP binding and hydrolyzing activity (15Walter C. Honer zu Bentrup K. Schneider E. J. Biol. Chem. 1992; 267: 8863-8869Abstract Full Text PDF PubMed Google Scholar). Other nucleotide-binding components have been purified by a variety of methods and demonstrated to hydrolyze ATP (16Koronakis V. Hughes C. Koronakis E. Mol. Microbiol. 1993; 8: 1163-1175Crossref PubMed Scopus (78) Google Scholar, 17Richarme G. El Yaagoubi A. Kohiyama M. J. Biol. Chem. 1993; 268: 9473-9477Abstract Full Text PDF PubMed Google Scholar). However, the activity of the purified subunits in general did not respond to stimulation by the respective soluble receptors. Thus, the interaction with receptors and substrates and the regulation of the ATPase activity must involve the integral membrane components; it is therefore necessary to characterize the properties of the intact membrane-bound complex. In this paper we describe a detailed study of the ATPase activity of the histidine permease reconstituted in PLS, using a novel assay in which the lipid bilayer has been rendered permeable to both ATP and the receptor. It is shown that HisQMP2 has an intrinsic low level ATPase activity in the absence of HisJ. The activity is stimulated slightly by unliganded HisJ and to a high level by liganded HisJ. Both the intrinsic and HisJ-stimulated activities display positive cooperativity for ATP. The biochemical characteristics of the activity have been defined. The following E. coli K12 strains were used: TA1889, which carries deletionunc702 (that eliminates the F0F1-ATPase) and harbors plasmid pFA17 (which contains the S. typhimurium hisQ, hisM, andhisP genes under the temperature-sensitive control of the λPL promoter (18Hobson A. Weatherwax R. Ames G.F.-L. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7333-7337Crossref PubMed Scopus (70) Google Scholar)); GA298 which is derived from TA1889 by loss of pFA17. Ether/acetone-precipitatedE. coli total phospholipids (Avanti Polar Lipids Inc.) were resuspended at a concentration of 50 mg/ml in argon-saturated, 2 mm 2-mercaptoethanol, briefly sonicated with a tip sonicator to obtain a viscous homogeneous suspension, and then stored in liquid nitrogen in aliquots. Before use, phospholipids were defrosted on ice and sonicated in 30-s intervals, with cooling in between, until the suspension became clear and no longer viscous. Phosphatidylethanolamine (PE; from E. coli; Avanti Polar Lipids), phosphatidylglycerol (16:0), phosphatidylserine (16:0), phosphatidic acid (16:0), lysophosphatidylethanolamine (16:0), and lysophosphatidic acid (18:1, Cis:9) (all from Sigma) were obtained as dry powder and treated in the same way as total phospholipids, except that the final concentration was 10 mg/ml. Cardiolipin (from E. coli; Sigma) was obtained as a suspension in chloroform; aliquots of cardiolipin were dried under a stream of nitrogen, resuspended in 2 mm 2-mercaptoethanol at a final concentration of 10 mg/ml, and sonicated to clarity. The concentration of phospholipids was determined by assaying total phosphate (19Ames B.N. Dubin D.T. J. Biol. Chem. 1960; 235: 769-775Abstract Full Text PDF PubMed Google Scholar, 20Bartlett G.R. J. Biol. Chem. 1959; 234: 466-468Abstract Full Text PDF PubMed Google Scholar). Membrane vesicles and PLS were prepared as described earlier (6Liu C.E. Ames G.F.-L. J. Biol. Chem. 1997; 272: 859-866Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), from strain TA1889 unless specified differently. ATPase reaction mixtures contained, in a final volume of 330 μl, either membrane vesicles (10 μl, containing 20 mg/ml protein) or PLS (100 μl, containing 0.4 and 10 mg/ml protein and phospholipid, respectively; or 20 μl of PLS reconstituted from purified HisQMP2, containing 0.2 and 10 mg/ml protein and phospholipid, respectively), and 50 mm MOPS/K+buffer, pH 7.5. Total E. coli phospholipids (3 mg/ml final concentration) were added when the assay was performed with either membrane vesicles or PLS reconstituted with purified HisQMP2. HisJ and histidine were added at the indicated concentrations. The reaction mixture was preincubated at 37 °C for 3 min in a water bath and the assay initiated by the addition of ATP together with MgSO4 (2 and 10 mm final, respectively, unless specified differently). At the indicated times, aliquots (100 μl) were mixed with equal volumes of 12% SDS. The rate of ATP hydrolysis was determined by assaying the Piliberated (21Chifflet S. Anal. Biochem. 1988; 168: 1-4Crossref PubMed Scopus (407) Google Scholar), using a NaH2PO4 calibration curve, up to 20 nmol of Pi. CF fluorescence was measured with a Perkin-Elmer LS50B Luminescence spectrofluorimeter, using excitation and emission wavelengths of 490 and 515 nm, respectively, and 5.0-nm slit widths for both excitation and emission. SDS-PAGE was on 12.5% gels with the pH of the resolving gel adjusted to 8.65 (18Hobson A. Weatherwax R. Ames G.F.-L. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7333-7337Crossref PubMed Scopus (70) Google Scholar, 22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). Protein concentration was determined by a modified Lowry procedure (23Peterson G.L. Anal. Biochem. 1977; 83: 346-356Crossref PubMed Scopus (7067) Google Scholar) using bovine serum albumin as a standard and without the trichloroacetic acid precipitation step. HisJ was purified as described (24Prossnitz E. Gee A. Ames G.F.-L. J. Biol. Chem. 1989; 264: 5006-5014Abstract Full Text PDF PubMed Google Scholar) and the unliganded and liganded forms were separated by high performance liquid chromatography (25Nikaido K. Ames G.F.-L. J. Biol. Chem. 1992; 267: 20706-20712Abstract Full Text PDF PubMed Google Scholar). Chemicals were purchased from the following sources: CF (high purity mixed isomers), Molecular Probes, Eugene, OR; protamine (free base, from salmon), Sigma; n-octyl-β-d-glucopyranoside,n-decanoylsucrose, and C12E8, Calbiochem; n-dodecyl-β-d-maltoside, Boehringer Mannheim; Deriphat, Miranol, and Hega-11 (undecanoyl-N-hydroxyethylglucamide), Anatrace, Maumee, OH; bafilomycin A1, Calbiochem; ouabain, andN-ethylmaleimide, Sigma; and AMP-PNP, AMP-PCP, and ATPγS, Calbiochem. The soluble receptor HisJ must initiate the ATPase activity of HisQMP2 in vivo by sending a signal across the membrane bilayer, because HisJ is located in the periplasm and the ATP-binding domain of HisP is located in the cytosol. It has already been shown that if HisJ and ATP are present on opposite sides of the PLS, either with ATP trapped internally and HisJ added externally (12Bishop L. Agbayani R.J. Ambudkar S.V. Maloney P.C. Ames G.F.-L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6953-6957Crossref PubMed Scopus (125) Google Scholar), or vice versa (6Liu C.E. Ames G.F.-L. J. Biol. Chem. 1997; 272: 859-866Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), the ATPase activity is stimulated by liganded HisJ. The finding that HisJ can stimulate the activity when present on either side of the PLS is due to the fact that under the conditions of reconstitution HisQMP2 is embedded in PLS in roughly equal amounts in the right side-out and inside-out orientations (6Liu C.E. Ames G.F.-L. J. Biol. Chem. 1997; 272: 859-866Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Because HisP has been shown to be accessible from the periplasmic side of the membrane bilayer (26Baichwal V. Liu D. Ames G.F.-L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 620-624Crossref PubMed Scopus (68) Google Scholar), it is possible that external ATP can reach the cytoplasmic ATP-hydrolyzing domain of HisP. To test this possibility, PLS containing HisQMP2 were prepared (6Liu C.E. Ames G.F.-L. J. Biol. Chem. 1997; 272: 859-866Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) and their ATPase activity examined when both liganded HisJ and ATP were presented to the outside of PLS. Liganded HisJ does not stimulate significantly the activity (Fig. 1 A, columns 1and 2), indicating that external ATP does not have access to internal hydrolyzing site(s). To analyze the HisJ-stimulated ATPase activity, HisJ and ATP can be placed on opposite sides of PLS, with either one being trapped internally. However, in both situations there are inherent problems. If ATP is trapped internally and HisJ is added externally, an accurate measurement of initial rates is very difficult because the internal ATP pool is rapidly exhausted 2Only a small number of ATP molecules can be trapped into PLS at ATP concentrations around itsKm value (20 molecules per PLS vesicle at 0.5 mm ATP). This calculation assumes an average PLS diameter of 50 nm (6Liu C.E. Ames G.F.-L. J. Biol. Chem. 1997; 272: 859-866Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) and therefore an average volume of 6.54 × 10−8 per vesicle. Note that not all of the trapped molecules are necessarily accessible because some PLS may be multilamellar.and the accumulated ADP inhibits the reaction (6Liu C.E. Ames G.F.-L. J. Biol. Chem. 1997; 272: 859-866Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Alternatively, trapping HisJ inside the PLS and supplying ATP externally (6Liu C.E. Ames G.F.-L. J. Biol. Chem. 1997; 272: 859-866Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 14Davidson A.L. Shuman H.A. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2360-2364Crossref PubMed Scopus (231) Google Scholar) presents several drawbacks: the procedure for trapping the receptor is cumbersome, a large amount of receptor protein is required, and a sizable fraction of the complexes must be reproducibly inserted in the inverted orientation; furthermore, because only one or two molecules of receptor can be trapped per PLS vesicle, 3At 10, 20, and 40 μm receptor concentration, an average of 0.4, 0.8, and 1.6 receptor molecules would be trapped inside each vesicle (same calculation and caveat as in Footnote 2). it would be difficult to study the kinetics of the interaction between the receptor and the complex. Therefore, a permeabilizing procedure that renders ATP and HisJ freely accessible to both sides of PLS is desirable. Total membrane solubilization would be a possible approach. We tested numerous detergents, 4n-Octyl-β-d-glucopyranoside,n-dodecyl-β-d-maltoside,n-decanoylsucrose, C12E8, Deriphat, Miranol, C-Hega-11, cholic acid, Triton X-100. but they all inhibited the ATPase activity. Mg2+ ions, at an appropriate concentration, were found to allow hydrolysis by PLS of externally added ATP in the presence of HisJ, thus suggesting that they permeabilize PLS. This possibility was explored using CF as an indicator of PLS permeability. CF can be trapped inside PLS at high concentration, which causes quenching of its fluorescence; if PLS are permeabilized, the fluorescence would be reactivated when the CF concentration drops as a consequence of its release into the medium (27Driessen A.J. Konings W.N. Methods Enzymol. 1993; 221: 394-408Crossref PubMed Scopus (27) Google Scholar). Fig. 1 Bshows that 10 mm MgSO4 induces 100% release of CF from PLS, which indicates that PLS have been permeabilized. The effect is due to Mg2+ ions rather than to SO42− ions, as shown by the fact that MgCl2 permeabilizes as well as MgSO4 (data not shown), while neither Na2SO4 nor NaCl have any effect. CaCl2 causes complete CF release at concentrations lower than those of MgSO4. 5CaCl2 is not routinely used for permeabilizing PLS because it interferes with the ATPase assay. The presence of protein is not required for permeabilization because similar results are obtained with liposomes as well (data not shown). ATP and histidine are also released by exposure to 10 mmMgSO4, demonstrating that this effect is not limited to CF. Large molecules, such as HisJ, can be released as well from PLS by 10 mm MgSO4 (up to 80% of the total), as determined by comparing the amounts of HisJ released from PLS before and after treatment with increasing concentrations of MgSO4, using SDS-PAGE. When Mg2+ ions are mixed with equimolar concentrations of EDTA or ATP before addition to PLS, no permeabilization is observed, indicating that free Mg2+ions are necessary for permeabilization. For the MgSO4 treatment to be useful for the ATPase assay, its effect should be very rapid. Within 9 s after the addition of 10 mm MgSO4, CF release is complete; this is probably an overestimate because it is technically impossible to take samples at earlier times. An analysis of the effect of temperature shows that permeabilization diminishes as the temperature is lowered below 37 °C (Fig. 1 C) and at 0 °C the permeabilization effect is eliminated (for unknown reasons). All experiments in this paper were conducted at 37 °C. In conclusion, although the basis of the Mg2+ permeabilizing effect was not understood, this method appeared to be very useful for assaying the ATPase activity in PLS and, as shown later, also in membrane vesicles. Fig. 1 D (squares) shows that the use of MgSO4 as a permeabilizing agent allows assay of ATP hydrolysis by PLS. The rate of hydrolysis in the presence of liganded HisJ is linear (up to at least 30 min). Fig. 1 E shows that the initial rate of hydrolysis is directly proportional to the volume of PLS added (with the phospholipid to protein ratio maintained constant at 50:1, w/w), and therefore, to PLS protein and phopholipid concentrations. This experiment also shows that a phospholipid concentration of 0.16 mg/ml is compatible with complete permeabilization. PLS were reconstituted with purified HisQMP2 to determine the effect of varying protein concentration while maintaining a constant phospholipid concentration (3 mg/ml). Fig. 1 F shows that the rate of hydrolysis is proportional to protein concentration also in this case, in which the ratio of phospholipid to protein varies from 350:1 to 7000:1 (w/w). Thus, under the standard assay conditions, the activity depends directly on the protein concentration and is not affected by the phospholipid concentration. The effect of varying the Mg2+ concentration on the rate of hydrolysis is shown in Fig. 1 A. When Mg2+ is in excess over ATP and in the absence of HisJ, the hydrolysis rate is 3-fold higher than when they are equimolar (compare columns 1 and 3). In the presence of liganded HisJ, the relative increase is 15-fold (compare columns 2 and4). In both cases, the increase in activity is due to the exposure of ATP to internal ATP-hydrolyzing sites by the permeabilization procedure. The larger increase in the presence of liganded HisJ is due to the fact that both internal ATP-hydrolyzing sites and internal HisJ contact sites become accessible to ATP (for hydrolysis) and HisJ (for signaling); thus, the stimulatory effect of liganded HisJ is the factor being measured. The increase in activity in the presence of MgSO4 is not due to Pi being released from PLS because the activity is dependent on the presence of ATP (data not shown). Fig. 1 D indicates that ATP is continuously replenished inside PLS, i.e. PLS are permeable throughout the length of the assay. If permeabilization were transient, the activity would rapidly decrease in time because of ATP exhaustion and accumulation of inhibitory ADP (as is the case in non-permeabilized PLS (6Liu C.E. Ames G.F.-L. J. Biol. Chem. 1997; 272: 859-866Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar)). Alternatively, permeabilization might be transient, while giant PLS might be formed which trap sufficient ATP for sustained hydrolysis. If this were true, chelation of Mg2+ by EDTA should have no effect. However, ATP hydrolysis stops promptly upon addition of EDTA (Fig. 1 D, diamonds). The latter experiment also indicates that permeabilization can be reversed. Three levels of ATPase activity are detected in PLS: 50, 100, and 370 nmol/min/mg protein in the absence of HisJ, and in the presence of HisJ and liganded HisJ, respectively (Fig. 2). The low level ATP hydrolysis observed in the absence of HisJ may be due either to contaminating ATPases or to an intrinsic activity of HisQMP2, or a combination of both. To differentiate between these possibilities, PLS containing various amounts of HisQMP2 in the presence of identical amounts of the other proteins were prepared. A culture of TA1889 was heat induced for different lengths of time and the concentration of HisQMP2in PLS present at each of the induction times was determined by SDS-PAGE and immunoblotting using antibodies raised against HisP and HisQ. Fig. 3 (A andB) shows that, as expected, the amount of HisQMP2 in PLS increases as the heat induction time increases (lanes 5–8). The ATPase activity in these PLS tested in the absence of HisJ increases in parallel (Fig. 3 C, open squares). To determine whether the heat induction of contaminating ATPases contributes to this activity, PLS were prepared from strain GA298 (which contains no HisQMP2) under the same conditions as for TA1889 (Fig. 3 A and B, lanes 1–4); they were shown to have a constant activity at all heat induction times (Fig. 3 C, open circles). Thus, the activity in TA1889 PLS in the absence of HisJ is due to the sum of an activity intrinsic to HisQMP2 (about 25 nmol/min/mg at 70 min induction time) and that of contaminating ATPases. That the complex has an intrinsic activity was further established by purifying it and showing that, upon reconstitution into PLS, it hydrolyzes ATP (140 nmol/min/mg) in the absence of HisJ (Fig. 3 D, squares). 6G. F-L. Ames, S. Zhang, C. E. Liu, P. Q. Liu, and S. Wolf, manuscript in preparation.Figure 3Intrinsic ATPase activity of HisQMP2. PLS were prepared from membrane vesicles obtained from either strain TA1889 or GA298, heat induced for different lengths of time as indicated. A, SDS-PAGE resolution of proteins in PLS (about 4 μg) from GA298 and TA1889 with heat induction times of 0, 20, 40, and 75 min (GA298, lanes 1–4; TA1889, lanes 5–8), respectively. The gel was stained with Coomassie Blue. B, same as A, but immunoblotted using antibodies against HisP and HisQ. C, ATPase activity of PLS preparations from TA1889 (squares) and GA298 (circles) was assayed in the absence or presence of 20 μm liganded HisJ. The abscissa indicates the length of time the cells were heat induced prior to preparing membrane vesicles. D, PLS prepared from purified HisQMP2were assayed for ATP hydrolysis in the presence (diamonds) or absence (squares) of 12 μm liganded HisJ.View Large Image Figure ViewerDownload Hi-res image Download (PPT) That the activity stimulated by liganded HisJ is due to HisQMP2 (as opposed to a contaminating activity which is stimulated by HisJ) is shown by its dependence on the HisQMP2 content in PLS (Fig. 3 C, solid squares). Purified HisQMP2 gave the same result (Fig. 3 D, diamonds), displaying an activity of 1.05 μmol/min/mg protein (an activity of 1.94 μmol/min/mg protein was obtained in the presence of a saturating concentration of liganded HisJ (50 μm)). The stimulation is specific for HisJ because a number of HisJ mutant proteins were found that are unable to stimulate the ATPase activity. 7C. E. Liu, A. Wolf, and G. F-L. Ames, unpublished data.HisQMP2 also interacts with another soluble receptor, LAO (the periplasmic lysine-, arginine-, ornithine-binding protein) (28Higgins C.F. Ames G.F.-L. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6038-6042Crossref PubMed Scopus (111) Google Scholar), which, therefore, would also be expected to stimulate the ATPase activity. Indeed, both unliganded and l-arginine-liganded LAO stimulate the activity. The affinity of LAO for HisQMP2and the maximum level of stimulated activity (purified HisQMP2) were determined to be 5.3 μm and 1.75 μmol/min/mg, respectively. These values are very similar to those obtained for HisJ (13Ames G.F.-L. Liu C.E. Joshi A.K. Nikaido K. J. Biol. Chem. 1996; 271: 14264-14270Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Purified HisQMP2 in PLS was used to accurately determine the affinity of HisQMP2 for ATP, both for the intrinsic activity and the activity stimulated by liganded HisJ. In both cases, the curves are sigmoidal with respect to ATP concentration (Fig. 4), implying a positive cooperativity between ATP-binding sites in HisQMP2, with identical Hill coefficients: napp = 1.9 ± 0.05. Thus, two binding sites in HisQMP2 are involved. TheK0.5 values for ATP are 0.5 and 0.6 mm, respectively, for the activity stimulated by liganded HisJ (8 μm) and for the intrinsic activity. TheVmax values are 1.9 and 0.14 μmol/min/mg, respectively, for the activity stimulated by liganded HisJ (11 μm) and for the intrinsic activity. To exclude the possibility that the cooperativity is due to the presence of excess Mg2+, PLS containing internally trapped liganded HisJ were assayed in the presence of external ATP; the activity showed positive cooperativity with a Hill coefficient and K0.5value of 2.0 and 1.7 mm, respectively. Nucleotides other than ATP have been shown to support histidine transport and to bind to HisP (18Hobson A. Weatherwax R. Ames G.F.-L. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7333-7337Crossref PubMed Scopus (70) Google Scholar, 29Ames G.F.-L. Nikaido K. Groarke J. Petithory J. J. Biol. Chem. 1989; 264: 3998-4002Abstract Full Text PDF PubMed Google Scholar), indicating that HisQMP2 has a relatively broad specificity for the hydrolysis of nucleotides and for their utilization in energizing transport. The relative rates of hydrolysis of nucleotides at concentrations between 0.2 and 2 mm are in the following order: ATP > GTP > UTP > CTP (Table I). This order of preference is the same as that obtained when nucleotides were tested for the energization of transport (12Bishop L. Agbayani R.J. Ambudkar S.V. Maloney P.C. Ames G.F.-L. Proc. Natl.