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• W2014746315 abstract "The nucleotide-binding subunit, HisP, of the histidine permease, a traffic ATPase (ABC transporter), has been purified as a soluble protein and characterized. Addition of a 6-histidine extension (HisP(His6)) allows a rapid and effective metal affinity purification, giving a 30-fold purification with a yield of 50%. HisP(his6) is indistinguishable from underivatized HisP when incorporated into the permease membrane-bound complex, HisQMP2. Purified HisP(his6) has a strong tendency to precipitate; 5 mm ATP and 20% glycerol maintain it in solution at a high protein concentration. HisP(his6) is active as a dimer, binds ATP with aK d value of 205 μm, and hydrolyzes it at a rate comparable to that of HisQMP2; in contrast to the latter, it does not display cooperativity for ATP. HisP(his6) has been characterized with respect to substrate and inhibitor specificity and various physico-chemical characteristics. Its pH optimum is 7 and it requires a cation for activity, with Co2+ and Mn2+ being more effective than Mg2+ at lower concentrations but inhibitory in the higher concentration range. In contrast to the intact complex, HisP(his6) is not inhibited by vanadate but is inhibited byN-ethylmaleimide. Neither the soluble receptor, HisJ, nor the transport substrate, histidine, has any effect on the activity. The nucleotide-binding subunit, HisP, of the histidine permease, a traffic ATPase (ABC transporter), has been purified as a soluble protein and characterized. Addition of a 6-histidine extension (HisP(His6)) allows a rapid and effective metal affinity purification, giving a 30-fold purification with a yield of 50%. HisP(his6) is indistinguishable from underivatized HisP when incorporated into the permease membrane-bound complex, HisQMP2. Purified HisP(his6) has a strong tendency to precipitate; 5 mm ATP and 20% glycerol maintain it in solution at a high protein concentration. HisP(his6) is active as a dimer, binds ATP with aK d value of 205 μm, and hydrolyzes it at a rate comparable to that of HisQMP2; in contrast to the latter, it does not display cooperativity for ATP. HisP(his6) has been characterized with respect to substrate and inhibitor specificity and various physico-chemical characteristics. Its pH optimum is 7 and it requires a cation for activity, with Co2+ and Mn2+ being more effective than Mg2+ at lower concentrations but inhibitory in the higher concentration range. In contrast to the intact complex, HisP(his6) is not inhibited by vanadate but is inhibited byN-ethylmaleimide. Neither the soluble receptor, HisJ, nor the transport substrate, histidine, has any effect on the activity. The superfamily of traffic ATPases (or ABC transporters) comprises both prokaryotic and eukaryotic transport proteins that share a conserved nucleotide-binding domain (1Ames G.F.-L. Mimura C. Holbrook S. Shyamala V. Adv. Enzymol. 1992; 65: 1-47PubMed 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 (957) Google Scholar). The superfamily includes, among others, bacterial periplasmic permeases, the yeast STE6 gene product, the mammalian P-glycoprotein (multidrug resistance protein or MDR), the human cystic fibrosis transmembrane conductance regulator (CFTR), 1The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; HisQMP2, the membrane-bound complex containing HisQ, HisM, and HisP; HisP(his6), HisP protein with a carboxyl-terminal extension of 8 amino acid residues: Leu-Glu-His-His-His-His-His-His; PAGE, polyacrylamide gel electrophoresis; TNP-ATP, 2′-(or -3′)-O-(trinitrophenyl)adenosine 5′-triphosphate; PE, phosphatidylethanolamine; NEM, N-ethylmaleimide; IPTG, isopropylthiogalactoside; AMP-PNP, 5′-adenylyl-imidodiphosphate; AMP-PCP, adenylyl (β,γ-methylene)-diphosphonate; ATPγS, adenosine 5′-O-(3-thio)triphosphate; MOPS, 4-morpholinepropanesulfonic acid; HPLC, high pressure liquid chromatography. and the mammalian heterodimeric transporter (TAP1/TAP2) involved in antigen processing (3Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3386) Google Scholar, 4Doige C.A. Ames G.F.-L. Annu. Rev. Microbiol. 1993; 47: 291-319Crossref PubMed Scopus (266) Google Scholar). The histidine permease of Salmonella typhimurium and the maltose permease of Escherichia coli have been extensively characterized; both are good model systems for understanding the mechanism of action of 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, 7Liu C.E. Liu P.-Q. Ames G.F.-L. J. Biol. Chem. 1997; 272: 21883-21891Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 8Nikaido H. FEBS Lett. 1994; 346: 55-58Crossref PubMed Scopus (69) Google Scholar). The histidine permease is composed of a soluble substrate-binding receptor, HisJ (the histidine-binding protein), and a membrane-bound complex, HisQMP2, comprising two integral membrane proteins, HisQ and HisM, and two copies of HisP that carries the ATP-binding motif (9Kerppola R.E. Shyamala V. Klebba P. Ames G.F.-L. J. Biol. Chem. 1991; 266: 9857-9865Abstract Full Text PDF PubMed Google Scholar, 10Mimura C.S. Admon A. Hurt K.A. Ames G.F.-L. J. Biol. Chem. 1990; 265: 19535-19542Abstract Full Text PDF PubMed Google Scholar, 11Mimura 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 (12Ames G.F.-L. Krulwich T.A. Bacterial Energetics. Academic Press, New York1990: 225-245Crossref Google Scholar). Our general approach toward understanding the mechanism of action of these transporters has been to purify and characterize biochemically (a) the intact complex, (b) the isolated nucleotide-binding subunit, HisP, and (c) a reassembled system in which the isolated nucleotide-binding subunit has been reconstituted into membrane vesicles and proteoliposomes containing only HisQ and HisM. Such an analysis will delineate the properties intrinsic to the individual subunits, relate them to those of the intact complex, and, in combination with the creation and analysis of mutant proteins, it will provide information of general use for understanding the mechanism of action of the traffic ATPases superfamily. In this article we present the purification and properties of the ATP-binding subunit of the histidine permease, HisP. HisP binds and hydrolyzes ATP and, presumably, is the subunit responsible for energizing the transport process. We show that HisP functions as a dimer and that under optimal conditions the rate at which it hydrolyzes ATP is comparable to that by the intact complex, HisQMP2. The properties of HisP are compared with those of the intact complex and of related subunits from other traffic ATPases. The construction of pFA351, a plasmid carrying the S. typhimurium hisP (His6) gene under the control of an IPTG-inducible T7 polymerase gene (13Studier F.W. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4842) Google Scholar), is described in the text. GA596, the E. coli strain containing plasmid pFA351 (freshly introduced by transformation) was grown at 30 °C until the culture reached anA 650 nm of 0.6. The slower growth at this temperature allows a higher percentage of HisP to remain in the soluble fraction. IPTG was added to a final concentration of 100 μm and growth continued for 2 h. Cells were harvested by centrifugation and stored at −80 °C as a paste. In a typical preparation, 8 g of cells harvested from 4 liters of culture were defrosted, washed with 40 ml of 50 mm Tris/Cl, pH 8.1, 1 mm MgCl2, resuspended in 40 ml of 50 mm Tris/Cl, pH 8.1, and disrupted twice at 10,000 p.s.i. in a French press apparatus. After centrifugation for 10 min at 1,300 × g, ATP was added to a final concentration of 5 mm to the supernatant fraction (crude extract), which was then centrifuged in a TLA 100.4 rotor at 100,000 rpm for 30 min in a Beckman ultracentrifuge (the supernatant is the cytoplasmic fraction). The cytoplasmic fraction (35 ml) was applied to a column (Bio-Rad glass Econo column; 2.5 × 10 cm) prepared from 10 ml of a slurry of TALON metal affinity resin (CLONTECH) equilibrated with 50 mm Tris/Cl, pH 8.1, 1 mmMgCl2, and gently shaken at 4 °C for 45 min. The column was allowed to drain (flow-through fraction) and washed with 40 ml of cold buffer A (50 mm Tris/Cl, pH 8.1, 100 mmNaCl, 5 mm ATP, 20% glycerol) and then with 20 ml of cold buffer A containing 6 mm imidazole. The resin was then transferred to a narrower column (1.5 × 13 cm) and eluted with 10 ml of buffer A containing 100 mm imidazole and 0.1 mm EDTA. Ten fractions (1.0 ml each) were collected and analyzed for protein content by the micro-BCA protein assay (14Wiechelman K. Braun R. Fitzpatrick J. Anal. Biochem. 1988; 175: 231-237Crossref PubMed Scopus (636) Google Scholar) (Pierce kit with bovine serum albumin as standard). Over 90% of the HisP retained was eluted within one or two fractions. If necessary, fractions were concentrated using an Amicon Centricon 10 centrifugal concentrator. Pure protein was aliquoted and stored in liquid nitrogen. A typical experiment yields 8–10 mg of pure HisP(his6). Whenever it was necessary to change the buffer, gel filtration through a PD-10 column (Pharmacia Biotech Inc.) equilibrated with the desired buffer was used. Pure HisP(his6) (2.1 mg/ml; 200 μl) was injected onto an HPLC column (TOSOHAAS G3000SW, 7.8 mm × 30 cm) equilibrated with 50 mm MOPS, pH 7, 4% glycerol, 100 mm NaCl, with a flow rate of 1 ml/min; fractions (∼0.5 ml each) were collected and the protein content analyzed by the BCA assay and by SDS-PAGE. Carbonic anhydrase and bovine serum albumin (molecular masses, 29 and 66 kDa, Sigma) were used as molecular weight standards. ATPase activity was assayed essentially as described (7Liu C.E. Liu P.-Q. Ames G.F.-L. J. Biol. Chem. 1997; 272: 21883-21891Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) with the following modifications to miniaturize it. Sixty μl of a HisP(his6) solution (final concentration: 0.7 mg/ml, 24 μm HisP(his6)) in assay buffer (60 mm MOPS, pH 7.0, 4% glycerol, 2 mm ATP, 0.1 mm EDTA) was equilibrated at 37 °C for 3 min, and the reaction was initiated by the addition of 2 μl of 30 mmMgCl2 (final concentration: 1 mm). Samples (10 μl) were taken at appropriate times (every 20 s at high enzyme concentrations) and placed into microtiter plate wells containing 40 μl of 7.5% SDS. The amount of Pi liberated was determined by a colorimetric assay (15Chifflet S. Torriglia A. Chiesa R. Tolosa S. Anal. Biochem. 1988; 168: 1-4Crossref PubMed Scopus (418) Google Scholar), using Na2HPO4 as a standard. HisP(his6) (150 μl, 6.8 mg/ml) was first applied to a Sephadex G-50 column (0.5 × 3.2 cm) equilibrated with 10 mm NaPi, pH 7.9, 10% glycerol, 30 mm NaCl, 0.5 mm ATP and eluted with the same buffer; 0.2-ml fractions were collected, and all of HisP(his6) appeared in two fractions, the first of which was used for the CD measurements, after dilution in the same buffer to a final concentration of 0.8 mg/ml. HisP(his6) prepared under these conditions stays in solution for several hours. The CD spectrum was determined at 25 °C using an AVIV 62DS spectropolarimeter, using a 0.01-cm path length cuvette. The final spectrum, representing an average of six scans, was corrected for the corresponding buffer and smoothed. Estimation of percentage of secondary structure was performed by unrestricted least squares fitting of the experimental spectrum to the sum of pure-component CD spectra characteristic of α-helical, β-sheet, turn, and non-regular structural elements (the LINCOMB algorithm (16Parczel A. Park K. Fasman G.D. Anal. Bichem. 1992; 203: 83-93Crossref PubMed Scopus (421) Google Scholar, 17Chang C.T. Wu C.-S.C. Yang J.T. Anal. Biochem. 1978; 91: 13-31Crossref PubMed Scopus (1026) Google Scholar, 18Chen Y.-H. Yang J.T. Biochem. Biophys. Res. Commun. 1971; 44: 1285-1291Crossref PubMed Scopus (328) Google Scholar, 19Greenfield N. Fasman G.D. Biochemistry. 1969; 8: 4108-4116Crossref PubMed Scopus (3332) Google Scholar, 20Yang J.T. Wu C.-S.C. Martinez H.M. Methods Enzymol. 1986; 130: 208-269Crossref PubMed Scopus (1740) Google Scholar)). SDS-PAGE and immunoblotting were performed as described (21Wolf A. Shaw E.W. Oh B.-H. De Bondt H. Joshi A.K. Ames G.F.-L. J. Biol. Chem. 1995; 270: 16097-16106Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), using polyclonal antibody raised against HisP (lot 1618), and quantitating the bands with an Alpha Imager (IS-1000; Alpha Innotech Corp.). Phospholipids, various nucleotides, and ATPase inhibitors were obtained and prepared as described (7Liu C.E. Liu P.-Q. Ames G.F.-L. J. Biol. Chem. 1997; 272: 21883-21891Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). To study the properties of HisP it is most convenient to have it in a soluble form. It had been established previously that a sizable portion of HisP overproduced in the absence of HisQ and HisM is located in the cytoplasmic fraction (9Kerppola R.E. Shyamala V. Klebba P. Ames G.F.-L. J. Biol. Chem. 1991; 266: 9857-9865Abstract Full Text PDF PubMed Google Scholar). Among several possible promoter systems, those producing large amounts of protein (such as the combined lambda PL/PR promoter) were discarded to avoid the risk of creating insoluble forms of HisP. Although inclusion bodies protein can be solubilized, e.g. by dissolving them in urea, and renatured (22Walter C. Honer zu Bentrup K. Schneider E. J. Biol. Chem. 1992; 267: 8863-8869Abstract Full Text PDF PubMed Google Scholar), a more gentle purification process was deemed more appropriate for future crystallization attempts. Reliably moderate amounts of soluble protein can be produced using the chosen T7 promoter system (23Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1074-1078Crossref PubMed Scopus (2459) Google Scholar) by controlling the induction time. A peptide extension containing leucine, glutamate, and six histidines ((His6) tail) was engineered into the carboxyl terminus of HisP. Plasmid pFA351 was constructed by introducing ahisP (His6)-containing DNA fragment obtained by polymerase chain reaction from pFA284 2Plasmid pFA284 carries the hisQ, hisM, and hisP (his6) genes under the temperature-sensitive control of the lambda PL promoter (strain GA500; laboratory strain collection). into theEcoRI/BamHI site of the PT7-5 vector (23Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1074-1078Crossref PubMed Scopus (2459) Google Scholar), followed by transformation into E. coli strain BL21(DE3) which carries an IPTG-inducible T7 polymerase gene on the chromosome (13Studier F.W. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4842) Google Scholar), resulting in final strain GA596. Fig. 1 (left) shows that strain GA596 produces large amounts of a protein upon induction with IPTG for 1 h (lanes 1 and 2); the amount increases with time of induction (lanes 2–4). This protein was identified as HisP(his6) by its cross-reaction with antibodies raised against HisP and by its size (slightly larger than that of HisP). HisP(his6) was shown to be physiologically indistinguishable from the wild type by its ability to transport d-histidine (a property completely dependent on a functional histidine permease (24Ames G.F.-L. Arch. Biochem. Biophys. 1964; 104: 1-18Crossref PubMed Scopus (78) Google Scholar)), as shown in a strain producing HisP(his6), which transports as well as a strain that is identical except for carrying a wild type hisP gene (Fig. 1,right). Furthermore, when HisP(his6) is present in a complex with HisQ and HisM, and it displays the same in vitro ATPase activity as wild type HisQMP2 (7Liu C.E. Liu P.-Q. Ames G.F.-L. J. Biol. Chem. 1997; 272: 21883-21891Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Thus, HisP(his6) is indistinguishable from HisP when associated with HisQ and HisM, both in vivo and in vitro, and is an appropriate candidate for further studies. The purification of HisP(his6) takes advantage of the metal-chelating properties of the (His6) tail. The procedure described under “Experimental Procedures” generally yields a final preparation that is over 95% pure, with a yield of about 50%. Fig.2 shows the results of a purification sequence. A crude extract (lane 2) contains high levels of HisP(his6), of which about 80% is in the cytoplasmic fraction (lanes 3 and 4), as judged by eye after SDS-PAGE and Coomassie Blue staining and by quantitation with antibodies against HisP in immunoblots of a similar SDS-PAGE resolution (data not shown). Upon application of the cytoplasmic fraction to a TALON column, most of the protein is not retained, together with a portion of HisP(his6) varying between 15 and 20% of the total cytoplasmic HisP(his6) (flow-through, lane 5). 3To exclude the possibility that this material did not stick to the column because of an overloading effect, it was reapplied to a new TALON column. None of it was retained. Therefore, it is assumed that this material has undergone some form of denaturation. With extensive washing, additional cytoplasmic protein is released together with a very small amount (about 4%) of HisP(his6) (lane 6). The elution buffer releases the bulk of HisP(his6)(lanes 7). 4A small amount of HisP(his6) (11%) is retained by the column after elution. TableI summarizes the quantitation of protein and HisP(his6) in the purification experiment shown in Fig.2. It can be seen that the yield is 48% after a 32-fold purification and HisP(his6) is estimated to be 96% pure.Table IPurification of HisP(His6)FractionTotal protein1-aDetermined using the BCA assay.HisP(His6)1-bQuantitated from an immunoblot using antibody raised against HisP and normalized against a standard curve of pure HisP(his6), the concentration of which had been determined by the BCA assay. In parentheses, % of total protein.mgmgCrude extract74122 (3)Cytoplasmic fraction57417.2 (3)Flow-through5183.4 (0.6)Wash I34.21.5 (4.4)Wash II3.2ND1-cND, not determined.Imidazole eluate11.210.6 (96)1-dAs determined from a Coomassie Blue-stained gel.1-a Determined using the BCA assay.1-b Quantitated from an immunoblot using antibody raised against HisP and normalized against a standard curve of pure HisP(his6), the concentration of which had been determined by the BCA assay. In parentheses, % of total protein.1-c ND, not determined.1-d As determined from a Coomassie Blue-stained gel. Open table in a new tab The purified HisP(his6) was found to be particularly inclined to precipitating, forming an insoluble white fluffy material that could not be redissolved by a variety of treatments, including numerous detergents. An eluate (2.2 mg/ml) from a TALON column in 2% glycerol, 0.5 mm ATP, 50 mm Tris/Cl, pH 8.1 buffer, 0.1 mm EDTA, 100 mm NaCl, and 100 mmimidazole starts precipitating within 2 h at 0 °C. Such a preparation was divided into aliquots, and one of the following detergents (1% final concentration) was added to each of them: Lubrol, Brij 35, Triton X-100, Tween 20, and sodium deoxycholate. After 5 days at 0 °C the samples were centrifuged, and the amount of HisP(his6) in the supernatant fraction was found to be 28, 40, 27, 29, and 54% of the total present, respectively, with 25% being present in the sample containing no detergent. Thus, none of these detergents is able to maintain HisP(his6) in solution at such a concentration. The precipitated HisP(his6) could be redissolved in 4 m urea (and found to be inactive, following a 10-fold dilution) or 2 m guanidine HCl. An SDS-PAGE analysis in the absence of β-mercaptoethanol showed that the insoluble form of HisP(his6), in contrast to the soluble form, has a mobility corresponding to that of dimeric HisP(his6), indicating the formation of a disulfide bond involving the single cysteine residue present in HisP (25Higgins C.F. Haag P.D. Nikaido K. Ardeshir F. Garcia G. Ames G.F.-L. Nature. 1982; 298: 723-727Crossref PubMed Scopus (192) Google Scholar). Several parameters were tested for improving the solubility of a crude extract in which about half of the HisP(his6) would precipitate upon storage at 0 °C for 5 days. Individually added ATP, ADP, and glycerol help somewhat to maintain solubility. Some detergents (0.5% n-octyl-β-d-glucopyranoside or 0.4% CHAPS) improve the solubility if added in combination with ATP. A more thorough analysis performed on HisP(his6) after purification on the TALON column indicated that both 20% glycerol and 5 mm ATP are essential if the purified protein is present at concentrations higher than 2 mg/ml. In the procedure finally adopted, 5 mm ATP is added to the crude extract prior to ultracentrifugation and 5 mm ATP, 20% glycerol, and 0.1 mm EDTA are present in the elution buffer. Final storage conditions also include 5 mm ATP and 20% glycerol, which allow HisP(his6) to remain in solution up to at least 8.5 mg/ml and for at least 1 week at 4 °C. If ATP hydrolysis is allowed to proceed by the addition of Mg2+, HisP(his6)starts precipitating as the ATP becomes depleted. Thus, it appears that an ATP-liganded conformation of HisP(his6) is necessary to maintain solubility. Therefore, EDTA (0.1 mm) is also added to eliminate a slow, continuous hydrolysis of ATP during storage. 5However, exhaustive removal of Mg2+by the addition of EDTA in several column washing steps before elution with 100 mm imidazole results in the disappearance of HisP(his6) from all column fractions (presumably it precipitates within the column matrix). The activity and solubility of HisP(his6) at 5–10 mg/ml in storage buffer and in liquid nitrogen do not change for at least several months and can withstand repeated freezing and thawing. HisP is known to bind ATP (and other nucleotides), and it has been shown that the intact complex, HisQMP2, hydrolyzes ATP under a variety of conditions (7Liu C.E. Liu P.-Q. Ames G.F.-L. J. Biol. Chem. 1997; 272: 21883-21891Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), and it has an intrinsic, low level ATPase activity that is stimulated somewhat by unliganded HisJ and to a high level by liganded HisJ. It was proposed that HisQ and/or HisM normally constrain the ability of HisP to hydrolyze ATP and that signaling by liganded HisJ allows hydrolysis to proceed by relieving such a constraint (7Liu C.E. Liu P.-Q. Ames G.F.-L. J. Biol. Chem. 1997; 272: 21883-21891Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). If this were true, isolated HisP should be able to hydrolyze ATP at a level considerably higher that the intrinsic activity of HisQMP2 in the absence of external stimuli. Fig.3 A (solid dots) shows that purified HisP(his6) indeed hydrolyzes ATP 6That this activity is due to HisP(his6) rather than to a contaminating ATPase is demonstrated by the fact that preparations obtained by an identical purification scheme from several hisP mutant strains defective in transport do not display any ATPase activity. at a high linear rate in the presence of Mg2+. The activity is dependent on the presence of a cation, and the efficiency of stimulation by various cations varies with their concentration (Fig. 3 B); Mn2+ is the best stimulator at higher concentrations (above 1.5 mm) and Co2+ is the best at lower concentrations (optimum at 1 mm, followed by an inhibition). 7Co2+ may stimulate the activity by interacting with the (His6) tail and inducing a conformational change. Ca2+ has a very poor stimulatory ability and Zn2+ has none (data not shown). Fig. 3 C shows that the pH optimum for activity is around 7, with a slow decline at higher pH values. Although HisP(his6) is dependent on the presence of 20% glycerol to stay in solution, the presence of glycerol above 7.5% during the assay inhibits the activity (Fig. 3 D), possibly by disruption of hydrophobic interactions. This inhibition is reversible, as shown by the fact that even though the protein is eluted from the TALON column in the presence of 20% glycerol, it is fully active upon dilution to glycerol concentrations lower than 7.5%. The activity of a preparation diluted to 0.7 mg/ml protein and 4% glycerol is stable at 0 °C for at least 1 h. The ATPase activity was observed to be non-linearly dependent on protein concentration (Fig. 4,circles), suggesting that the active form of the enzyme is a multimer. The specific activity, as calculated along the concentration curve in Fig. 4, indicates that the activity of the monomer, if any, would be extremely low. Because HisP is present in two copies in HisQMP2 and its ATPase activity displays positive cooperativity for ATP with a Hill coefficient of 2 (7Liu C.E. Liu P.-Q. Ames G.F.-L. J. Biol. Chem. 1997; 272: 21883-21891Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), the most likely form of the active enzyme would be expected to be a dimer. 8The membrane-bound complex of the maltose permease, which contains two copies of the ATP-binding subunit, MalK, also displays positive cooperativity for ATP (26Davidson A.L. Laghaeian S.S. Mannering D.E. J. Biol. Chem. 1996; 271: 4858-4863Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Fig. 4 provides support for this hypothesis because the experimental data match well the curve of activity versus protein concentration, as calculated for the active form being a dimer (solid line). The dimeric state of HisP(his6) could also be demonstrated by physical methods. A preparation of HisP(his6) (1 ml, 1 mg/ml) was applied to a molecular sieve column (0.5 × 75 cm; ACA54, LKB). A large peak of HisP was eluted at the position corresponding approximately to a molecular mass of 29.8 kDa (which is the calculated molecular mass of a single copy of HisP (25Higgins C.F. Haag P.D. Nikaido K. Ardeshir F. Garcia G. Ames G.F.-L. Nature. 1982; 298: 723-727Crossref PubMed Scopus (192) Google Scholar) containing the 8-residue extension), and a small peak appeared in the position corresponding to about 55 kDa, indicating that both monomer and dimer forms might be present. The small amount of the presumed dimer form suggested that the two forms are in rapid equilibrium with each other. Therefore, a more rapid separation was performed using HPLC (Fig.5 A). HisP(his6)was present both in the large peak corresponding to the monomer position and in small amount (about 3% total protein) in the position corresponding to about 60 kDa. An additional small peak appeared in the void volume; considering the propensity of HisP(his6) to precipitate, it was assumed that this latter peak contains higher aggregates and was not investigated further. A fraction from the presumed dimer peak was rechromatographed on the same column, yielding a peak at the position of the monomer, which is consistent with its being a dimer in equilibrium with the monomer. Evidence that the equilibrium between the monomer and the dimer is rapid was obtained by diluting a solution of 5.6 mg/ml HisP(his6) (which is estimated to contain 60% of the protein as a dimer; see inset in Fig. 4) 8-fold in assay buffer to a final concentration of 0.7 mg/ml (which contains 26% of the protein as dimer); one aliquot was assayed immediately and another aliquot was kept at 0 °C, and the ATPase activity was assayed at several time intervals (up to 1 h). The specific activity did not change in time, indicating that the protein reaches final equilibrium within the earliest time interval tested (5 min at 0 °C). A more rapid procedure was utilized by diluting 30- and 80-fold the same HisP(his6) solution into prewarmed assay buffer containing Mg2+ and immediately (within 5 s) assaying for activity. Fig. 5 B shows that the rates do not change significantly in time after dilution and that they correspond to what is expected for the amount of HisP(his6) dimer that is present at those final concentrations (10 and 4%, respectively). This indicates that the protein reaches equilibrium within seconds after dilution. The affinity for ATP, measured following dilution of a concentrated preparation to final concentrations of HisP(his6) and ATP of 167 μg/ml and 100 μm, respectively, gave a K d value for ATP of 205 μm (Fig. 6). In contrast to the results obtained with the intact complex, HisQMP2 (7Liu C.E. Liu P.-Q. Ames G.F.-L. J. Biol. Chem. 1997; 272: 21883-21891Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), there is no evidence of cooperativity. Although Fig. 4 showed that the measurable activity is ascribable to the dimeric form and that the monomeric form, if active at all, has a very low specific activity, it is possible that the low level, non-cooperative, (hypothetical) activity of monomeric HisP(his6) contributed slightly to the measurements. Therefore, the K d value was also measured at high HisP(his6)concentration. 9This measurement requires the elimination of the ATP obligatorily present in solutions of HisP(his6) at high concentrations. The ATP analog, TNP-ATP, which has a high affinity for the enzyme and is hydrolyzed very slowly, maintains high concentrations of HisP(his6) in solution when present at 10 μm, for 14 days at 4 °C. HisP(his6) was purified as usual and the ATP was replaced with TNP-ATP by passage through a gel filtration column containing 10 μm TNP-ATP. This concentration of TNP-ATP does not significantly interfere with the assay for ATPase activity (Table II). The ATPase activity of such a preparation (containing 0.8 mg/ml HisP(his6), which is calculated to contain about 29% d" @default.
• W2014746315 created "2016-06-24" @default.
• W2014746315 creator A5004561226 @default.
• W2014746315 creator A5046685224 @default.
• W2014746315 creator A5062198870 @default.
• W2014746315 date "1997-10-01" @default.
• W2014746315 modified "2023-10-18" @default.
• W2014746315 title "Purification and Characterization of HisP, the ATP-binding Subunit of a Traffic ATPase (ABC Transporter), the Histidine Permease of Salmonella typhimurium" @default.
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