Kinetic Study of the Antiport Mechanism of an Escherichia coli Zinc Transporter, ZitB
2004; Elsevier BV; Volume: 279; Issue: 13 Linguagem: Inglês
10.1074/jbc.m313510200
ISSN1083-351X
Autores Tópico(s)Aluminum toxicity and tolerance in plants and animals
ResumoZitB is a member of the cation diffusion facilitator (CDF) family that mediates efflux of zinc across the plasma membrane of Escherichia coli. We describe the first kinetic study of the purified and reconstituted ZitB by stopped-flow measurements of transmembrane fluxes of metal ions using a metal-sensitive fluorescent indicator encapsulated in proteoliposomes. Metal ion filling experiments showed that the initial rate of Zn2+ influx was a linear function of the molar ratio of ZitB to lipid and was related to the concentration of Zn2+ or Cd2+ by a hyperbola with a Michaelis-Menten constant (Km) of 104.9 ± 5.4 μm and 90.1 ± 3.7 μm, respectively. Depletion of proton stalled Cd2+ transport down its diffusion gradient, whereas tetraethylammonium ion substitution for K+ did not affect Cd2+ transport, indicating that Cd2+ transport is coupled to H+ rather than to K+. H+ transport was inferred by the H+ dependence of Cd2+ transport, showing a hyperbolic relationship with a Km of 19.9 nm for H+. Applying H+ diffusion gradients across the membrane caused Cd2+ fluxes both into and out of proteoliposomes against the imposed H+ gradients. Likewise, applying outwardly oriented membrane electrical potential resulted in Cd2+ efflux, demonstrating the electrogenic effect of ZitB transport. Taken together, these results indicate that ZitB is an antiporter catalyzing the obligatory exchange of Zn2+ or Cd2+ for H+. The exchange stoichiometry of metal ion for proton is likely to be 1:1. ZitB is a member of the cation diffusion facilitator (CDF) family that mediates efflux of zinc across the plasma membrane of Escherichia coli. We describe the first kinetic study of the purified and reconstituted ZitB by stopped-flow measurements of transmembrane fluxes of metal ions using a metal-sensitive fluorescent indicator encapsulated in proteoliposomes. Metal ion filling experiments showed that the initial rate of Zn2+ influx was a linear function of the molar ratio of ZitB to lipid and was related to the concentration of Zn2+ or Cd2+ by a hyperbola with a Michaelis-Menten constant (Km) of 104.9 ± 5.4 μm and 90.1 ± 3.7 μm, respectively. Depletion of proton stalled Cd2+ transport down its diffusion gradient, whereas tetraethylammonium ion substitution for K+ did not affect Cd2+ transport, indicating that Cd2+ transport is coupled to H+ rather than to K+. H+ transport was inferred by the H+ dependence of Cd2+ transport, showing a hyperbolic relationship with a Km of 19.9 nm for H+. Applying H+ diffusion gradients across the membrane caused Cd2+ fluxes both into and out of proteoliposomes against the imposed H+ gradients. Likewise, applying outwardly oriented membrane electrical potential resulted in Cd2+ efflux, demonstrating the electrogenic effect of ZitB transport. Taken together, these results indicate that ZitB is an antiporter catalyzing the obligatory exchange of Zn2+ or Cd2+ for H+. The exchange stoichiometry of metal ion for proton is likely to be 1:1. Zinc is a micronutrient essential for the growth, development, and differentiation of cells by contributing to a number of important biological processes, including gene expression, DNA synthesis, enzymatic catalysis, hormone storage and release, neurotransmission, memory, and apoptosis (1Beyersmann D. Haase H. Biol. Metals. 2001; 14: 331-341Google Scholar). The function of zinc in metalloenzymes attributes to its inherent chemical properties as an acid catalyst, a structural ion, and a regulatory co-factor (2Frausto da Silva J. Williams R. The Biological Chemistry of the Elements. Oxford University Press, Oxford2001: 315-340Google Scholar, 3Kimura E. Kikuta E. J. Biol. Inorg. Chem. 2000; 5: 139-155Crossref PubMed Scopus (121) Google Scholar, 4Outten C. O'Halloran T. Science. 2001; 292: 2488-2492Crossref PubMed Scopus (1159) Google Scholar). Consequently, cellular processes are critically dependent on the maintenance of zinc at optimal physiological levels, ranging from about 10–11m in the cytoplasm of many cells to 10–3m in some vesicles (5Choi D. Koh J. Annu. Rev. Neuroscience. 1998; 21: 347-375Crossref PubMed Scopus (678) Google Scholar). In bacteria and eukaryotic cells, zinc homeostatic control mechanisms have evolved based on a complex network of transport, chelation, and sequestration processes to maintain zinc within narrow physiological ranges and sustain zinc concentration gradients across membranes of different cellular compartments (6Blencowe D. Morby A. FEMS Microbiol. Rev. 2003; 27: 291-311Crossref PubMed Scopus (173) Google Scholar, 7Eide D. J. Nutr. 2003; 133: 1532-1535Crossref PubMed Google Scholar, 8Vasak M. Hasler D. Curr. Opin. Chem. Biol. 2000; 4: 177-183Crossref PubMed Scopus (371) Google Scholar). Several families of membrane proteins have been identified as zinc uptake or efflux transporters, responsible for zinc homeostasis by moving zinc into and out of cells or intracellular vesicles (9Harris E. Nutr. Rev. 2002; 60: 121-124Crossref PubMed Scopus (45) Google Scholar). Of all zinc transporters identified so far, mechanisms by which zinc transporters bind and transport metal ions have not yet been defined in molecular detail. In Escherichia coli, zinc homeostasis is accomplished largely through the transcriptional control of four zinc transporter systems, including two uptake transporters, the high affinity ABC transporter ZnuABC (10Patzer S. Hantke K. Mol. Microbiol. 1998; 28: 1199-1210Crossref PubMed Scopus (385) Google Scholar), and the ZIP transporter ZupT (11Grass G. Wong M. Rosen B. Smith R. Rensing C. J. Bacteriol. 2002; 184: 864-866Crossref PubMed Scopus (136) Google Scholar). The efflux of zinc is mediated by two efflux transporters, the P-type ATPase ZntA (12Beard S. Hashim R. Membrillo-Hernandez J. Hughes M. Poole R. Mol. Microbiol. 1997; 25: 883-891Crossref PubMed Scopus (159) Google Scholar, 13Rensing C. Mitra B. Rosen B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14326-14331Crossref PubMed Scopus (344) Google Scholar) and the CDF 1The abbreviations used are: CDF, cation diffusion facilitator; TEA, tetraethylammonium; DDM, n-dodecyl-β-d-maltopyranoside; BTM, Bis-Tris-MES; β-OG, n-octyl-β-d-glucoside; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; β-ME, β-mercaptoethanol; HPLC, high pressure liquid chromatography. transporter ZitB (14Grass G. Fan B. Rosen B. Franke S. Nies D. Rensing C. J. Bacteriol. 2001; 183: 4664-4667Crossref PubMed Scopus (125) Google Scholar). CDF is a ubiquitous family of metal transporters found in prokaryotes and eukaryotes (15Paulsen I. Saier M.J. J. Membr. Biol. 1997; 156: 99-103Crossref PubMed Scopus (295) Google Scholar). Sequence analysis suggests that CDF proteins contain a hydrophobic N-terminal domain followed by a hydrophilic C-terminal domain with large variations both in size and sequence. The number of transmembrane domains and the oligomeric structure of CDF proteins have not yet been established experimentally, but hydropathy profiles of CDF sequences suggest that this family of transporters may contain five to six membrane-spanning domains. Mammalian and yeast CDF transporters have been localized to the plasma and vesicular membranes. They are involved in the removal of cytoplasmic zinc through efflux out of cells or sequestration into intracellular vesicular compartments (16Palmiter R. Findley S. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (639) Google Scholar, 17Palmiter R. Cole T. Findley S. EMBO J. 1996; 15: 1784-1791Crossref PubMed Scopus (397) Google Scholar, 18Li L. Kaplan J. J. Biol. Chem. 2001; 276: 5036-5043Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 19MacDiarmid C. Gaither L. Eide D. EMBO J. 2000; 19: 2845-2855Crossref PubMed Scopus (306) Google Scholar). Like many homologous eukaryotic CDF transporters, the expression of E. coli ZitB was found to be inducible by zinc (14Grass G. Fan B. Rosen B. Franke S. Nies D. Rensing C. J. Bacteriol. 2001; 183: 4664-4667Crossref PubMed Scopus (125) Google Scholar). ZitB was implicated in a role of an efflux pump by the observations that overexpression of ZitB increased zinc tolerance and reduced zinc uptake, whereas double ΔzitB and ΔzntA deletion resulted in zinc hypersensitivity (14Grass G. Fan B. Rosen B. Franke S. Nies D. Rensing C. J. Bacteriol. 2001; 183: 4664-4667Crossref PubMed Scopus (125) Google Scholar). A Zn2+/K+/H+ antiport mechanism was proposed for CDF based on genetic complementation experiments in which ZitB and CzcD, a Bacillus subtilis CDF transporter, were shown to complement K+ uptake deficiency of a mutant E. coli stain in the presence of zinc (20Guffanti A. Wei Y. Rood S. Krulwich T. Mol. Microbiol. 2002; 45: 145-153Crossref PubMed Scopus (111) Google Scholar, 21Lee S. Grass G. Haney C. Fan B. Rosen B. Anton A. Nies D. Rensing C. FEMS Microbiol. Lett. 2002; 215: 273-278Crossref PubMed Google Scholar). However, evidence for an antiport mechanism is indirect, because the observed Zn2+ efflux could simply reflect a mixed contribution of different transport systems residing in the plasma membrane. The apparent K+ and H+ dependences of CzcD and ZitB functions could result from the interplay of many H+ and K+-linked pumps in cells. Therefore, the transport mechanism of CDF remains obscure. An explicit mechanistic description can only come from a direct kinetic study using a purified reconstitution system. Here we describe the kinetic analysis of the purified and reconstituted ZitB using Zn2+-sensitive fluorescent indicator fluozin-1 to monitor the Zn2+ transport in response to transmembrane chemical gradients and electrical potentials. Fluozin-1 is a single wavelength fluorophore that exhibits more than 200-fold fluorescence enhancement upon Zn2+ or Cd2+ binding with essentially no change in absorption or emission wavelengths (Molecular Probes Handbook). It has been used for detection of rapid synaptic Zn2+ transients in the 0.1–100 μm range (22Gee K. Zhou Z. Ton-That D. Sensi S. Weiss J. Cell Calcium. 2002; 31: 245-251Crossref PubMed Scopus (220) Google Scholar). To quantify the transport kinetics of ZitB, we have established a linear relationship between the fluozin-1 response and the total intravesicular Zn2+ concentration and used a stopped-flow apparatus to determine kinetic parameters of ZitB transport under controlled conditions of proton gradients and membrane electrical potentials. These experiments led to the conclusion that ZitB is an obligatory Zn2+/H+ antiporter. Cloning and Expression Plasmid Construct—The entire open reading frame sequence of ZitB was obtained by PCR using the genomic DNA of E. coli BL21 strain as a template and a pair of ZitB-specific primers with an XhoI and a BamHI site incorporated into the 5′-ends of the forward and reverse primer, respectively (forward primer, 5′-ATGCTCGAGGCGCACTCACACTCAC-3′; reverse primer, 5′-CCGGATCCTTAATGGTGATGATGTGAATG-3′). The resulting PCR product was double-digested using XhoI and BamHI (New England BioLabs, Beverly, MA) and subsequently inserted between the same sites in an expression vector, pET15b (Novagen Inc., Madison, WI), in frame with an N-terminal His6 affinity tag followed by a thrombin proteolytic cleavage site. The resulting expression construct, pHis-TB-ZitB, was verified by automatic sequencing of both strands. Overexpression of ZitB—pHis-TB-ZitB was transformed into a host strain BL21(DE3)pLysS for overexpression of ZitB (Novagen Inc., Madison, WI). A single colony of transformed cells was inoculated into the LB broth containing 100 μg/ml ampicillin. The culture was grown overnight at 37 °C and diluted 100-fold to a 6-liter fresh LB broth. The large culture was grown to an A600 of 0.17 absorbance units, whereupon expression was induced by the addition of isopropyl-β-d-thiogalactoside to a final concentration of 0.2 mm. Cells were incubated at 37 °C with vigorous shaking for an additional 4 h and then harvested by centrifugation. Membrane Preparation and Solubilization—Cell pellets were resuspended in a Tris buffer (20 mm Tris, 500 mm NaCl, pH 8.0) containing 0.1% protease inhibitor mixture (Sigma) and 0.1% ethanol-saturated phenylmethanesulfonyl fluoride. Cells were lysed by three passages through an ice-chilled microfluidizer (Microfluidics Co., Newton, MA) at 1000 p.s.i., and the resulting membrane vesicles were collected by centrifugation at 140,000 × g for 45 min. The membrane pellet was washed with Tris buffer, and then membrane proteins were extracted by a detergent buffer (100 mm NaCl, 20 mm HEPES, pH 7.5, 7% n-dodecyl-β-d-maltopyranoside (DDM; Anatrace, Maumee, OH), 20% (w/v) glycerol, 2.0 mm β-ME). Insoluble cellular debris was pelleted from the supernatant by an additional centrifugation step at 140,000 × g for 30 min. Purification and Thrombin Digestion—Solubilized materials were applied to a DEAE-Sepharose fast flow column (Amersham Biosciences), pre-equilibrated with 100 mm NaCl, 20 mm HEPES, pH 7.5, 20% glycerol, 2 mm β-ME, and 0.05% DDM. The flow-through from the DEAE column was loaded to a Ni2+-nitrilotriacetic acid superflow column (Qiagen, Valencia, CA) at a flow rate of 2 ml/min. The Ni2+ column was washed exhaustively using a wash buffer (20 mm HEPES, pH 7.5, 300 mm NaCl, 20% (w/v) glycerol, 0.05% DDM, 2.0 mm β-ME, 30 mm imidazole), and then His-ZitB was eluted by the same wash buffer with additional imidazole added to 500 mm. The imidazole in the purified His-ZitB sample was removed by gel filtration chromatography using a Sephadex G-50 desalting column (Amersham Biosciences) that was preequilibrated with a desalting buffer (20 mm HEPES, pH 7.0, 100 mm NaCl, 20% glycerol, 0.05% DDM, 2.0 mm β-ME). The N-terminal His tag of the purified His-ZitB was proteolytically cleaved by overnight incubation with thrombin at a ratio of one unit of thrombin for each mg of ZitB (Novagen, Madison, WI). The resulting ZitB was concentrated to 10–20 mg/ml and then further purified by size exclusion HPLC using a TSK 3000SWXL column (TosoHaas, Montgomeryville, PA), preequilibrated with a detergent mobile phase (20 mm HEPES, pH 7.0, 100 mm NaCl, 12.5% glycerol, 0.05% DDM, 2.0 mm β-ME). The protein peaks were detected using a System-Gold HPLC system (Beckman Coulter, Fullerton, CA), and ZitB was collected as a discrete peak using a Beckman SC100 fraction collector. The concentration of the purified ZitB was determined using the BCA protein assay (Pierce). Mass Spectrometric Analysis—α-Cyano-4-hydroxycinnamic acid matrix was prepared as a saturated solution in a 2:1 mixture of acetonitrile and water. Aliquots of protein in a series of 1:2 dilutions were mixed with matrix solution and then spotted onto the sample plate using the sandwich method (23Beavis R. Chait B. Methods Enzymol. 1996; 270: 519-551Crossref PubMed Google Scholar). MALDI-TOF spectrometric data were collected using a Voyager Biospectrotometry work station, operated in linear mode. The spectra were externally calibrated with a calibrant mixture containing cytochrome c and bovine serum albumin. Reconstitution—Liposomes were prepared from E. coli polar lipid extract (Avanti Polar Lipids Inc., Alabaster, AL) hydrated to a final concentration of 50 mg/ml in a BTM buffer (2 mm Bis-Tris-MES (BTM), 2 mm β-ME, pH 6.8) by vigorous mixing on a vortex mixer. The suspension of multilamellar liposomes was sonicated under argon in an ice-chilled bath sonicator (Misonix Inc., Farmingdale, NY) until a clear lipid suspension was obtained. Depending on experimental designs, the purified ZitB, typically at a concentration of 10 mg/ml, was diluted to a final concentration ranging from 0.01 to 0.25 mg/ml, using an n-octyl-β-d-glucoside (β-OG) buffer (20 mm BTM, pH 6.8, 1% β-OG, 2 mm β-ME, and 50 mm either K2SO4 or TEA2SO4 when indicated). Using the freshly prepared sonicated lipid and ZitB dilution, reconstitution of ZitB proceeded with sequential additions of 100 μl of 10% β-OG to 130 μl of sonicated lipid, followed by the addition of 770 μl of ZitB. At this ratio, lipids were completely solubilized by β-OG, as indicated by the disappearance of turbidity. The resulting ZitB-lipid-detergent mixture was incubated at room temperature for 20 min and then applied to an Econo-Pac 10 DG desalting column (Bio-Rad), preequilibrated with an assay buffer (20 mm BTM or 20 mm Tris2SO4 with 50 mm K2SO4 or 50 mm TEA2SO4, when indicated). The cloudy void faction was collected and subjected to ultracentrifugation at 140,000 g for 45 min to pellet proteoliposomes. The control liposomes were prepared following exactly the same procedure without adding ZitB. Encapsulation of Membrane-impermeable Fluorescent Indicators— Proteoliposome and control liposome pellets were resuspended in 200 μl of assay buffer. An aliquot of 2 mm Zn2+-sensitive indicator fluozin-1 (Molecular Probes, Inc., Eugene, OR) or pH-sensitive indicator pyranine (Molecular Probes) was added to the proteoliposome resuspension to a final concentration of 200 μm. The samples were sonicated for 10 s and subjected to one cycle of freeze-thaw (liquid nitrogen, room temperature), followed by an additional 10-s sonication. The untrapped indicator exterior to proteoliposomes was removed using an Econo-Pac 10 DG desalting column, preequilibrated with assay buffer. The indicator-loaded proteoliposomes were eluted in the void fraction. Stopped-flow Fluorescence Measurement—Kinetic experiments were performed in florescence mode on a stopped-flow apparatus (KinTeK, Clarence, PA). The dead time of the instrument was about 2 ms. The reaction temperature was set at 8 °C using a circulating water bath to maintain a constant temperature through jackets surrounding mixing syringes and the mixing cell. Proteoliposome samples and an assay buffer containing varying concentrations of ZnSO4 or CdSO4 were loaded into two separate mixing syringes of equal volume, and transport reactions were initiated by pushing 60 μl of fresh reactants at a 1:1 ratio through the 12-μl mixing cell at a flow rate of 20 ml/s. Fluorescence was monitored at 90° to the incident excitation beam. For measurements of fluozin-1 fluorescence changes, samples were excited at 490 nm, and emissions were monitored at 525 nm using a 10-nm band pass cut-off filter. For measurements of intravesicular pH changes by the ratiometric fluorescent indicator pyranine, proteoliposome samples were excited alternately by a pair of wavelengths at 450 and 410 nm, and emissions were recorded at 525 nm. The ratio of emission intensities (F450/F405) at two excitation wavelengths was calculated. For measurements of transmembrane potential changes, the voltage-sensitive lipophilic indicator oxonol VI (Molecular Probes) was added directly to the proteoliposome sample to a final concentration of 1 μm. Oxonol fluorescence was excited at 615 nm, and the emission was recorded at 646 nm. The base-line fluorescence for each indicator was set by flushing reactants through the mixing cell and adjusting the photomultiplier tube voltage to a level that would give a 5-V signal in the middle of the 0–10-V detectable range. Data were collected by an IBM work station with sensitivity, offset, and gain kept constant for the duration of each set of experiments. Kinetic traces were recorded using two equal time partitions, with the first half allocated for the fast kinetic component (0–500 ms) and the second half for the slow kinetic component (0.5–500 s). All traces were the cumulative average of 5–10 successive recordings. Data Analysis—A concentration of fluozin-1 at 200 μm was chosen to achieve a linear fluorescent response (ΔF) to the total intravesicular metal concentration (Δ[M]). This linear relationship is derived by the following equations. The concentration of the entrapped metal indicator complex ([I-M]) (24Dineley K. Malaiyandi L. Reynolds I. Mol. Pharmacol. 2002; 62: 618-627Crossref PubMed Scopus (90) Google Scholar) is given by Equation 1, [I-M]=([I]+Kd+[M])-([I]+Kd+[M])2-4[I][M]2(Eq. 1) where [I] represents the total indicator concentration, [M] is the total metal ion concentration, and Kd is the binding affinity of fluozin-1 for the metal ion. The differential of Equation 1 produces Equation 2. Δ[I-M]=(I-[I]-[M]-Kd([I]+Kd+[M])2-4[I][M])×Δ[M]2(Eq. 2) If it is assumed that [I] ≫ Kd, Equation 2 can be simplified to the following. Δ[I-M]≈(1+[I]-[M]|[I]-[M]|)×Δ[M]2(Eq. 3) When[M]<[I],Δ[I-M]≈Δ[M](Eq. 4) When[M]>[I],Δ[I-M]≈0(Eq. 5) Kd of M binding to fluozin-1 is about 8 μm according to the Molecular Probes handbook. [I] at 200 μm is more than 20-fold higher than the Kd of fluozin-1. Therefore, the approximation Δ[I-M] ≈ Δ[M] holds. The observed ΔF is predominantly determined by Δ[I-M], which approximately equals Δ[M] when [M] < [I]. Hence, Δ[M] is given by Equation 6, Δ[M]≈kΔF/ΔFmax(Eq. 6) where k is a converting factor. ΔF is normalized to the maximum fluorescence change (ΔFmax) when the entrapped indicator is fully exposed to 2 mm external metal ion by detergent solubilization of proteoliposomes. In principal, the useful metal ion concentration range for this linear relationship, as suggested by Equation 5, is up to the concentration of the encapsulated fluozin-1 (200 μm). Calibration of Equation 6 was performed by exposing fluozin-1-loaded proteoliposomes to a series of controlled Zn2+ concentrations in the presence of 20 μm zinc ionophore pyrithione. Linear regression of the calibration curve yielded the converting factor k. All stopped-flow traces were normalized either to a maximum response elicited by detergent solubilization or to a quasistationary response when indicated. Background traces collected from liposome samples were subtracted, yielding net fluorescence responses that were fit to a biexponential function, A1(1 – exp(–k1t)) + A2(1 – exp(–k2t)), where A1, A2, k1, and k2 are amplitudes and rate constants, respectively. The initial rate of transport, Vi = (ΔF/ΔFmax)/Δt(t→0)), was calculated as A1k1 + A2k2. Concentration dependence data were analyzed by least squares fits of the normalized initial transport rate, Vi/Vmax, to a hyperbola defined by Equation 7, ViVmax=[M][M]+Km(Eq. 7) where [M] represents the metal ion concentration, Vmax is the maximum initial transport rate when the rate of transport approaches a quasistationary state, and Km is the Michaelis-Menten constant. Fits of experimental data were preformed using the data analysis software SIGMAPLOT 4.0 (SPSS Inc., Chicago, IL). Overexpression and Purification—The overexpression of ZitB was driven by a T7 promoter in E. coli BL21 (DE3) pLysS cells. The optimal A600 for isopropyl-β-d-thiogalactoside induction was found to be 0.17, and the log phase of growth was arrested 4 h after isopropyl-β-d-thiogalactoside induction to reach an A600 of 1.8. Cells were harvested, and the membrane fraction was isolated and then solubilized using DDM at a detergent-to-cell ratio of 1:10 (w/w). His-tagged ZitB could be purified to homogeneity by a single step nickel chelate affinity chromatography (Fig. 1A). Overnight incubation of the purified His-ZitB with thrombin at 20 °C resulted in a complete removal of the His tag, as confirmed by Western blot analysis using a His tag-specific antibody (data not shown). The molecular identity of the purified ZitB was confirmed by MALDI-TOF mass spectrometric measurements. Molecular masses of 37,067 ± 7 and 35,179 ± 9 Da were obtained for the purified His-ZitB before and after thrombin digestion. The expected and experimentally measured molecular masses agreed within 23 Da (expected mass: 37,084 and 35,202 Da, respectively). The thrombin-digested ZitB was concentrated to about 10–20 mg/ml, and further purification was achieved by preparative size exclusion HPLC. Analytical size exclusion HPLC analysis indicated that the purified ZitB was free of high molecular weight aggregates, showing a major monodisperse species followed by a minor species with retention times corresponding to apparent molecular masses of 140 and 45 kDa, respectively (Fig. 1B). MALDI-TOF mass spectrometric analysis of these two peak fractions revealed a single mass peak at 35179 Da for the major species and a cluster of mass peaks between 700–800 Da for the minor species, indicating the lipidic nature of the minor species. The monodispersity of the 140-kDa fraction was confirmed by dynamic light scattering analysis. These results indicated that ZitB was purified as a single oligomeric species; however, the order of ZitB oligomerization is yet to be determined. Functional Reconstitution—The detergent-mediated reconstitution of ZitB was achieved using a detergent removal method as described under "Experimental Procedures." DDM in the purified ZitB sample was diluted 40–1000-fold with 1% β-OG prior to reconstitution. Encapsulation of fluozin-1 in reconstituted vesicles was found to be stable over the period of a few days. Exposing ZitB proteoliposomes to 2 mm Zn2+ exterior to proteoliposomes caused a rapid rise of fluozin-1 fluorescence. The background Zn2+ leakage across the membrane was measured using control liposomes and was subtracted to yield a net Zn2+ response attributable to the ZitB-catalyzed Zn2+ influx (Fig. 2A). The initial rate of fluozin-1 response increased linearly with the ZitB-to-lipid ratio (Fig. 2B), demonstrating a linear relationship between the amount of the reconstituted ZitB and the initial rate of the fluorescence response. At a ZitB-to-lipid molar ratio of 8:11,517 (equal to 1:1440), the background Zn2+ leakage is negligible. Hence, this ZitB-to-lipid ratio was used thereafter for all stopped-flow measurements unless otherwise indicated. Furthermore, the quasistationary fluorescence responses also increased with ZitB-to-lipid ratios (Fig. 2A), indicative of the presence of a mixed population of proteoliposomes and liposomes in the reconstituted suspension. Increasing the ZitB-to-lipid ratio resulted in an increase of the proteoliposome population and a consequent increase of the quasistationary response. The quasistationary fluorescence was normalized to the maximum florescence, measured by adding β-OG to 1% at the end of each experiment to solubilize both proteoliposomes and liposomes. The resulting relative fluorescence varied from 0.05 to 0.5 depending on ZitB-to-lipid ratios (Fig. 2A). To determine the specific activity of ZitB, the relationship between the intravesicular Zn2+ concentration and the fluorescent response was calibrated based on Equation 6 as described under "Experimental Procedures." Proteoliposomes were prepared at three ZitB-to-lipid ratios: 4:11,517, 8:11,517, and 16: 11,517. Correspondingly, relative quasistationary responses were 0.43, 0.61, and 0.52. The maximum quasistationary response occurred at a ZitB-to-lipid ratio of 8:11,517. The reduced response at a lower ratio was due to the presence of empty liposomes, whereas the decline at a higher ratio might be caused by the presence of multiple copies of ZitB in one proteoliposome. A calibration curve was obtained using the optimal ZitB-to-lipid ratio of 8:11,517 (Fig. 2C). Under this condition, ΔF/ΔFmax was linearly related to [Zn2+] up to a value of 0.28, corresponding to a zinc concentration of 50 μm. Although this experimentally determined linear range was narrower compared with the theoretical prediction, it was sufficient to quantify initial zinc responses that fell within the range of 0–20% of the ΔFmax. The specific activity was calculated by converting the normalized initial Zn2+ response (ΔF/ΔFmax)/Δt to Δ[Zn2+]/Δt (μm/s), multiplying by the total proteoliposome internal volume (μl) and then normalizing to the total ZitB (μmol) in the reconstitution system. Using the calibration curve obtained from the same sample, the specific activity was determined to be 2.36 ± 0.42 (n = 3) μmol of Zn2+/s/μmol of ZitB, giving an average turnover number of 2.36 s–1 at 8 °C. Substrate Concentration Dependence—The initial rate of Zn2+ influx was measured in a zinc concentration range between 0 and 4 mm exterior to proteoliposomes. Proteoliposomes preloaded with fluozin-1 were rapidly mixed with an assay buffer containing various concentrations of Zn2+, resulting in an inward Zn2+ diffusion gradient that drove Zn2+ influx (Fig. 2A). Due to the low internal volume of membrane vesicles, Zn2+ influx may rapidly build up a transmembrane electrochemical gradient against the imposed Zn2+ diffusion gradient. Therefore, only the initial rate of the Zn2+ filling kinetics was analyzed. The relative initial rate of the fluorescence responses increased in a hyperbolic manner with the Zn2+ concentration and a fit of the relative rate data to a single hyperbolic function yielded a Km of 104.9 ± 5.4 μm (Fig. 3A). This kinetic behavior is consistent with a two-step process shown in Scheme 1, M+T1↔k-1/k1MT1→k2T2+MScheme 1 where T1 and T2 are different conformational states of ZitB, and M is the substrate. The first step is a rapid equilibrium with a binding constant k–1/k1, followed by a rate-limiting conformational transition from T1 to T2 with a rate constant k2. The relationship among k1, k–1, and k2 is defined as Km = (k2 + k–1)/k1. This kinetic scheme suggests that ZitB catalyzes Zn2+ transport with a stoichiometry of one ZitB for one Zn2+. In a parallel set of experiments, the Cd2+ concentration dependence of ZitB transport was investigated. The ZitB-mediated Cd2+ influx showed a rapid rise of fluozin-1 fluorescence upon the mixing of proteoliposomes with an assay buffer containing varying concentrations of Cd2+ ranging form 0 up to 4 mm. The initial rate of the fluorescence response increased in a hyperbolic manner with the Cd2+ concentration. The concentration dependence of the relative initial rate, when fitted to a single hyperbolic function, yielded a Km of 90.1 ± 3.7 μm (Fig. 3B). Therefore, Cd2+ transport can also be interpreted with a two-step process as described above for the Zn2+ transport. These results indi
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