David H. Petering

University of Wisconsin - Milwaukee, Milwaukee, Wisconsin, United States

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Publications (215)756.82 Total impact

  • David H. Petering · Mohammad Razaul Karim
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    ABSTRACT: Newport Green (NPG) is a recognized sensor of cellular Zn(2+) that displays fluorescence enhancement upon binding to Zn(2+). Because of its modest affinity for Zn(2+), the extent of its capacity to bind cellular Zn(2+) is unclear. The present study investigated the range of reactivity of NPGESTER with cells, isolated (Zn)-proteome, and model Zn-proteins. The sensor accumulated in pig kidney LLC-PK1 cells and was slowly (>40 min) hydrolyzed to the fluorescent, acid form, NPGACID. The powerful, cell permeant Zn(2+) chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)-ethane-1,2-diamine (TPEN) failed to quench the growing fluorescence emission, indicating that Zn-NPGACID had not formed and NPG-Zn-protein adduct species probably were not present. Furthermore, NPGACID did not bind to Zn-carbonic anhydrase or Zn-alcohol dehydrogenase, two proteins that form adducts with some other sensors. Strikingly, most of the NPGACID that had been converted from NPGESTER was detected in the extracellular medium not the cells. As a result, after cells were incubated with NPGESTER and then Zn-pyrithione to raise the internal concentration of mobile Zn(2+), Zn-NPGACID was only observed in the external medium. Residual cellular NPGACID was unable to bind extra intracellular Zn(2+) delivered by pyrithione. Proteome isolated from the sonicated cell supernatant was also unreactive with NPGACID. Titration of proteome or glutathione with Zn(2+) in the presence of NPGACID revealed that NPGACID only weakly competes for mobile Zn(2+) in the presence of these cellular components. In addition, when proteomic Zn(2+) was released by a nitric oxide donor or N-ethyl-maleimide, little Zn(2+) was detected by NPGACID. However, exposure to nitric oxide independently enhanced the fluorescence properties of NPGACID. Thus, the biochemical properties of NPG related to cellular Zn(2+) chelation deepen the question of how it functions as a Zn(2+) sensor.
    No preview · Article · Dec 2015 · Metallomics
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    ABSTRACT: Fluorescent zinc sensors are the most commonly used tool to study the intracellular mobile zinc status within cellular systems. Previously, we have shown that the quinoline-based sensors Zinquin and 6-methoxy-8-p-toluenesulfonamido-quinoline (TSQ) predominantly form ternary adducts with members of the Zn-proteome. Here, the chemistries of these sensors are further characterized, including how Zn(sensor)2 complexes may react in an intracellular environment. We demonstrate that these sensors are typically used in higher concentrations than needed to obtain maximum signal. Exposing cells to either Zn(Zinquin)2 or Zn(TSQ)2 resulted in efficient cellular uptake and the formation of sensor-Zn-protein adducts as evidenced by both a fluorescence spectral shift toward that of ternary adducts and the localization of the fluorescence signal within the proteome after gel filtration of cellular lysates. Likewise, reacting Zn(sensor)2 with the Zn-proteome from LLC-PK1 cells resulted in the formation of sensor-Zn-protein ternary adducts that could be inhibited by first saturating the Zn- proteome with excess sensor. Further, a native SDS-PAGE analysis of the Zn-proteome reacted with either the sensor or the Zn(sensor)2 complex revealed that both reactions result in the formation of a similar set of sensor-Zn-protein fluorescent products. The results of this experiment also demonstrated that TSQ and Zinquin react with different members of the Zn-proteome. Reactions with the model apo-Zn-protein bovine serum albumin showed that both Zn(TSQ)2 and Zn(Zinquin)2 reacted to form ternary adducts with its apo-Zn-binding site. Moreover, incubating Zn(sensor)2 complexes with non-zinc binding proteins failed to elicit a spectral shift in the fluorescence spectrum, supporting the premise that blue-shifted emission spectra are due to sensor-Zn-protein ternary adducts. It was concluded that Zn(sensors)2 species do not play a significant role in the overall reaction between these sensors and intact cells. In turn, this study further supports the formation of sensor-Zn-protein adducts as the principal observed fluorescent product during experiments employing these two sensors.
    No preview · Article · Dec 2015 · Inorganic Chemistry
  • Andrew Nowakowski · Mohammad Karim · David Petering

    No preview · Chapter · Sep 2015
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    ABSTRACT: The hypothesis was tested that Cd2 + undergoes measureable reaction with the Zn-proteome through metal ion exchange chemistry. The Zn-proteome of pig kidney LLC-PK1 cells is relatively inert to reaction with competing ligands, including Zinquin acid, EDTA, and apo-metallothionein. Upon reaction of Cd2 + with the Zn-proteome, Cd2 + associates with the proteome and near stoichiometric amounts of Zn2 + become reactive with these chelating agents. The results strongly support the hypothesis that Cd2 + displaces Zn2 + from native proteomic binding sites resulting in the formation of a Cd-proteome. Mobilized Zn2 + becomes adventitiously bound to proteome and available for reaction with added metal binding ligands. Cd-proteome and Zn-metallothionein readily exchange metal ions, raising the possibility that this reaction restores functionality to Cd-proteins. In a parallel experiment, cells were exposed to Cd2 + and pyrithione briefly to generate substantial proteome-bound Cd2 +. Upon transition to a Cd2 + free medium, the cells generated new metallothionein protein over time that bound most of the proteomic Cd2 + as well as additional Zn2 +.
    No preview · Article · Jul 2014 · Journal of inorganic biochemistry
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    ABSTRACT: Abstract Developing zebrafish embryos were used as a model system for high school students to conduct scientific investigations that reveal features of normal development and to test how different environmental toxicants impact the developmental process. The primary goal of the module was to engage students from a wide range of socio-economic backgrounds, with particular focus on underserved inner-city high schools, in inquiry-based learning and hands-on experimentation. In addition, the module served as a platform for both teachers and students to design additional inquiry-based experiments. In this module, students spawned adult zebrafish to generate developing embryos, exposed the embryos to various toxicants, then gathered, and analyzed data obtained from control and experimental embryos. The module provided a flexible, experimental framework for students to test the effects of numerous environmental toxicants, such as ethanol, caffeine, and nicotine, on the development of a model vertebrate organism. Students also observed the effects of dose on experimental outcomes. From observations of the effects of the chemical agents on vertebrate embryos, students drew conclusions on how these chemicals could impact human development and health. Results of pre-tests and post-tests completed by participating students indicate statistically significant changes in awareness of the impact of environmental agents on fish and human beings In addition, the program's evaluator concluded that participation in the module resulted in significant changes in the attitude of students and teachers toward science in general and environmental health in particular.
    Full-text · Article · Jun 2014 · Zebrafish
  • Andrew B Nowakowski · William J Wobig · David H Petering
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    ABSTRACT: Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is commonly used to obtain high resolution separation of complex mixtures of proteins. The method initially denatures the proteins that will undergo electrophoresis. Although covalent structural features of resolved proteins can be determined with SDS-PAGE, functional properties are destroyed, including the presence of non-covalently bound metal ions. To address this shortcoming, blue-native (BN)-PAGE has been introduced. This method retains functional properties but at the cost of protein resolving power. To address the need for a high resolution PAGE method that results in the separation of native proteins, experiments tested the impact of changing the conditions of SDS-PAGE on the quality of protein separation and retention of functional properties. Removal of SDS and EDTA from the sample buffer together with omission of a heating step had no effect on the results of PAGE. Reduction of SDS in the running buffer from 0.1% to 0.0375% together with deletion of EDTA also made little impact on the quality of the electrophoretograms of fractions of pig kidney (LLC-PK1) cell proteome in comparison with that achieved with the SDS-PAGE method. The modified conditions were called native (N)SDS-PAGE. Retention of Zn(2+) bound in proteomic samples increased from 26 to 98% upon shifting from standard to modified conditions. Moreover, seven of nine model enzymes, including four Zn(2+) proteins that were subjected to NSDS-PAGE retained activity. All nine were active in BN-PAGE, whereas all underwent denaturation during SDS-PAGE. Metal retention after electrophoresis was additionally confirmed using laser ablation-inductively coupled plasma-mass spectrometry and in-gel Zn-protein staining using the fluorophore TSQ.
    No preview · Article · Apr 2014 · Metallomics
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    ABSTRACT: Understanding human environmental health is difficult for high school students, as is the process of scientific investigation. This module provides a framework to address both concerns through an inquiry-based approach using a hypothesis-driven set of experiments that draws upon a real-life concern, environmental exposures to lead (Pb2+). Students learn how scientists use model organisms to understand basic biological concepts, and how these models relate to human and environmental health. Students observe how Pb2+ alters fish behaviors. Because many levels of biological organization are involved, this module has application for multiple units within general and advanced biology classes. Beginning with what is known about Pb2+ toxicity, students develop testable hypotheses about how it may affect behavior, apply this knowledge to human populations, and identify the "next experiment."
    Full-text · Article · Mar 2013 · The American Biology Teacher
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    ABSTRACT: Abstract This article presents a detailed guide for high school through graduate level instructors that leads students to write effective and well-organized scientific papers. Interesting research emerges from the ability to ask questions, define problems, design experiments, analyze and interpret data, and make critical connections. This process is incomplete, unless new results are communicated to others because science fundamentally requires peer review and criticism to validate or discard proposed new knowledge. Thus, a concise and clearly written research paper is a critical step in the scientific process and is important for young researchers as they are mastering how to express scientific concepts and understanding. Moreover, learning to write a research paper provides a tool to improve science literacy as indicated in the National Research Council's National Science Education Standards (1996), and A Framework for K-12 Science Education (2011), the underlying foundation for the Next Generation Science Standards currently being developed. Background information explains the importance of peer review and communicating results, along with details of each critical component, the Abstract, Introduction, Methods, Results, and Discussion. Specific steps essential to helping students write clear and coherent research papers that follow a logical format, use effective communication, and develop scientific inquiry are described.
    Full-text · Article · Oct 2012 · Zebrafish
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    Andrew Nowakowski · David Petering
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    ABSTRACT: The impact of the thiol binding reagent N-ethylmaleimide (NEM) on proteomic Zn(2+) availability was investigated in rat glioma cells. Zinquin (ZQ) or TSQ, two related fluorescent sensors, were used to observe reactive Zn(2+). Control cells contained proteomic Zn(2+) but no detectable low molecular weight (LMW) Zn(2+). With either sensor, basal cellular fluorescence emission centered near 470 nm, indicative of sensor-Zn-proteins. ZQ sequestered 13% of proteomic Zn(2+) as Zn(ZQ)(2); TSQ reacted only with the Zn-proteome. NEM (100 μM) abolished LMW thiols, including glutathione (GSH) and lowered proteomic sulfhydryl content about 30%. In ZQ-treated cells, NEM exposure enhanced fluorescent intensity and the formation of Zn(ZQ)(2) (λ(MAX), 492 nm). Cells incubated with TSQ and NEM also displayed increased fluorescence without a spectral shift in wavelength maximum, consistent with increased formation of TSQ-Zn-protein adducts but not Zn(TSQ)(2). In neither experiment was Zn(2+) lost from cells. NEM altered Zn(2+) accessibility to sensors in membrane-nuclear and cytosolic fractions, but Zn(ZQ)(2) was only generated in the cytosol. Similar results were obtained when cell supernatant replaced cells. In contrast, when isolated proteome was reacted with ZQ and 100 μM NEM in the absence of GSH, 70% of the proteomic thiols underwent reaction. As a consequence, most of the ZQ-Zn-protein adducts were converted to Zn(ZQ)(2). Substituting TSQ for ZQ, only increased TSQ-Zn-proteins were observed. Evidently, the results of imaging cells with Zn(2+) sensors are dependent upon the specific chemical properties of the sensors and can only be understood after detailed chemical analysis.
    Preview · Article · Apr 2012 · Metallomics
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    ABSTRACT: Methylmercury (MeHg) exposure at high concentrations poses significant neurotoxic threat to humans worldwide. The present study investigated the mechanisms of glutathione-mediated attenuation of MeHg neurotoxicity in primary cortical culture. MeHg (5 μM) caused depletion of mono- and disulfide glutathione in neuronal, glial and mixed cultures. Supplementation with exogenous glutathione, specifically glutathione monoethyl ester (GSHME) protected against the MeHg induced neuronal death. MeHg caused increased reactive oxygen species (ROS) formation measured by dichlorodihydrofluorescein (DCF) fluorescence with an early increase at 30 min and a late increase at 6h. This oxidative stress was prevented by the presence of either GSHME or the free radical scavenger, trolox. While trolox was capable of quenching the ROS, it showed no neuroprotection. Exposure to MeHg at subtoxic concentrations (3 μM) caused an increase in system x(c)(-) mediated (14)C-cystine uptake that was blocked by the protein synthesis inhibitor, cycloheximide (CHX). Interestingly, blockade of the early ROS burst prevented the functional upregulation of system x(c)(-). Inhibition of multidrug resistance protein-1 (MRP1) potentiated MeHg neurotoxicity and increased cellular MeHg. Taken together, these data suggest glutathione offers neuroprotection against MeHg toxicity in a manner dependent on MRP1-mediated efflux.
    No preview · Article · Mar 2012 · NeuroToxicology
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    Jeffrey W Meeusen · Andrew Nowakowski · David H Petering
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    ABSTRACT: The commonly used Zn(2+) sensors 6-methoxy-8-p-toluenesulfonamidoquinoline (TSQ) and Zinquin have been shown to image zinc proteins as a result of the formation of sensor-zinc-protein ternary adducts not Zn(TSQ)(2) or Zn(Zinquin)(2) complexes. The powerful, cell-permeant chelating agent N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) is also used in conjunction with these and other Zn(2+) sensors to validate that the observed fluorescence enhancement seen with the sensors depends on intracellular interaction with Zn(2+). We demonstrated that the kinetics of the reaction of TPEN with cells pretreated with TSQ or Zinquin was not consistent with its reaction with Zn(TSQ)(2) or Zn(Zinquin)(2). Instead, TPEN and other chelating agents extract between 25 and 35% of the Zn(2+) bound to the proteome, including zinc(2+) from zinc metallothionein, and thereby quench some, but not all, of the sensor-zinc-protein fluorescence. Another mechanism in which TPEN exchanges with TSQ or Zinquin to form TPEN-zinc-protein adducts found support in the reactions of TPEN with Zinquin-zinc-alcohol dehydrogenase. TPEN also removed one of the two Zn(2+) ions per monomer from zinc-alcohol dehydrogenase and zinc-alkaline phosphatase, consistent with its ligand substitution reactivity with the zinc proteome.
    Full-text · Article · Mar 2012 · Inorganic Chemistry
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    Andrew B Nowakowski · David H Petering
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    ABSTRACT: Zinquin (ZQ) is a commonly used sensor for cellular Zn(2+) status. It has been assumed that it measures accessible Zn(2+) concentrations in the nanomolar range. Instead, this report shows a consistent pattern across seven mammalian cell and tissue types that ZQ reacts with micromolar concentrations of Zn(2+) bound as Zn-proteins. The predominant class of products were ZQ-Zn-protein adducts that were characterized in vivo and in vitro by a fluorescence emission spectrum centered at about 470 nm, by their migration over Sephadex G-75 as protein not low molecular weight species, by the exclusion of reaction with lipid vesicles, and by their large aggregate concentration. In addition, variable, minor formation of Zn(ZQ)(2) with a fluorescence band at about 490 nm was observed in vivo in each case. Because incubation of isolated Zn-proteome with ZQ also generated similar amounts of Zn(ZQ)(2), it was concluded that this species had formed through direct ligand substitution in which ZQ had successfully competed for protein-bound Zn(2+). Parallel studies with the model Zn-proteins, alcohol dehydrogenase (ADH), and alkaline phosphatase (AP) revealed a similar picture of reactivity: ZQ(ACID) (Zinquin acid, (2-methyl-8-p-toluenesulfonamido-6-quinolyloxy)acetate)) able to bind to one Zn(2+) and extract the other in Zn(2)-ADH, whereas it removed one Zn(2+) from Zn(2)-AP and did not bind to the other. Zinquin ethyl ester (ethyl(2-methyl-8-p-toluenesulfonamido-6-quinolyloxy)acetate); ZQ(EE)) bound to both proteins without sequestering Zn(2+) from either one. In contrast to a closely related sensor, 6-methoxy-8-p-toluenesulfonamido-quinoline (TSQ), neither ZQ(ACID) nor ZQ(EE) associated with Zn-carbonic anhydrase. A survey of reactivity of these sensors with partially fractionated Zn-proteome confirmed that ZQ and TSQ bind to distinct, overlapping subsets of the Zn-proteome.
    Preview · Article · Sep 2011 · Inorganic Chemistry
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    ABSTRACT: Zn(2+) is a necessary cofactor for thousands of mammalian proteins. Research has suggested that transient fluxes of cellular Zn(2+) are also involved in processes such as apoptosis. Observations of Zn(2+) trafficking have been collected using Zn(2+) responsive fluorescent dyes. A commonly used Zn(2+) fluorophore is 6-methoxy-8-p-toluenesulfonamido-quinoline (TSQ). The chemical species responsible for TSQ's observed fluorescence in resting or activated cells have not been characterized. Parallel fluorescence microscopy and spectrofluorometry of LLC-PK(1) cells incubated with TSQ demonstrated punctate staining that concentrated around the nucleus and was characterized by an emission maximum near 470 nm. Addition of cell permeable Zn-pyrithione resulted in greatly increased, diffuse fluorescence that shifted the emission peak to 490 nm, indicative of the formation of Zn(TSQ)(2). TPEN (N,N,N'N'-tetrakis(-)[2-pyridylmethyl]-ethylenediamine), a cell permeant Zn(2+) chelator, largely quenched TSQ fluorescence returning the residual fluorescence to the 470 nm emission maximum. Gel filtration chromatography of cell supernatant from LLC-PK(1) cells treated with TSQ revealed that TSQ fluorescence (470 nm emission) eluted with the proteome fractions. Similarly, addition of TSQ to proteome prior to chromatography resulted in 470 nm fluorescence emission that was not observed in smaller molecular weight fractions. It is hypothesized that Zn-TSQ fluorescence, blue-shifted from the 490 nm emission maximum of Zn(TSQ)(2), results from ternary complex, TSQ-Zn-protein formation. As an example, Zn-carbonic anhydrase formed a ternary adduct with TSQ characterized by a fluorescence emission maximum of 470 nm and a dissociation constant of 1.55 × 10(-7) M. Quantification of TSQ-Zn-proteome fluorescence indicated that approximately 8% of cellular Zn(2+) was imaged by TSQ. These results were generalized to other cell types and model Zn-proteins.
    Full-text · Article · Aug 2011 · Inorganic Chemistry
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    ABSTRACT: The present paper centers on mammalian metallothionein 1 and 2 in relationship to cell and tissue injury beginning with its reaction with Cd²⁺ and then considering its role in the toxicology and chemotherapy of both metals and non-metal electrophiles and oxidants. Intertwined is a consideration of MTs role in tumor cell Zn²⁺ metabolism. The paper updates and expands on our recent review by Petering et al. (Met Ions Life Sci 5:353-398, 2009).
    No preview · Article · Aug 2011 · European Journal of Biochemistry
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    ABSTRACT: Diabetes may alter renal glucose reabsorption by sodium (Na(+))-dependent glucose transporters (SGLTs). Radiolabeled substrates are commonly used for in vitro measurements of SGLT activity in kidney cells. We optimized a method to measure glucose uptake using a fluorescent substrate, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG). Uptake buffers for 2-NBDG were the same as for (14)C-labeled α-methyl-d-glucopyranoside ([(14)C]AMG). Cell lysis buffer was optimized for fluorescence of 2-NBDG and Hoechst DNA stain. Uptake was performed on cultures of primary mouse kidney cells (PMKCs), the LLC-PK(1) proximal tubule cell line, or COS-7 cells transiently overexpressing mouse SGLT1 or SGLT2 by incubating cells at 37°C in buffer containing 50-200 μM 2-NBDG. Microscopy was performed to visualize uptake in intact cells, while a fluorescence microplate reader was used to measure intracellular concentration of 2-NBDG ([2-NBDG](i)) in cell homogenates. Fluorescent cells were observed in cultures of PMKCs and LLC-PK(1) cells exposed to 2-NBDG in the presence or absence of Na(+). In LLC-PK(1) cells, 2-NBDG transport in the presence of Na(+) had a maximum rate of 0.05 nmol/min/μg of DNA. In these cells, Na(+)-independent uptake of 2-NBDG was blocked with the GLUT inhibitor, cytochalasin B. The Na(+)-dependent uptake of 2-NBDG decreased in response to co-exposure to the SGLT substrate, AMG, and it could be blocked with the SGLT inhibitor, phlorizin. Immunocytochemistry showed overexpression of SGLT1 and SGLT2 in COS-7 cells, in which, in the presence of Na(+), [2-NBDG](i) was fivefold higher than in controls. Glucose transport in cultured kidney cells can be measured with the fluorescence method described in this study.
    Preview · Article · Jul 2011 · Diabetes Technology & Therapeutics
  • S. Seal · S. Krezoski · T. L. Barr · D. H. Petering

    No preview · Article · Jan 2011 · MRS Online Proceeding Library
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    ABSTRACT: Subsistence fishing is a common strategy for ethnic communities to obtain high quality protein that partially compensates for economic inequalities that they experience. At the same time, subsistence fishing provides a way to transmit cultural traditions across generations. While fish is a source of high quality, low fat protein and health-promoting omega-3-fatty acids, fish also contain varying concentrations of environmental contaminants. Our Children's Environmental Health Sciences Core Center has conducted several fish benefit/risk communication projects with ethnic communities in Milwaukee and Anishinaabe tribal members. For our current project, we collaborated with community partners to understand patterns of fishing, cooking, and consumption of fish (by species, size, and specific waterways) through key informants and focus group participants in the urban African American community. We used these data together with existing published data to estimate the concentration of omega-3-fatty acids and data from creel surveys, provided by the Department of Natural Resources, to estimate the uptake of mercury and other contaminants for men, women of childbearing age, and children. We describe the process and outcomes of estimating benefits and risks of eating locally caught fish for community members. In addition, we discuss multiple methods that we have employed to develop culturally sensitive communication processes and tools that involve community members, scientists, communication and outreach specialists, and videographers.
    No preview · Conference Paper · Nov 2010
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    Rajendra Kothinti · Niloofar M Tabatabai · David H Petering
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    ABSTRACT: The electrophoretic mobility shift assay (EMSA) offers a principal method to detect specific DNA-protein interactions. As commonly conducted, the reaction and electrophoresis running buffers contain large concentrations of EDTA. EDTA has large affinity for Zn(2+) and readily competes with zinc finger peptides for Zn(2+) resulting in protein unfolding. Nevertheless, EMSA is routinely used to detect zinc finger protein-DNA adducts. This paper examines the chemistry that permits the detection of zinc finger-DNA complexes in the presence of EDTA, using Zn(3)-Sp1 and a cognate DNA binding site, GC1. Twice as much adduct was detected when the reaction was conducted in the absence than in the presence of EDTA. The observation of Zn-Sp1-GC1 was shown to depend on three properties: the inertness of Zn-Sp1-GC1 to reaction with EDTA and the comparatively similar rates of reaction of EDTA and GC1 with Zn(3)-Sp1 under the conditions of the assay that permit some Zn(3)-Sp1-GC1 to form. Inquiring about the mechanism of stabilization of Zn(3)-Sp1 by GC1, EDTA readily reacted with Zn(3)-Sp1 bound to a non-specific DNA, (polydI-dC). Two structurally similar but oppositely charged chelators, nitrilotriacetate (NTA) and tris-(2-ethylaminoethyl) amine (TREN), that react with free Zn(3)-Sp1 failed to compete for zinc bound in the Zn(3)-Sp1-GC-1 adduct. On the basis of these, other results indicated that the stability of Zn(3)-Sp1-GC-1 has a thermodynamic, not a kinetic origin. It is concluded that the observation of zinc finger proteins in the EMSA rests on a fortuitous set of chemical properties that may vary depending on the structures involved.
    Preview · Article · Aug 2010 · Journal of inorganic biochemistry
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    John Ejnik · C Frank Shaw · David H Petering
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    ABSTRACT: Cellular metallothionein (MT) protects against Cd(2+) exposure through direct binding of the metal ion. The model reaction between rabbit liver Zn(7)-MT-2 with Cd(2+) was studied with stopped flow kinetics. Four kinetic steps were observable. Comparison of this reaction with an analog utilizing the MT Zn(4)-alpha domain revealed that only the fastest step involved the Zn(3)-beta domain. Each step of the Zn(4)-alpha domain reaction with Cd(2+) displayed hyperbolic dependence of the observed rate constant on Cd(2+) concentration, with the first step comprising 50% of the total reaction and each of the other two, 25%. The two constants extracted from each of these relationships were interpreted as the equilibrium constant for the initial binding of Cd(2+) to the Zn((4-n)),Cd(n)-thiolate cluster (n = 0-3) of the alpha domain and the first order rate constant for the exchange of Cd(2+) for Zn(2+) in the cluster. Activation enthalpies and entropies were determined for each constant. A suite of Zn((4-n)),Cd(n)-thiolate clusters (n = 0-3) was prepared by titration of the Zn(4)-alpha domain with (113)Cd(2+). The products were analyzed by one-dimensional (113)Cd(2+) NMR spectroscopy to define the distribution of (113)Cd(2+) among the four cluster binding sites. Each of these species was also reacted with Cd(2+). The properties of these reactions were similar to those extracted from the reaction of Cd(2+) with the overall domain. Thus, the kinetic results were linked to (113)Cd(2+) occupancy among the cluster metal binding sites. In turn, this linkage permitted the interpretation of the various constants determined for the reaction of Cd(2+) with the Zn(4)-alpha domain in relation to the alpha domain cluster structure.
    Preview · Article · Jul 2010 · Inorganic Chemistry
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    ABSTRACT: Cadmium (Cd) exposure causes glucosuria (glucose in the urine). Previously, it was shown that Cd exposure of primary cultures of mouse kidney cells (PMKC) decreased mRNA levels of the glucose transporters, SGLT1 and SGLT2 and that Sp1 from Cd-exposed cells displayed reduced binding to the GC boxes of the mouse SGLT1 promoter in vitro. Here, we identified a GC box upstream of mouse SGLT2 gene. ChIP assays on PMKC revealed that exposure to 5 microM Cd abolished Sp1 binding to SGLT1 GC box while it decreased Sp1 binding to SGLT2 GC sequence by 30% in vivo. The in vitro DNA binding assay, EMSA, demonstrated that binding of Sp1 from Cd (7.5 microM)-treated PMKC to the SGLT2 GC probe was 86% lower than in untreated cells. Sp1 is a zinc finger protein. Compared to PMKC exposed to 5 microM Cd alone, inclusion of 5 microM Zn restored SGLT1 and 2 mRNA levels by 15% and 30%, respectively. Cd (10 microM) decreased the binding of recombinant Sp1 (rhSp1) to SGLT1 and SGLT2 GC probes to 12% and 8% of untreated controls. Cd exerted no effect on GC-bound rhSp1. Co-treatment with Cd and Zn showed that added Zn significantly restored rhSp1 binding to the SGLT1 and SGLT2. Addition of Zn post Cd treatment was not stimulatory. We conclude that Cd can replace Zn in Sp1 DNA binding domain to reduce its binding to GC sites in mouse SGLT1 and SGLT2 promoters.
    Preview · Article · May 2010 · Toxicology and Applied Pharmacology

Publication Stats

5k Citations
756.82 Total Impact Points

Institutions

  • 1974-2015
    • University of Wisconsin - Milwaukee
      • • Department of Chemistry and Biochemistry
      • • College of Nursing
      Milwaukee, Wisconsin, United States
  • 1972-2003
    • Northwestern University
      • Department of Chemistry
      Evanston, Illinois, United States
  • 2000
    • Chuo University
      • Department of Applied Chemistry
      Tokyo, Tokyo-to, Japan
  • 1998
    • Medical College of Wisconsin
      • Department of Medicine
      Milwaukee, Wisconsin, United States