Name: simultaneous measurement of superoxide/hydrogen peroxide and nadh production by flavin-containing mitochondrial dehydrogenases
Uploaded: 2018-02-24T14:00:00
Description: Watch this Scientific Journal Video about Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases at JoVE.com

This work was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC). Video production was carried out in collaboration with the Center for Innovation in Teaching and Learning (CITL) at Memorial University of Newfoundland.

1. Chemicals and Purified Enzymes
<ol>
	<li>Procure the following materials: PDHC and KGDHC of porcine heart origin (or another purified mitochondrial flavoenzyme); H2O2 (30% solution), pyruvate, &#945;-ketoglutarate, NAD+, NADH, CoASH, thiamine pyrophosphate (TPP), mannitol, HEPES, sucrose, EGTA, 3-methyl-2-oxo valeric acid (KMV), superoxide dismutase (SOD), horseradish peroxidase (HRP), 10-Acetyl-3,7-dihydroxyphenoxazine reagent (AUR), and CPI-613.</li>
</ol>
2. ...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+mitochondrial+reactive+oxygen+species+production+by+GSH+mediated+S-glutathionylation+of+2-oxoglutarate+dehydrogenase.">Induction of mitochondrial reactive oxygen species production by GSH mediated S-glutathionylation of 2-oxoglutarate dehydrogenase.</a> Redox Biol. 8, 285-297 (2016).</li><li>Mailloux, R. J., Gardiner, D., O'Brien, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2-Oxoglutarate+dehydrogenase+is+a+more+significant+source+of+O2(.-)/H2O2+than+pyruvate+dehydrogenase+in+cardiac+and+liver+tissue.">2-Oxoglutarate dehydrogenase is a more significant source of O2(.-)/H2O2 than pyruvate dehydrogenase in cardiac and liver tissue.</a> Free Radic Biol Med. 97, 501-512 (2016).</li><li>Ambrus, A., Adam-Vizi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dihydrolipoamide+dehydrogenase+(E3)+deficiency:+Novel+insights+into+the+structural+basis+and+molecular+pathomechanism.">Human dihydrolipoamide dehydrogenase (E3) deficiency: Novel insights into the structural basis and molecular pathomechanism.</a> Neurochem Int. , (2017).</li><li>Young, A., Gardiner, D., Brosnan, M. E., Brosnan, J. T., Mailloux, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+levels+of+formate+activate+mitochondrial+superoxide/hydrogen+peroxide+release+from+mouse+liver+mitochondria.">Physiological levels of formate activate mitochondrial superoxide/hydrogen peroxide release from mouse liver mitochondria.</a> FEBS Lett. 591 (16), 2426-2438 (2017).</li><li>Fisher-Wellman, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+glutathione+depletion+reveals+a+novel+role+for+the+pyruvate+dehydrogenase+complex+as+a+key+H2O2-emitting+source+under+conditions+of+nutrient+overload.">Mitochondrial glutathione depletion reveals a novel role for the pyruvate dehydrogenase complex as a key H2O2-emitting source under conditions of nutrient overload.</a> Free Radic Biol Med. 65, 1201-1208 (2013).</li><li>Kussmaul, L., Hirst, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+superoxide+production+by+NADH:ubiquinone+oxidoreductase+(complex+I)+from+bovine+heart+mitochondria.">The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria.</a> Proc Natl Acad Sci U S A. 103 (20), 7607-7612 (2006).</li><li>Huang, L. 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This protocol is advantageous since, 1) it eliminates any competing reactions that may otherwise interfere with H2O2 detection (e.g., antioxidant systems or other sources of ROS), 2) provides a direct assessment of the native rate of ROS release by a flavin-containing mitochondrial dehydrogenase, 3) allows the comparison of the native ROS release rates of two or more purified flavin-based dehydrogenases, 4) can allow for a direct comparison of the rate of ROS release and enzyme activity, an...

The ultimate goal of nutrient metabolism is to make adenosine triphosphate (ATP). In mammalian cells, this occurs in mitochondria, double-membraned organelles that convert the energy stored in carbon into ATP. The production of ATP begins when carbon is combusted by mitochondria forming two electron carriers, NADH and flavin adenine dinucleotide (FADH2)1. NADH and FADH2 are then oxidized by multi-subunit respiratory complexes I and II, respectively, and the liberated electrons are ferried to the terminal electron acceptor molecular oxygen (O2) at complex IV1. The thermodynamically favorable &#34;downhill&#34; transfer of electrons to O2 at the end of the chain is coupled to the export of protons into the intermembrane space by complexes I, III, and IV. This creates a transmembrane electrochemical gradient of protons (proton-motive force), a temporary form of Gibbs free energy that is tapped by complex V to make ATP2. Electron transfer reactions in mitochondria are not perfectly coupled to ATP production. At various points in the Krebs cycle and the respiratory chain, electrons can prematurely interact with O2 to form ROS3. The most biologically relevant ROS generated by mitochondria are O2&#9679;- and H2O2. Although O2&#9679;- is often considered the proximal ROS formed by mitochondria, it is now evident that sites of production form a mixture of O2&#9679;- and H2O2, which is associated with the free radical chemistry of flavin prosthetic groups4,5. At high levels, ROS can be dangerous, damaging biological constituents required for cell function resulting in oxidative distress6. However, at low amounts, mitochondrial ROS fulfill vital signaling functions. For instance, H2O2 release from mitochondria has been implicated in controlling T-cell activation, stress signaling (e.g., induction of Nrf2 signaling pathways), the induction of cell proliferation and differentiation, insulin signaling and release, and feeding behavior7. Considerable progress has been made in understanding the signaling function of ROS. However, important questions still remain in regard to which enzymes in mitochondria serve as the most important sources and how production is controlled.
ROS release by a site of production depends on several factors: 1) the concentration of the electron donating site, 2) the redox state of the electron donating site, 3) access to oxygen, and 4) the concentration and type of oxidation substrate3,8,9. In mitochondria, other factors like the concentration of NADH and membrane potential strength also influence ROS production8,10. For example, the rate of O2&#9679;-/H2O2 production by purified PDHC or KGDHC increases with increasing NADH availability5,11. In this scenario, electrons are flowing backwards from NADH to the FAD center in the E3 subunit of PDHC or KGDHC, the site for O2&#9679;-/H2O2 production12. Similarly, the provision of NAD+ has the opposite effect, decreasing ROS release from KGDHC12. Thus, controlling entry or exit of electrons from sites of ROS production can alter how much O2&#9679;-/H2O2 is formed. For instance, blocking the E2 subunit of PDHC or KGDHC with CPI-613, a lipoic acid analog, results in an almost 90% decrease in O2&#9679;-/H2O2 production13. Similar results can be obtained with the chemical S-glutathionylation catalysts, diamide and disulfiram, which almost abolish O2&#9679;-/H2O2 production by PDHC or KGDHC via the conjugation of glutathione to the E2 subunit14. The trade-off for blocking electron flow through the E2 subunit of PDHC and KGDHC is a decrease in NADH production which diminishes ROS formation by the electron transport chain (e.g., complex III). This can also decrease ATP output by the electron transport chain. Overall, blocking electron entry to sites of ROS production can be a highly effective means of controlling O2&#9679;-/H2O2 production.
Mitochondria can contain up to 12 potential O2&#9679;-/H2O2 sources6. Most of these sites are flavin-containing enzymes which generate a mixture of O2&#9679;- and H2O2. Use of different substrate and inhibitor combinations have allowed for the identification of which respiratory complexes and mitochondrial dehydrogenases serve as high capacity O2&#9679;-/H2O2 forming sites in different tissues3. PDHC and KGDHC have been shown to serve as high capacity O2&#9679;-/H2O2 emitting sites in muscle and liver mitochondria13,15. However, some difficulties remain in regard to the examination of the O2&#9679;-/H2O2 forming potential of individual sites in mitochondria and the impact that different substrate and inhibitor combinations have on the activities of the enzymes. This is due to the presence of unwanted side reactions (e.g., formation of O2&#9679;-/H2O2 by sites other than the enzyme of interest), contaminating endogenous nutrients (e.g.,fatty acids) or organelles (e.g., peroxisomes which also form O2&#9679;-/H2O2), and use of inhibitors that lack selectivity, and/or use of compounds that do not fully inhibit ROS production. Certain inhibitors can also alter the mitochondrial bioenergetics and direction of electron flow, and this alters ROS release from other sites of production and confounds results. Absolute rates for O2&#9679;-/H2O2 release from individual sites in mitochondria are also difficult to quantify due to the high concentration of O2&#9679;- and H2O2 eliminating enzymes in the matrix and intermembrane space. Therefore, elimination of any competing reactions that can interfere with O2&#9679;-/H2O2 release measurements can be useful when identifying high capacity O2&#9679;-/H2O2 forming sites.
Here, we present a simple method that allows for the simultaneous examination of O2&#9679;-/H2O2 production and NADH formation by purified flavin-dependent dehydrogenases. By using purified enzymes, unwanted ROS forming side reactions and ROS degrading enzymes can be eliminated allowing for a more accurate measure of native O2&#9679;-/H2O2 production rates for individual flavoenzymes. This method can be used to directly compare the O2&#9679;-/H2O2 forming capacity of different purified dehydrogenases or to screen potential site-specific inhibitors for O2&#9679;-/H2O2 release. Finally, measuring O2&#9679;-/H2O2 and NADH production simultaneously can allow for a real-time assessment of the relationship between enzyme activity and ROS release capacity.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pyruvate dehydrogenase complex</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">P7032-10UN</td>
			<td style="width:167px;">purified flavoenzyme</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">alpha-ketoglutarate dehydrogenase complex</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">K1502-20UN</td>
			<td>purified flavoenzyme</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">30% hydrogen peroxide solution</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">HX0640-5</td>
			<td>reagent, standard curves</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NAD+</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">N0632-1G</td>
			<td>reagent, activity/ROS release assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NADH</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">N4505-100MG</td>
			<td>reagent, standard curves</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pyruvate</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">P2256-5G</td>
			<td>reagent, activity/ROS release assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">alpha-ketoglutarate</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">75892-25G</td>
			<td>reagent, activity/ROS release assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CoASH</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">C3019-25MG</td>
			<td>reagent, activity/ROS release assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">thiamine pyrophosphate</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">C8754-1G</td>
			<td>reagent, activity/ROS release assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mannitol</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">M4125-100G</td>
			<td>buffer component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hepes</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">H3375-25G</td>
			<td>buffer component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sucrose</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">S7903-250G</td>
			<td>buffer component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EGTA</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">E3889-10G</td>
			<td>buffer component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KMV</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">198978-5G</td>
			<td>reagent, ROS release inhibitor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CPI-613</td>
			<td style="width:71px;">Santa Cruz</td>
			<td style="width:119px;">sc-482709</td>
			<td>reagent, ROS release inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SOD</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">S9697-15KU</td>
			<td>reagent, ROS release detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">horseradish peroxidase</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">P8375-1KU</td>
			<td>reagent, ROS release detection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Amplex Ultra Red</td>
			<td style="width:71px;">Thermofisher</td>
			<td style="width:119px;">A36006</td>
			<td>reagent, ROS release detection</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Biotech Synergy 2 microplate reader</td>
			<td style="width:71px;">BioTek Instruments</td>
			<td style="width:119px;"></td>
			<td>microplate reader for assays</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Gen5 software</td>
			<td style="width:71px;">BioTek Instruments</td>
			<td style="width:119px;"></td>
			<td>software, used for collection of raw RFU</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Graphpad Prism</td>
			<td style="width:71px;">Graphpad software</td>
			<td style="width:119px;"></td>
			<td>software, data analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microsoft EXCEL</td>
			<td style="width:71px;">Microsoft</td>
			<td style="width:119px;"></td>
			<td>software, data analysis</td>
		</tr>
	</tbody>
</table>

simultaneous measurement of superoxide/hydrogen peroxide and nadh production by flavin-containing mitochondrial dehydrogenases

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include flavin-dependent respiratory complexes and dehydrogenases that produce a mixture of superoxide (O2&#9679;-) and hydrogen peroxide (H2O2). Accurate quantification of the ROS-producing potential of individual sites in isolated mitochondria can be challenging due to the presence of antioxidant defense systems&#160;and side reactions that also form O2&#9679;-/H2O2. Use&#160;of nonspecific inhibitors that can disrupt mitochondrial bioenergetics can also compromise measurements by altering&#160;ROS release from other sites of production. Here, we present an easy method for the simultaneous measurement of H2O2 release and nicotinamide adenine dinucleotide (NADH) production by purified flavin-linked dehydrogenases. For our purposes here, we have used purified pyruvate dehydrogenase complex (PDHC) and &#945;-ketoglutarate dehydrogenase complex (KGDHC) of porcine heart origin as examples. This method allows for an accurate measure of native H2O2 release rates by individual sites of production by eliminating other potential sources of ROS and antioxidant systems. In addition, this method allows for a direct comparison of the relationship between H2O2 release and enzyme activity and the screening of the effectiveness and selectivity of inhibitors for ROS production. Overall, this approach can allow for the in-depth assessment of native rates of ROS release for individual enzymes prior to conducting more sophisticated experiments with isolated mitochondria or permeabilized muscle fiber.

Figure 3A provides a representative trace for the RFU collected during the simultaneous measurement of H2O2 and NADH production by purified KGDHC. The raw RFU data for each time interval is depicted in Figure 3B. The raw RFU data are then exported for analysis. By extrapolating from standard curves presented in Figure 2, the absolute amount of NADH and H2O2...

Watch this Scientific Journal Video about Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases at JoVE.com

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

Mitochondria contain several flavin-dependent enzymes that can produce reactive oxygen species (ROS). Monitoring ROS release from individual sites in mitochondria is challenging due to unwanted side reactions. We present an easy, inexpensive method for direct assessment of native rates for ROS release using purified flavoenzymes and microplate fluorometry.

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include ...

The overall goal of this procedure is to quantify the superoxide/hydrogen peroxide and NADH production rate of purified flavin-containing mitochondrial dehydrogenases using microplate fluorometry. Mitrochondria can contain up to 12 different superoxide/hydrogen peroxide forming sites which have been identified using different substrate and inhibitor combinations with isolated mitochondria. However, some difficulties still remain in regard to accurately quantifying the release capacity of individual sites of production in isolated mitochondria.And this is associated with unwanted side reactions that also produce reactive oxygen species, but also the presence of antioxidants and the use of inhibitors that lack specificity in addition to the use of additives that can interfere with measurements. Here we present the method for the simultaneous measure of superoxide/hydrogen peroxide and NADH production by purified flavin-dependent mitochondrial dehydrogenases. This method is advantageous since it can allow for the direct and accurate quantification of the ROS-producing potential of individual sites of production by eliminating unwanted side reactions and any reactive oxygen species quenching systems.The simultaneous measure of both ROS and NADH production also allows for direct comparison of the superoxide/hydrogen peroxide forming potential and activity of individual enzymes which can be valuable for screening inhibitors for ROS production prior to conducting assays with mitochondria. For our purposes here, we will be using pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex of porcine heart origin as examples. Both are flavin-containing enzymes and known sources for reactive oxygen species.In addition, these two enzymes can be easily purified. The simultaneous measurement of superoxide/hydrogen peroxide and NADH is achieved by tracking changes in florescence. NADH has its own intrinsic florescence properties which allows for easy measurement of its production.Changes in superoxide/hydrogen peroxide meanwhile requires superoxide dismutatase, horseradish peroxidase and amplex ultrared reagent. In the presence of horseradish peroxidase and hydrogen peroxide, the amplex ultrared is converted from a non-fluorescent molecule to fluorescent resorufin. Superoxide dismutase ensures that any superoxide formed by the dehydrogenase being studied is converted to hydrogen peroxide.Prior to setting up the assay, working solutions are prepared. To streamline the assay, all reagents are added to the wells of a 96-well black plate in 20-microliter aliquots. The volume for each reaction in the well is 200 microliters, therefore working solutions are 10 times the concentration found in the reaction well.Next, program the microplate reader to run a kinetic assay for the dual measurement of NADH autoflorescence and resorufin florescence. This is done by first selecting Procedure and setting it to 25 degrees Celsius. Next, click Start Kinetic.Set the time to run for approximately five minutes followed by selection of a fluorescent read interval of 30 seconds. Next, click Read and set the fluorescent measurement parameters for your assay. Here the position of the probe, the wavelengths used for tracking changes in florescence and the wells that will be read are inputed into the software.The different channels for the fluorescent reads are also selected with one being dedicated to the autoflorescence of NADH, which is set to 376 nanometers excitation and 450 nanometer emission wavelengths. Changes in resorufin florescence are tracked at 565 nanometers and 600 nanometers. Once the assay conditions have been set up, individual wells are loaded with buffer and different reagents required to measure both NADH and superoxide/hydrogen peroxide production at the same time.The order for loading the wells with different reagents is as follows. First, the requisite amount of buffer is added to the well followed by the addition of 20 microliters of purified enzyme solution. The enzyme is equilibrated in the buffer for a few minutes inside the instrument.And then, the remaining reagents are added in the following order. Once the substrate has been added, press Start. The plate carrier will enter the reader and the protocol will be initiated.During the assay, realtime outputs for raw relative fluorescent units, or RFUs, can be tracked using the software. The user can flip between the different fluorescent reads by using the Data tab above the well layout. Note that curves for the different fluorescent reads can be selected.In addition, by holding Control on the keyboard and left-clicking on the wells, one can yield a composite graph allowing for the realtime comparisons of the different reactions. Upon completion of the experiment, the microplate carrier will exit the instrument. At this point, results in the representative graph for changes in RFU are exported to Excel.Raw RFU values are exported by first clicking on Data on the representative RFU graph. This will yield a table of raw RFU values for NADH and resorufin florescence. Not that raw RFU numbers corresponding to changes in NADH or resorufin florescence cannot be exported simultaneously.Once exported, results are organized for analysis. Using standard curves for NADH and resorufin florescence generated prior to conducting the assay, the amount of NADH in micromole and superoxide/hydrogen peroxide in picomole formed over time can be calculated. This can be followed by calculating the rate of NADH and superoxide/hydrogen peroxide production.For this procedure, it is vital to check the purity of your isolated flavin-containing dehydrogenase prior to conducting assays. This can be achieved by conducting a simple coomassie stain for proteins or by assaying the activity of the enzyme in the presence of different substrates. Note that it is advised that reducing agents like DTT should be omitted from the assay since it can serve as a substrate for superoxide/hydrogen peroxide production by flavin-linked dehydrogenases.Overall, this method is highly flexible in that it can be used to screen inhibitors for superoxide/hydrogen peroxide production and also allow for a direct comparison of the ROS-forming capacity of different flavin-linked mitochondrial dehydrogenases. After watching this video, you should have an intimate understanding of how to measure ROS and NADH production by purified dehydrogenases. This assay can serve as a stepping stone for more sophisticated experiments with isolated mitochondria.

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Research

JoVE Journal

Biochemistry

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human organs, possess highly conserved metabolic pathways and have a relatively short lifespan that allows the study of different fundamental mechanisms in a short period of time. It is, however, surprising that one of the mechanisms essential for cellular metabolism, the mitochondrial respiration, has not been thoroughly investigated in this model. It is likely because the measure of the mitochondrial respiration in Drosophila usually requires a very large number of individuals and the results obtained are not highly reproducible. Here, a method allowing the precise measurement of mitochondrial oxygen consumption using minimal amounts of tissue from Drosophila is described. In this method, the thoraxes are dissected and permeabilized both mechanically with sharp forceps and chemically with saponin, allowing different compounds to cross the cell membrane and modulate the mitochondrial respiration. After permeabilization, a protocol is performed to evaluate the capacity of the different complexes of the electron transport system (ETS) to oxidize different substrates, as well as their response to an uncoupler and to several inhibitors. This method presents many advantages compared to methods using mitochondrial isolations, as it is more physiologically relevant because the mitochondria are still interacting with the other cellular components and the mitochondrial morphology is conserved. Moreover, sample preparations are faster, and the results obtained are highly reproducible. By combining the advantages of Drosophila as a model for the study of metabolism with the evaluation of mitochondrial respiration, important new insights can be unveiled, especially when the flies are experiencing different environmental or pathophysiological conditions.

In this paper, a method to measure oxygen consumption using high-resolution respirometry in permeabilized thoraxes of Drosophila is described. This technique requires a minimal amount of tissue compared to the classic mitochondrial isolation technique and the results obtained are more physiologically relevant.

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

Measuring Liver Mitochondrial Oxygen Consumption and Proton Leak Kinetics to Estimate Mitochondrial Respiration in Holstein Dairy Cattle

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert NADH and FADH into ATP. Since mitochondria are also the primary site for oxygen use and nutrient oxidation to carbon dioxide and water, how efficiently they use oxygen and produce ATP directly relates to the efficiency of nutrient metabolism, nutrient requirements of the animal, and health of the animal. The purpose of this method is to examine mitochondrial respiration, which can be used to examine the effects of different drugs, diets and environmental effects on mitochondrial metabolism. Results include oxygen consumption measured as proton dependent respiration (State 3) and proton leak dependent respiration (State 4). The ratio of State 3 / State 4 respiration is defined as respiratory control ratio (RCR) and can represent mitochondrial energetic efficiency. Mitochondrial proton leak is a process that allows dissipation of mitochondrial membrane potential (MMP) by uncoupling oxidative phosphorylation from ADP decreasing the efficiency of ATP synthesis. Oxygen and TRMP+ sensitive electrodes with mitochondrial substrates and electron transport chain inhibitors are used to measure State 3 and State 4 respiration, mitochondrial membrane PMF (or the potential to produce ATP) and proton leak. Limitations to this method are that liver tissue must be as fresh as possible and all biopsies and assays must be performed in less than 10 h. This limits the number of samples that can be collected and processed by a single person in a day to approximately 5. However, only 1 g of liver tissue is needed, so in large animals, such as dairy cattle, the amount of sample needed is small relative to liver size and there is little recovery time needed.

Here, we share methods for measuring mitochondrial oxygen consumption, a defining concept of nutritional energetics, and proton leak, the primary cause of inefficiency in mitochondrial generation of ATP. These results can account for 30% of the energy lost in nutrient utilization to help evaluate mitochondrial function.

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, &#947;-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

We describe a method for the characterization of proton-driven membrane transporters in membrane vesicle preparations produced by heterologous expression in E. coli and lysis of cells using a French press.

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked&#160;bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple causes, including autoimmune diseases, diabetes, infections, inherited disorders, and tumors. Assessing mitochondrial function in mouse peripheral nerves can be challenging due to the small sample size, a limited number of mitochondria present in the tissue, and the presence of a myelin sheath. The technique described in this work minimizes these challenges by using a unique permeabilization protocol adapted from one used for muscle fibers, to assess sciatic nerve mitochondrial function instead of isolating the mitochondria from the tissue. By measuring fluorimetric reactive species production with Amplex Red/Peroxidase and comparing different mitochondrial substrates and inhibitors in saponin-permeabilized nerves, it was possible to detect mitochondrial respiratory states, reactive oxygen species (ROS), and the activity of mitochondrial complexes simultaneously. Therefore, the method presented here offers advantages compared to the assessment of mitochondrial function by other techniques.

High-resolution respirometry coupled to fluorescence sensors determines mitochondrial oxygen consumption and reactive oxygen species (ROS) generation. The present protocol describes a technique to assess mitochondrial respiratory rates and ROS production in the permeabilized sciatic nerve.

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple ...