Name: a fluorescence-based assay of phospholipid scramblase activity
Uploaded: 2016-09-20T08:00:00
Description: Watch this Scientific Journal Video about A Fluorescence-based Assay of Phospholipid Scramblase Activity at JoVE.com

This study was supported by the Velux Stiftung (A.K.M.), NIH grant EY024207 (A.K.M.) and the Austrian Science Fund (FWF) project J3686 (B.P.).

1. Preparation of Liposomes and Proteoliposomes
<ol>
	<li>Liposome Formation
	<ol>
		<li>Using a glass syringe, add 1,435 &#181;l POPC (25 mg/ml, in chloroform) and 160 &#181;l POPG (25 mg/ml, in chloroform) to a round bottom flask to obtain 52.5 &#181;mol lipids in a molar ratio of POPC:POPG = 9:1.</li>
		<li>Dry the lipids for 30 min using a rotary evaporator at a rotation speed of 145 rpm (no water bath is needed for this volume of solvent), then transfer the flask to a vacuum desiccator for at least 3 hr, or overni...

<ol>
	<li>Ernst, O. P., 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=Microbial+and+animal+rhodopsins:+structures,+functions,+and+molecular+mechanisms.">Microbial and animal rhodopsins: structures, functions, and molecular mechanisms.</a> Chem. Rev. 114, 126-163 (2014).</li><li>Menon, I., 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=Opsin+is+a+phospholipid+flippase.">Opsin is a phospholipid flippase.</a> Curr. Biol. CB. 21, 149-153 (2011).</li><li>Goren, M. A., 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=Constitutive+phospholipid+scramblase+activity+of+a+G+protein-coupled+receptor.">Constitutive phospholipid scramblase activity of a G protein-coupled receptor.</a> Nat. Commun. 5, 5115 (2014).</li><li>Ernst, O. P., Menon, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+scrambling+by+rhodopsin.">Phospholipid scrambling by rhodopsin.</a> Photochem. Photobiol. Sci. Off. J. Eur. Photochem. Assoc. Eur. Soc. Photobiol. , (2015).</li><li>Sanyal, S., Menon, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flipping+lipids:+why+an'+what's+the+reason+for?.">Flipping lipids: why an' what's the reason for?.</a> ACS Chem. Biol. 4, 895-909 (2009).</li><li>Bevers, E. M., Williamson, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+scramblase:+an+update.">Phospholipid scramblase: an update.</a> FEBS Lett. 584, 2724-2730 (2010).</li><li>Leventis, P. A., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distribution+and+function+of+phosphatidylserine+in+cellular+membranes.">The distribution and function of phosphatidylserine in cellular membranes.</a> Annu. Rev. Biophys. 39, 407-427 (2010).</li><li>Quazi, F., Lenevich, S., Molday, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ABCA4+is+an+N-retinylidene-phosphatidylethanolamine+and+phosphatidylethanolamine+importer.">ABCA4 is an N-retinylidene-phosphatidylethanolamine and phosphatidylethanolamine importer.</a> Nat. Commun. 3, 925 (2012).</li><li>Quazi, F., Molday, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP-binding+cassette+transporter+ABCA4+and+chemical+isomerization+protect+photoreceptor+cells+from+the+toxic+accumulation+of+excess+11-cis-retinal.">ATP-binding cassette transporter ABCA4 and chemical isomerization protect photoreceptor cells from the toxic accumulation of excess 11-cis-retinal.</a> Proc. Natl. Acad. Sci. U. S. A. 111, 5024-5029 (2014).</li><li>Ben-Shabat, S., 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=Formation+of+a+nonaoxirane+from+A2E,+a+lipofuscin+fluorophore+related+to+macular+degeneration,+and+evidence+of+singlet+oxygen+involvement.">Formation of a nonaoxirane from A2E, a lipofuscin fluorophore related to macular degeneration, and evidence of singlet oxygen involvement.</a> Angew. Chem. Int. Ed Engl. 41, 814-817 (2002).</li><li>Sparrow, J. R., Wu, Y., Kim, C. Y., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+meets+all-trans-retinal:+the+making+of+RPE+bisretinoids.">Phospholipid meets all-trans-retinal: the making of RPE bisretinoids.</a> J. Lipid Res. 51 (2010), 247-261 (2010).</li><li>Sch&#252;tt, F., Davies, S., Kopitz, J., Holz, F. G., Boulton, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodamage+to+human+RPE+cells+by+A2-E,+a+retinoid+component+of+lipofuscin.">Photodamage to human RPE cells by A2-E, a retinoid component of lipofuscin.</a> Invest. Ophthalmol. Vis. Sci. 41, 2303-2308 (2000).</li><li>Picollo, A., Malvezzi, M., Accardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TMEM16+proteins:+unknown+structure+and+confusing+functions.">TMEM16 proteins: unknown structure and confusing functions.</a> J. Mol. Biol. 427, 94-105 (2015).</li><li>Mohammadi, T., 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=Identification+of+FtsW+as+a+transporter+of+lipid-linked+cell+wall+precursors+across+the+membrane.">Identification of FtsW as a transporter of lipid-linked cell wall precursors across the membrane.</a> EMBO J. 30, 1425-1432 (2011).</li><li>Meeske, A. J., 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=MurJ+and+a+novel+lipid+II+flippase+are+required+for+cell+wall+biogenesis+in+Bacillus+subtilis.">MurJ and a novel lipid II flippase are required for cell wall biogenesis in Bacillus subtilis.</a> Proc. Natl. Acad. Sci. 112, 6437-6442 (2015).</li><li>Ruiz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+Flippases+for+Bacterial+Peptidoglycan+Biosynthesis.">Lipid Flippases for Bacterial Peptidoglycan Biosynthesis.</a> Lipid Insights. 8, 21-31 (2015).</li><li>Rick, P. D., 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=Evidence+that+the+wzxE+gene+of+Escherichia+coli+K-12+encodes+a+protein+involved+in+the+transbilayer+movement+of+a+trisaccharide-lipid+intermediate+in+the+assembly+of+enterobacterial+common+antigen.">Evidence that the wzxE gene of Escherichia coli K-12 encodes a protein involved in the transbilayer movement of a trisaccharide-lipid intermediate in the assembly of enterobacterial common antigen.</a> J. Biol. Chem. 278, 16534-16542 (2003).</li><li>Suzuki, J., Imanishi, E., Nagata, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+of+phosphatidylserine+by+Xk-related+protein+family+members+during+apoptosis.">Exposure of phosphatidylserine by Xk-related protein family members during apoptosis.</a> J. Biol. Chem. 289, 30257-30267 (2014).</li><li>Suzuki, J., Nagata, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+scrambling+on+the+plasma+membrane.">Phospholipid scrambling on the plasma membrane.</a> Methods Enzymol. 544, 381-393 (2014).</li><li>Alaimo, C., 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=Two+distinct+but+interchangeable+mechanisms+for+flipping+of+lipid-linked+oligosaccharides.">Two distinct but interchangeable mechanisms for flipping of lipid-linked oligosaccharides.</a> EMBO J. 25, 967-976 (2006).</li><li>Ernst, C. M., Peschel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broad-spectrum+antimicrobial+peptide+restistance+by+MprF-mediated+aminoacylation+and+flipping+of+phospholipids.">Broad-spectrum antimicrobial peptide restistance by MprF-mediated aminoacylation and flipping of phospholipids.</a> Mol. Microbial. 80 (2), 290-299 (2011).</li><li>Vehring, S., 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=Flip-flop+of+fluorescently+labeled+phospholipids+in+proteoliposomes+reconstituted+with+Saccharomyces+cerevisiae+microsomal+proteins.">Flip-flop of fluorescently labeled phospholipids in proteoliposomes reconstituted with Saccharomyces cerevisiae microsomal proteins.</a> Eukaryot. Cell. 6, 1625-1634 (2007).</li><li>Rouser, G., 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=Two+dimensional+then+[sic]+layer+chromatographic+separation+of+polar+lipids+and+determination+of+phospholipids+by+phosphorus+analysis+of+spots.">Two dimensional then [sic] layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots.</a> Lipids. 5, 494-496 (1970).</li><li>Rigaud, J. -. L., L&#233;vy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+membrane+proteins+into+liposomes.">Reconstitution of membrane proteins into liposomes.</a> Methods Enzymol. 372, 65-86 (2003).</li><li>Geertsma, E. R., Nik Mahmood, N. A. B., Schuurman-Wolters, G. K., Poolman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+reconstitution+of+ABC+transporters+and+assays+of+translocator+function.">Membrane reconstitution of ABC transporters and assays of translocator function.</a> Nat. Protoc. 3, 256-266 (2008).</li><li>Mimms, L. T., Zampighi, G., Nozaki, Y., Tanford, C., Reynolds, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+vesicle+formation+and+transmembrane+protein+incorporation+using+octyl+glucoside.">Phospholipid vesicle formation and transmembrane protein incorporation using octyl glucoside.</a> Biochemistry. 20, 833-840 (1981).</li><li>Malvezzi, M., 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=Ca2+-dependent+phospholipid+scrambling+by+a+reconstituted+TMEM16+ion+channel.">Ca2+-dependent phospholipid scrambling by a reconstituted TMEM16 ion channel.</a> Nat. Commun. 4, 2367 (2013).</li><li>Eckford, P. D. W., Sharom, F. 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+reconstituted+P-glycoprotein+multidrug+transporter+is+a+flippase+for+glucosylceramide+and+other+simple+glycosphingolipids.">The reconstituted P-glycoprotein multidrug transporter is a flippase for glucosylceramide and other simple glycosphingolipids.</a> Biochem. J. 389, 517-526 (2005).</li><li>Marek, M., G&#252;nther-Pomorski, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+Flippase+Activity+in+Proteoliposomes+Using+Fluorescent+Lipid+Derivatives.">Assay of Flippase Activity in Proteoliposomes Using Fluorescent Lipid Derivatives.</a> Methods Mol. Biol. 1377, 181-191 (2016).</li><li>Coleman, J. A., Kwok, M. C. M., Molday, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+purification,+and+functional+reconstitution+of+the+P4-ATPase+Atp8a2,+a+phosphatidylserine+flippase+in+photoreceptor+disc+membranes.">Localization purification, and functional reconstitution of the P4-ATPase Atp8a2, a phosphatidylserine flippase in photoreceptor disc membranes.</a> J. Biol. Chem. 284, 32670-32679 (2009).</li><li>Coleman, J. A., Quazi, F., Molday, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+P4-ATPases+and+ABC+transporters+and+their+role+in+phospholipid+transport.">Mammalian P4-ATPases and ABC transporters and their role in phospholipid transport.</a> Biochim. Biophys. Acta. 1831, 555-574 (2013).</li><li>Romsicki, Y., Sharom, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+flippase+activity+of+the+reconstituted+P-glycoprotein+multidrug+transporter.">Phospholipid flippase activity of the reconstituted P-glycoprotein multidrug transporter.</a> Biochemistry. 40, 6937-6947 (2001).</li><li>Zhou, X., Graham, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+phospholipid+translocase+activity+with+purified+Drs2p,+a+type-IV+P-type+ATPase+from+budding+yeast.">Reconstitution of phospholipid translocase activity with purified Drs2p, a type-IV P-type ATPase from budding yeast.</a> Proc. Natl. Acad. Sci. U. S. A. 106, 16586-16591 (2009).</li></ol>

The scramblase activity assay enabled us originally to determine that opsin has phospholipid scramblase activity 2. The assay also allowed us to characterize opsin&#39;s scramblase activity by testing specificity (we used a variety of NBD-labeled reporter lipids such as NBD-phosphatidylethanolamine, labeled with NBD on an acyl chain as shown for NBD-PC in Figure 1A, or on the headgroup, NBD-sphingomyelin or NBD- phosphatidylserine 2), the effect of vesicle lipid composition (e.g...

The photoreceptor rhodopsin, a prototypical G protein-coupled receptor (reviewed for example in reference 1), is the first phospholipid scramblase to be identified and biochemically verified 2,3. Scramblases are phospholipid transporters that increase the intrinsically slow rate of transbilayer phospholipid movement to physiologically appropriate levels in a bidirectional, ATP-independent manner 4-6. Examples of their actions can be found in the endoplasmic reticulum and bacterial cytoplasmic membrane where constitutive scrambling is needed for membrane homeostasis and growth, as well as for a variety of glycosylation pathways 5. Regulated phospholipid scrambling is needed to expose phosphatidylserine (PS) on the surface of apoptotic cells where it acts as an &#34;eat-me&#34;-signal for macrophages 7 and provides a procoagulant surface on activated blood platelets to catalyze the production of protein factors needed for blood clotting. In photoreceptor disc membranes, rhodopsin&#39;s scrambling activity has been suggested to counteract the phospholipid imbalance between the two membrane leaflets of the bilayer that is generated by the ATP-dependent, unidirectional lipid flippase ABCA4 4,8,910-12.
Despite the physiological importance of scramblases, their identity remained elusive until rhodopsin was reported as a scramblase in photoreceptor discs 2, members of the TMEM16 protein family were identified as Ca2+-dependent scramblases needed for PS exposure at the plasma membrane (reviewed in reference 13), and the bacterial protein FtsW was proposed as a Lipid II scramblase required for peptidoglycan synthesis 14. These discoveries were based on the reconstitution of purified proteins in liposomes and demonstration of scramblase activity in the resulting proteoliposomes using the methodology described here. Other potential scramblases 15-21&#160;&#8212; the MurJ and AmJ proteins implicated in peptidoglycan biosynthesis, WzxE and related proteins implicated in scrambling O-antigen precursors, MprF protein needed to translocate aminoacylated phosphatidylglycerol across the bacterial cytoplasmic membrane, and Xkr8 family members that have been proposed to expose PS on the surface of apoptotic cells&#160;&#8212; remain to be tested biochemically. This highlights the importance of a robust assay to identify and characterize scramblase activity.
Here, we describe the reconstitution of purified opsin, the apoprotein of the photoreceptor rhodopsin, into large unilamellar vesicles (LUVs), and subsequent analysis of scramblase activity in the resulting proteoliposomes using a fluorescence-based assay. There are several well-described protocols available in the literature for the heterologous expression and purification of opsin, therefore we will not describe it in this protocol; we use the protocols described in Goren et al. 3 which yields FLAG-tagged, thermostable opsin at about 100 ng/&#181;l in 0.1% (w/v) dodecylmaltoside (DDM).
Reconstitution is achieved by treating LUVs with sufficient detergent so that they swell but do not dissolve. Under these conditions, a membrane protein&#160;&#8212; supplied in the form of protein-detergent micelles&#160;&#8212; will integrate into the liposomes and become reconstituted into the liposome membrane upon detergent removal, resulting in proteoliposomes. To reconstitute opsin (obtained as a purified protein in 0.1% (w/v) DDM), LUVs are prepared from a mixture of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]) and saturated with DDM before adding opsin and NBD-PC. The detergent is then removed by treating the sample with polystyrene beads.
The principle underlying the fluorescence-based assay is shown in Figure 1B. LUVs are symmetrically reconstituted with a trace amount of NBD-PC or other NBD-labeled fluorescent phospholipid reporter (Figure 1A). On adding dithionite, a membrane-impermeant dianion, NBD-PC molecules in the outer leaflet of the LUVs are rendered non-fluorescent as the nitro-group of NBD is reduced to a non-fluorescent amino-group. As neither NBD-PC molecules nor dithionite are able to traverse the membrane on the time-scale of the experiment (&#60;10 min), this results in 50% reduction of the fluorescent signal. However, if the liposomes are reconstituted with a scramblase, NBD-PC molecules in the inner leaflet can scramble rapidly to the outside where they are reduced. This results in the total loss of fluorescence in the ideal case (Figure 1C).
<img alt="Figure 1" src="/files/ftp_upload/54635/54635fig1.jpg" /> 
Figure 1: Schematic representation of the scramblase activity assay. The assay uses a fluorescent NBD-labeled reporter lipid; NBD-PC is shown (A). Large unilamellar vesicles are reconstituted with a trace amount of NBD-PC. Reconstitution produces symmetric vesicles, with NBD-PC distributed equally in the outer and inner leaflets. Dithionite (S2O42-) chemically reduces the nitro-group of NBD to a non-fluorescent amino-group. Treatment of protein-free liposomes with dithionite (B, top) causes a 50% reduction of fluorescence since only the NBD-PC molecules in the outer leaflet are reduced: dithionite is negatively charged and cannot cross the membrane to react with NBD-PC molecules in the inner leaflet. Dithionite treatment of opsin-containing proteoliposomes (B, bottom), i.e., scramblase-active proteoliposomes, results in 100% loss of fluorescence as opsin facilitates movement of NBD-PC between the inner and the outer leaflet. (C) shows idealized fluorescence traces obtained on treating protein-free liposomes and opsin-containing proteoliposomes with dithionite. The rate of fluorescence loss is the same in both cases indicating that the chemical reduction of NBD by dithionite is rate-limiting, and that scrambling occurs at a rate equal to or greater than the rate of the chemical reaction. Traces obtained from an actual experiment are shown in Figure 3. <a href="https://www.jove.com/files/ftp_upload/54635/54635fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The methods we describe can be used to reconstitute and assay other purified proteins, as well as mixtures of membrane proteins obtained, for example, by extracting microsomes with detergent 22.

<table border="0" cellpadding="0" cellspacing="0" width="1148">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:683px;">1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine</td>
			<td style="width:141px;">Avanti Polar Lipids</td>
			<td style="width:132px;">850457C</td>
			<td style="width:193px;">POPC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (sodium salt)</td>
			<td>Avanti Polar Lipids</td>
			<td>840457C</td>
			<td>POPG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-an-glycero-3-phosphocholine</td>
			<td>Avanti Polar Lipids</td>
			<td>810130C</td>
			<td>NBD-PC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid</td>
			<td>VWR Scientific</td>
			<td>EM-5330</td>
			<td>HEPES</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma</td>
			<td>S7653-1KG</td>
			<td>NaCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dodecyl-&#946;-d-maltoside&#160;</td>
			<td>Anatrace</td>
			<td>D310 5 GM</td>
			<td>DDM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorimeter cuvettes</td>
			<td>sigma</td>
			<td>C0918-100EA</td>
			<td>cuvettes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectrofluorometer</td>
			<td colspan="2">Photon Technology International, Inc.</td>
			<td>fluorimeter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydrosulfite technical grade, 85%</td>
			<td>Sigma</td>
			<td>157953-5G</td>
			<td>dithionite</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GraphPad Prism 5 software</td>
			<td></td>
			<td></td>
			<td>Prism</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Base</td>
			<td>VWR</td>
			<td>JTX171-3</td>
			<td>Tris</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIPEX 10 mL extruder&#160;</td>
			<td>Northern Lipids, Inc.</td>
			<td></td>
			<td>Extruder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:683px;">Whatman, Drain disc, PE, 25 mm</td>
			<td>Sigma</td>
			<td style="width:132px;">28156-243</td>
			<td>Disc support</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:683px;">Whatman Nuclepore Track-Etched Membranes, 0.4 &#181;m, 25 mm diameter</td>
			<td>Sigma</td>
			<td style="width:132px;">WHA110607</td>
			<td>400 nm membrane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:683px;">Whatman Nucleopore Track-Etched Membranes, 0.2 &#181;m, 25 mm diameter</td>
			<td>Sigma</td>
			<td style="width:132px;">WHA110606</td>
			<td>200 nm membrane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium phosphate</td>
			<td>Sigma</td>
			<td>S3264-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VWR Culture Tubes, Disposable, Borosilicate Glass, 13x100 mm</td>
			<td>VWR Scientific</td>
			<td>47729-572</td>
			<td>glass tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perchloric acid</td>
			<td>Sigma</td>
			<td>30755-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium Molybdate Tetrahydrate</td>
			<td>Sigma</td>
			<td>A-7302</td>
			<td>ammonium molybdate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(+)-Sodium L-ascorbate</td>
			<td>Sigma</td>
			<td>A7631-25G</td>
			<td>sodium ascorbate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bio-Beads SM2 adsorbents</td>
			<td>Bio Rad</td>
			<td>1523920</td>
			<td>polystyrene beads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;2.0 mL Microtubes clear</td>
			<td>VWR Scientific</td>
			<td>10011-742</td>
			<td>Reconstitution tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reconstitution glass tube</td>
			<td>VWR Scientific</td>
			<td style="width:132px;">53283-800</td>
			<td>Reconstitution glass tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zetasizer&#160;</td>
			<td>Malvern&#160;</td>
			<td></td>
			<td>DLS</td>
		</tr>
	</tbody>
</table>

a fluorescence-based assay of phospholipid scramblase activity

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and biochemically verified was opsin, the apoprotein of the photoreceptor rhodopsin. Rhodopsin is a G protein-coupled receptor localized in rod photoreceptor disc membranes of the retina where it is responsible for the perception of light. Rhodopsin&#39;s scramblase activity does not depend on its ligand 11-cis-retinal, i.e., the apoprotein opsin is also active as a scramblase. Although constitutive and regulated phospholipid scrambling play an important role in cell physiology, only a few phospholipid scramblases have been identified so far besides opsin. Here we describe a fluorescence-based assay of opsin&#39;s scramblase activity. Opsin is reconstituted into large unilamellar liposomes composed of phosphatidylcholine, phosphatidylglycerol and a trace quantity of fluorescent NBD-labeled PC (1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazole-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine). Scramblase activity is determined by measuring the extent to which NBD-PC molecules located in the inner leaflet of the vesicle are able to access the outer leaflet where their fluorescence is chemically eliminated by a reducing agent that cannot cross the membrane. The methods we describe have general applicability and can be used to identify and characterize scramblase activities of other membrane proteins.

We describe the reconstitution of opsin into LUVs to characterize its scramblase activity using a fluorescence-based assay. We analyze the results to place a lower limit on the rate of opsin-mediated phospholipid scrambling and to determine the oligomeric state in which opsin functionally reconstitutes into the vesicles.
To identify optimal reconstitution conditions, it is necessary to determine empirically the amount of deterge...

Watch this Scientific Journal Video about A Fluorescence-based Assay of Phospholipid Scramblase Activity at JoVE.com

A Fluorescence-based Assay of Phospholipid Scramblase Activity

We describe a fluorescence-based assay to measure phospholipid scrambling in large unilamellar liposomes reconstituted with opsin.

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and ...

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