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. Fremstilling af liposomer og proteoliposomer <ol><li> liposomdannelse <ol><li> Ved hjælp af en glassprøjte, tilsættes 1.435 pi POPC (25 mg / ml, i chloroform) og 160 pi POPG (25 mg / ml, i chloroform) til en rundbundet kolbe til opnåelse 52,5 pmol lipider i et molforhold på POPC: POPG = 9: 1. </li><li> Tør lipiderne i 30 minutter under anvendelse af en rotationsfordamper ved en rotationshastighed på 145 rpm (der ikke er brug vandbad i dette volumen opløsningsmiddel), og derefter overføre kolben til et vaku...

<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>

Den scramblase aktivitet assay muligt for os oprindeligt at fastslå, at opsin har phospholipid scramblase aktivitet 2. Assayet også tilladt os at karakterisere opsin s scramblase aktivitet ved at teste specificiteten (vi anvendt en række forskellige NBD-mærkede reporter lipider, såsom NBD-phosphatidylethanolamin, mærket med NBD på en acylkæde som vist for NBD-PC i figur 1A, eller på hovedgruppe, NBD-sphingomyelin eller NBD- phosphatidylserin 2), virkningen af vesikel lipid...

Fotoreceptor rhodopsin, en prototypisk G-protein-koblede receptorer (gennemgået for eksempel i reference 1), er den første phospholipid scramblase der skal identificeres og biokemisk verificeret 2,3. Scramblases er phospholipid transportører, der øger uløseligt langsomme transbilayer phospholipid bevægelse til fysiologisk relevante niveauer i en tovejs, ATP-uafhængig måde 4-6. Eksempler på deres handlinger kan findes i det endoplasmatiske reticulum og bakteriel cytoplasmatiske membran, hvor konstitutiv scrambling er behov for membran homeostase og vækst, samt til en række forskellige glycosyleringsveje 5. Reguleret phospholipid scrambling er nødvendig for at eksponere phosphatidylserin (PS) på overfladen af apoptotiske celler, hvor det virker som en &quot;spise-me&quot; -signal for makrofager 7 og tilvejebringer et prokoagulant overflade på aktiverede blodplader til at katalysere produktion af protein faktors behov for blodpropper. I fotoreceptor disc membraner, har rhodopsin s scrambling aktivitet blevet foreslået at modvirke phospholipidet ubalance mellem de to membran foldere af dobbeltlaget, der genereres af den ATP-afhængige, ensrettet lipid flippase ABCA4 4,8,9 10-12. På trods af den fysiologiske betydning scramblases, deres identitet forblev undvigende indtil rhodopsin blev rapporteret som en scramblase i fotoreceptor skiver 2 blev medlemmer af TMEM16 protein familien identificeret som Ca 2 + -afhængige scramblases nødvendige for PS eksponering ved plasmamembranen (revideret i henvisning 13), og det bakterielle protein FtsW blev foreslået som et lipid II scramblase påkrævet til peptidoglycansyntese 14. Disse opdagelser var baseret på rekonstituering af oprensede proteiner i liposomer og demonstration af scramblase aktivitet i de resulterende proteoliposomer anvendelse af metoden described her. Andre potentielle scramblases 15-21 - de MurJ og AMJ proteiner impliceret i peptidoglycan biosyntesen, WzxE og beslægtede proteiner impliceret i scrambling O-antigen forstadier, MprF protein er nødvendig for at translokere aminoacylerede phosphatidylglycerol tværs den bakterielle cytoplasmatiske membran, og Xkr8 familiemedlemmer, der er blevet foreslået at eksponere PS på overfladen af ​​apoptotiske celler - forbliver der skal testes biokemisk. Dette understreger betydningen af ​​en robust assay at identificere og karakterisere scramblase aktivitet. Her beskriver vi rekonstituering af oprensede opsin, apoproteinet af fotoreceptor rhodopsin, i store unilamellare vesikler (LUV&#39;er), og efterfølgende analyse af scramblase aktivitet i de resulterende proteoliposomer bruger fluorescens-baseret assay. Der er flere velbeskrevne protokoller tilgængelige i litteraturen til heterolog ekspression og oprensning af opsin, derfor vil vi ikkebeskrive det i denne protokol; vi bruger protokollerne der er beskrevet i Goren et al. 3 som giver FLAG-mærket, termostabil opsin ved ca. 100 ng / pl i 0,1% (vægt / volumen) dodecylmaltoside (DDM). Rekonstituering opnås ved at behandle LUV&#39;er med tilstrækkelig vaskemiddel, således at de kvælder, men ikke opløses. Under disse betingelser, et membranprotein - leveres i form af protein-detergent-miceller - vil integrere i liposomerne og blive rekonstitueret i liposommembranen ved fjernelse detergent, hvilket resulterer i proteoliposomer. Til rekonstituering opsin (opnået som et oprenset protein i 0,1% (vægt / volumen) DDM), er LUV fremstilles ud fra en blanding af POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholin) og POPG (1- palmitoyl-2-oleoyl-sn-glycero-3- [phospho-rac- (1-glycerol)]) og mættet med DDM før tilsætning opsin og NBD-PC. Vaskemidlet fjernes derefter ved behandling af prøven med polystyrenkugler. tøs = &quot;jove_content&quot;&gt; Princippet for den fluorescens-assay er vist i figur 1B. LUV&#39;er er symmetrisk rekonstitueres med en spormængde af NBD-PC eller anden NBD-mærket fluorescerende phospholipid reporter (figur 1A). Ved tilsætning dithionit, er en membran-impermeabel dianion, er NBD-PC-molekyler i den ydre folder af LUV&#39;er gøres ikke-fluorescerende som nitro-gruppen af ​​NBD reduceret til en ikke-fluorescerende amino-gruppe. Da hverken NBD-PC-molekyler eller dithionit er i stand til at krydse membranen på tidsskalaen af ​​forsøget (&lt;10 min), resulterer dette i 50% reduktion af det fluorescerende signal. Men hvis liposomerne rekonstitueres med en scramblase, kan NBD-PC-molekyler i indre blad forvrænge hurtigt til ydersiden, hvor de er reduceret. Dette resulterer i fuldstændigt tab af fluorescens i det ideelle tilfælde (figur 1C). es / ftp_upload / 54.635 / 54635fig1.jpg &quot;/&gt; . Figur 1: Skematisk afbildning af scramblase aktivitetsassay Assayet anvender et fluorescerende NBD-mærket reporter lipid; NBD-PC er vist (A). Store unilamellære vesikler rekonstitueres med en spormængde af NBD-PC. Rekonstituering producerer symmetriske blærer, med NBD-PC ligeligt i de ydre og indre foldere. Dithionit (S 2 O 4 2-) reducerer kemisk nitro-gruppen af NBD til en ikke-fluorescerende amino-gruppe. Behandling af protein-frie liposomer med dithionit (B, øverst) bevirker en 50% reduktion af fluorescens, da kun de NBD-PC-molekyler i den ydre indlægsseddel reduceres: dithionit er negativt ladet og kan ikke krydse membranen og reagerer med NBD-PC-molekyler i den indre folder. Dithionit behandling af opsin-holdige proteoliposomer (B, bund), dvs. scramblase-aktive proteoliposomer, resulterer i 100% tab af fluorescence som opsin letter flytning af NBD-pc mellem den indre og den ydre folder. (C) viser idealiserede fluorescens spor opnået på at behandle proteinfrie liposomer og opsin-holdige proteoliposomer med dithionit. Hastigheden af ​​fluorescens tab er den samme i begge tilfælde indikerer, at den kemiske reduktion af NBD ved dithionit er hastighedsbegrænsende, og at scrambling sker ved en hastighed lig med eller større end hastigheden af ​​den kemiske reaktion. Spor opnået fra en faktiske forsøg er vist i figur 3. <a href="https://www.jove.com/files/ftp_upload/54635/54635fig1large.jpg" target="_blank">Klik her for at se en større version af dette tal.</a> De metoder, vi beskriver, kan anvendes til at rekonstituere og analysere andre oprensede proteiner, såvel som blandinger af membranproteiner fremstillet for eksempel ved ekstraktion mikrosomer med 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 translokerer fosfolipider over membranen dobbeltlag bidirektionalt i en ATP-uafhængig måde. Den første scramblase der skal identificeres og biokemisk verificeret blev opsin, apoproteinet af fotoreceptor rhodopsin. Rhodopsin er en G-protein-koblede receptorer lokaliseret i stangen fotoreceptor plademembranerne i nethinden, hvor den er ansvarlig for opfattelsen af ​​lys. Rhodopsin s scramblase aktivitet ikke afhænger af dens ligand 11- cis -retinal, dvs. apoproteinet opsin er også aktiv som scramblase. Selvom konstitutiv og reguleret phospholipid scrambling spiller en vigtig rolle i celle fysiologi, har kun få phospholipid scramblases blevet identificeret hidtil udover opsin. Her beskriver vi en fluorescens-baseret assay af opsin s scramblase aktivitet. Opsin rekonstitueres i store unilamellare liposomer bestående af phosphatidylcholin, phosphatidylglycerol og et spor mængde fluorescerende NBD-mærket PC (1-palmitoyl-2- {6- [7-nitro-2-1,3-benzoxadiazol-4-yl) amino] hexanoyl} - sn-glycero-3-phosphocholin). Scramblase aktivitet bestemmes ved måling i hvilket omfang NBD-PC-molekyler placeret i indre blad af vesiklen kan få adgang den ydre folder, hvor deres fluorescens kemisk elimineres med et reduktionsmiddel, som ikke kan krydse membranen. De metoder vi beskriver har generel anvendelighed og kan anvendes til at identificere og karakterisere scramblase aktiviteter i andre membranproteiner.

Vi beskriver rekonstituering af opsin i LUV at karakterisere sin scramblase med anvendelse af fluorescens-baserede assay. Vi analyserer resultaterne til at placere en nedre grænse for hastigheden af ​​opsin-medieret phospholipid scrambling og at bestemme den oligomere tilstand, hvor opsin funktionelt genopretter i vesiklerne. For at identificere optimale rekonstitution betingelser, er det nødvendigt empirisk at bestemme ...

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.

En Fluorescens-baseret assay phospholipid Scramblase Aktivitet

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

Research

JoVE Journal

Biochemistry

Expression, Purification, and Antimicrobial Activity of S100A12

Ekspression, oprensning og antimikrobiell aktivitet af S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

DNA Polymerase aktivitet Assay ved hjælp af nær-infrarødt fluorescerende mærket DNA visualiseret af acrylamid Gel elektroforese

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

En In Vitro Assay at opdage tRNA-Isopentenyl-Transferase aktivitet

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

Antistatiske Microgravimetry at studere bindende dynamikken i Phospholipid bindende Protein Annexin A2 til fast-understøttet Lipid dobbeltlag ved hjælp af et Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis

Hurtig fluorescens-baserede karakterisering af enkelt ekstracellulære vesikler i humant blod med nanopartikler-tracking analyse

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released ...

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

Et Fluorogenic peptid kavalergang Assay til at screene for proteolytiske aktivitet: ansøgninger om coronavirus spike protein aktivering

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

En kolorimetrisk analyse af citrat syntase aktivitet i Drosophila melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Enkelt trin berigelse af en TAP-Tagged Histon Deacetylase af Filamentous svamp Aspergillus nidulans for enzymatisk aktivitetsanalyse

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Kinetisk screening af Nukleaseaktivitet ved hjælp af nukleinsyre sonder

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

Fluorescensbaserede målinger af fosfatylserin/Fosfatylinositol 4-fosfatudveksling mellem membraner

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found ...