Name: quantification of coenzyme a in cells and tissues
Uploaded: 2019-09-27T14:00:00
Description: Watch this Scientific Journal Video about Quantification of Coenzyme A in Cells and Tissues at JoVE.com

Forfatterne anerkender finansiering til sponsoreret forskning leveret af CoA Therapeutics, Inc., et datterselskab af BridgeBio LLC, de nationale institutter for sundhed Grant GM34496, og den amerikanske libanesiske syriske associerede velgørende organisationer.

Den i denne protokol omhandlede dyre procedure blev udført i overensstemmelse med protokollerne 323 og 556 og specielt godkendt af St. Jude children's Research Hospital institutionel dyrepleje-og Brugsudvalg.
1. forberedelse af løsninger
Bemærk: Brug ultrarent vand til alle opløsninger, og når det er angivet i procedurer.
<ol><li>Forbered 1 mM kaliumhydroxid (KOH) i vand.</li>
	<li>Forbered 0,25 M KOH i vand.</li>
	<li>Forbered 1 M Trizma-HCl i vand og justé...

<ol>
	<li>Abiko, Y., Greenburg, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Metabolic Pathways. 7, 1-25 (1975).</li><li>Leonardi, R., Zhang, Y. -. M., Rock, C. O., Jackowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coenzyme+A:+Back+in+action.">Coenzyme A: Back in action.</a> Progress in Lipid Research. 44, 125-153 (2005).</li><li>Jackowski, S., Rock, C. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+coenzyme+A+biosynthesis.">Regulation of coenzyme A biosynthesis.</a> Journal of Bacteriology. 148, 926-932 (1981).</li><li>Robishaw, J. D., Berkich, D. A., Neely, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rate-limiting+step+and+control+of+coenzyme+A+synthesis+in+cardiac+muscle.">Rate-limiting step and control of coenzyme A synthesis in cardiac muscle.</a> Journal of Biological Chemistry. 257, 10967-10972 (1982).</li><li>Di Meo, I., Carecchio, M., Tiranti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inborn+errors+of+coenzyme+A+metabolism+and+neurodegeneration.">Inborn errors of coenzyme A metabolism and neurodegeneration.</a> Journal of Inherited Metabolic Disease. 42, 49-56 (2019).</li><li>Zhou, B., 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=A+novel+pantothenate+kinase+gene+(PANK2)+is+defective+in+Hallervorden-Spatz+syndrome.">A novel pantothenate kinase gene (PANK2) is defective in Hallervorden-Spatz syndrome.</a> Nature Genetics. 28, 345-349 (2001).</li><li>Dusi, 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=Exome+sequence+reveals+mutations+in+CoA+synthase+as+a+cause+of+neurodegeneration+with+brain+iron+accumulation.">Exome sequence reveals mutations in CoA synthase as a cause of neurodegeneration with brain iron accumulation.</a> American Journal of Human Genetics. 94, 11-22 (2014).</li><li>Dansie, L. E., 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=Physiological+roles+of+the+pantothenate+kinases.">Physiological roles of the pantothenate kinases.</a> Biochemical Society Transactions. 42, 1033-1036 (2014).</li><li>Leonardi, R., Rehg, J. E., Rock, C. O., Jackowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pantothenate+kinase+1+is+required+to+support+the+metabolic+transition+from+the+fed+to+the+fasted+state.">Pantothenate kinase 1 is required to support the metabolic transition from the fed to the fasted state.</a> PLoS ONE. 5, 11107 (2015).</li><li>Leonardi, R., Rock, C. O., Jackowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pank1+deletion+in+leptin-deficient+mice+reduces+hyperglycaemia+and+hyperinsulinaemia+and+modifies+global+metabolism+without+affecting+insulin+resistance.">Pank1 deletion in leptin-deficient mice reduces hyperglycaemia and hyperinsulinaemia and modifies global metabolism without affecting insulin resistance.</a> Diabetologia. 57, 1466-1475 (2014).</li><li>Israel, B. C., Smith, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+acute+and+chronic+ethanol+ingestion+on+pantothenate+and+CoA+status+of+rats.">Effects of acute and chronic ethanol ingestion on pantothenate and CoA status of rats.</a> The Journal of Nutrition. 117, 443-451 (1987).</li><li>Garcia, M., Leonardi, R., Zhang, Y. M., Rehg, J. E., Jackowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+deletion+of+pantothenate+kinases+1+and+2+reveals+the+key+roles+for+CoA+in+postnatal+metabolism.">Germline deletion of pantothenate kinases 1 and 2 reveals the key roles for CoA in postnatal metabolism.</a> PLoS One. 7, 40871 (2012).</li><li>Corbin, D. R., 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=Excess+coenzyme+A+reduces+skeletal+muscle+performance+and+strength+in+mice+overexpressing+human+PANK2.">Excess coenzyme A reduces skeletal muscle performance and strength in mice overexpressing human PANK2.</a> Molecular Genetics and Metabolism. 120, 350-362 (2017).</li><li>Shumar, S. A., Kerr, E. W., Fagone, P., Infante, A. M., Leonardi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+Nudt7+decreases+bile+acid+levels+and+peroxisomal+fatty+acid+oxidation+in+the+liver.">Overexpression of Nudt7 decreases bile acid levels and peroxisomal fatty acid oxidation in the liver.</a> Journal of Lipid Research. 60, 1005-1019 (2019).</li><li>Shumar, S. 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=Induction+of+neuron-specific+degradation+of+Coenzyme+A+models+pantothenate+kinase-associated+neurodegeneration+by+reducing+motor+coordination+in+mice.">Induction of neuron-specific degradation of Coenzyme A models pantothenate kinase-associated neurodegeneration by reducing motor coordination in mice.</a> PLoS ONE. 10, 0130013 (2015).</li><li>Zano, S. P., Pate, C., Frank, M., Rock, C. O., Jackowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+a+genetic+deficiency+in+pantothenate+kinase+1+using+phosphopantothenate+replacement+therapy.">Correction of a genetic deficiency in pantothenate kinase 1 using phosphopantothenate replacement therapy.</a> Molecular Genetics and Metabolism. 16, 281-288 (2015).</li><li>Sharma, L. K., 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=A+therapeutic+approach+to+pantothenate+kinase+associated+neurodegeneration.">A therapeutic approach to pantothenate kinase associated neurodegeneration.</a> Nature Communications. 9, 4399 (2018).</li><li>Alvarez-Cordoba, 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=Pantothenate+Rescues+Iron+Accumulation+in+Pantothenate+Kinase-Associated+Neurodegeneration+Depending+on+the+Type+of+Mutation.">Pantothenate Rescues Iron Accumulation in Pantothenate Kinase-Associated Neurodegeneration Depending on the Type of Mutation.</a> Molecular Neurobiology. 56, 3638-3656 (2019).</li><li>Arber, 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=iPSC-derived+neuronal+models+of+PANK2-associated+neurodegeneration+reveal+mitochondrial+dysfunction+contributing+to+early+disease.">iPSC-derived neuronal models of PANK2-associated neurodegeneration reveal mitochondrial dysfunction contributing to early disease.</a> PLoS One. 12, 0184104 (2017).</li><li>Di Meo, 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=Acetyl-4'-phosphopantetheine+is+stable+in+serum+and+prevents+phenotypes+induced+by+pantothenate+kinase+deficiency.">Acetyl-4'-phosphopantetheine is stable in serum and prevents phenotypes induced by pantothenate kinase deficiency.</a> Scientific Reports. 7, 11260 (2017).</li><li>Nishikawa, T., Edelstein, D., Brownlee, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+missing+link:+a+single+unifying+mechanism+for+diabetic+complications.">The missing link: a single unifying mechanism for diabetic complications.</a> Kidney International Supplements. 77, 26-30 (2000).</li><li>Tsuchiya, Y., Pham, U., Gout, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+measuring+CoA+and+CoA+derivatives+in+biological+samples.">Methods for measuring CoA and CoA derivatives in biological samples.</a> Biochemical Society Transactions. 42, 1107-1111 (2014).</li><li>Demoz, A., Netteland, B., Svardal, A., Mansoor, M. A., Berge, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+and+Detection+of+Tissue+Coash+and+Long-Chain+Acyl-Coa+by+Reversed-Phase+High-Performance+Liquid-Chromatography+after+Precolumn+Derivatization+with+Monobromobimane.">Separation and Detection of Tissue Coash and Long-Chain Acyl-Coa by Reversed-Phase High-Performance Liquid-Chromatography after Precolumn Derivatization with Monobromobimane.</a> Journal of Chromatography. 635, 251-256 (1993).</li><li>Shimada, K., Mitamura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivatization+of+Thiol-Containing+Compounds.">Derivatization of Thiol-Containing Compounds.</a> Journal of Chromatography B. 659, 227-241 (1994).</li><li>Minkler, P. E., Kerner, J., Ingalls, S. T., Hoppel, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+isolation+procedure+for+short-,+medium-,+and+long-chain+acyl-coenzyme+A+esters+from+tissue.">Novel isolation procedure for short-, medium-, and long-chain acyl-coenzyme A esters from tissue.</a> Analytical Biochemistry. 376, 275-276 (2008).</li><li>Newton, G. L., Fahey, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+biothiols+by+bromobimane+labeling+and+high-performance+liquid+chromatography.">Determination of biothiols by bromobimane labeling and high-performance liquid chromatography.</a> Methods in Enzymology. 251, 148-166 (1995).</li><li>Radkowsky, A. E., Kosower, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimanes+.17.+(Haloalkyl)-1,5-Diazabicyclo[3.3.0]Octadienediones+(Halo-9,10-Dioxabimanes)+-+Reactivity+toward+the+Tripeptide+Thiol,+Glutathione.">Bimanes .17. (Haloalkyl)-1,5-Diazabicyclo[3.3.0]Octadienediones (Halo-9,10-Dioxabimanes) - Reactivity toward the Tripeptide Thiol, Glutathione.</a> Journal of the American Chemical Society. 108, 4527-4531 (1986).</li><li>Zhang, Y. 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=Chemical+knockout+of+pantothenate+kinase+reveals+the+metabolic+and+genetic+program+responsible+for+hepatic+coenzyme+A+homeostasis.">Chemical knockout of pantothenate kinase reveals the metabolic and genetic program responsible for hepatic coenzyme A homeostasis.</a> Chemistry &amp; Biology. 14, 291-302 (2007).</li><li>Tokutake, Y., Onizawa, N., Katoh, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toyoda+A,Chohnan+S.+Coenzyme+A+and+its+thioester+pools+in+fasted+and+fed+rat+tissues.">Toyoda A,Chohnan S. Coenzyme A and its thioester pools in fasted and fed rat tissues.</a> Biochemical and Biophysical Research Communications. 402, 158-162 (2010).</li><li>Shibata, K., Nakai, T., Fukuwatari, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+high-performance+liquid+chromatography+determination+of+coenzyme+A,+dephospho-coenzyme+A,+and+acetyl-coenzyme+A+in+normal+and+pantothenic+acid-deficient+rats.">Simultaneous high-performance liquid chromatography determination of coenzyme A, dephospho-coenzyme A, and acetyl-coenzyme A in normal and pantothenic acid-deficient rats.</a> Analytical Biochemistry. 430, 151-155 (2012).</li><li>Chohnan, S., Takamura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+micromethod+for+measurement+of+CoASH+and+its+use+in+measuring+the+intracellular+levles+of+CoASH+and+short+chain+acyl-CoAs+in+Escherichia+coli+K12+cells.">A simple micromethod for measurement of CoASH and its use in measuring the intracellular levles of CoASH and short chain acyl-CoAs in Escherichia coli K12 cells.</a> Agricultural and Biological Chemistry. 55, 87-94 (1991).</li></ol>

Her viser vi en pålidelig, trinvis procedure for kvantificering af total CoA i celler og animalsk væv med en bred vifte af lineær detektion, der er tilgængelig i mange laboratorier, der har en HPLC med enten en absorbans eller fluorescens udgang detektor. Alternativt, massespektrometri er en fælles teknik til evaluering af CoA og CoA thioesters, men er ikke almindeligt tilgængelige på grund af omkostningerne ved instrumentering og den specialiserede viden, der kræves for udvikling af metodologi og fortolkning af ...

Coenzym A (CoA) er en essentiel cofaktor i alle levende organismer og syntetiseres fra pantothensyre, også kaldet pantothenat (salt af Pantotensyre) eller vitamin B5. CoA er den største intracellulære bærer af organiske syrer, herunder kortkædede syrer såsom acetat og succinat, gren-kæde syrer såsom propionat og methylmalonat, langkædede fedtsyrer såsom palmitat og oleat, meget langkædede fedtsyrer såsom flerumættede fedtsyrer og xenobiotika såsom valproinsyre. Den organiske syre danner en thioester-forbindelse enzymatisk med CoA for at gøre det muligt at bruge det som substrat i over 100 reaktioner i intermediær metabolisme1. CoA-thiostere er også allosteriske regulatorer og transkriptionelle aktiverere. Det er nu værdsat2 , at den cellulære samlede COA-forsyning er reguleret3,4; således kan CoA tilgængelighed være begrænsende, og at ægthedsbeviset mangler kan være katastrofale, som eksemplificeret ved nedarvede genetiske lidelser, der påvirker CoA biosyntese5. Pantothenate kinase katalyserer det første trin i CoA-biosyntesen (figur 1) og Pantothenate kinase associeret neurodegeneration, kaldet pkan, er forårsaget af mutationer i PANK2 -genet6. CoA syntase, kodet af coasyn genet, katalyserer de sidste to trin i COA biosyntese (figur 1) og Coasy protein-associeret neurodegeneration, kaldet Copan, er forårsaget af en mutation i coasyn gen7. Både pkan og Copan er nedarvede neurodegenerative sygdomme forbundet med jernophobning i hjernen og COA mangler bagvedlig sygdoms patologier.
Cellulære niveauer af total CoA varierer blandt væv8 og total COA kan stige eller falde under en række fysiologiske, patologiske og farmakologiske tilstande. Leveren CoA stiger under brændstof skifte fra fed til fastende tilstand9, og lever COA niveauer er unormalt højt i leptin-mangel overvægtige mus10. Lever CoA falder som reaktion på kronisk ethanol indtagelse11. Hjernen CoA niveauer i Pank2 knockout musemodel er deprimeret i perinatal periode, men senere i voksen scenen hjernen COA indhold svarer til vild-type niveauer, hvilket indikerer en adaptiv COA respons under udvikling12. Manipulation af vævs COA-indhold ved transgenese eller genleveringsmetoder påvirker metaboliske og neurale funktioner13,14,15. Præklinisk udvikling af potentielle terapier for pkan eller Copan omfatter celle-eller vævs-COA-målinger som indikatorer for effekt16,17,18,19,20 . Evaluering af alle disse betingelser og deres metaboliske eller funktionelle konsekvenser kræver en kvantitativ metode til måling af det samlede ægthedsbevis.
En nøjagtig, pålidelig analyse til måling af CoA i biologiske prøver er en teknisk udfordring i mange laboratorier. Desværre er der ingen prøver til rådighed til at evaluere eller kvantificere CoA eller CoA thioesters i intakte celler, selv om analoner af naturlige CoA thioesters har været meget anvendt som mekaniske sonder i undersøgelser af CoA ester udnytter enzymer21. Omdannelsen af COA, med en fri sulfgidrile (-sh), til en COA thioester (eller omvendt) er hurtig i celler eller animalsk væv under overførsel til et andet miljø og under cellelyse. Talrige acyl-CoA-synthetaser og acyl-CoA-thioesteraser i celler formidler interkonverteringerne inden for CoA-puljen, og yderligere enzymer, der anvender CoA-thiostere som substrater, forbliver aktive i biologiske prøver, indtil de slukkes af kemiske eller fysiske Betyder. Den off-loading af acyl-grupper fra CoA til carnitin af acyl-transferaser er et eksempel inden for netværket af reaktioner, der kan ændre CoA/CoA thioester fordeling. Radioaktive røbestoffer kan bruges til at måle satserne for CoA syntese i celler. De nuværende metoder til måling af CoA-og CoA-derivater i biologiske prøver er blevet revideret22 og omfatter koblede enzymatiske spektrofotometriske analyser, højtryksvæskekromatografi og massespektrometri-baserede procedurer. Men disse metoder er ofte fokuseret på bestemte CoA molekyle arter og er blinde for variation af den samlede CoA pulje. De koblede enzymatiske analyser kræver generelt større mængder inputmateriale på grund af lav detekteringsfølsomhed og har et begrænset område af linearitet.
Vores laboratorium har udviklet en pålidelig procedure for kvantificering af total CoA i dyrkede celler og animalsk væv. Strategien omfatter hydrolyse af alle CoA thioesters at give kun gratis CoA under prøveforberedelse, snarere end at gøre en indsats for at vedligeholde og analysere hele spektret af CoA-arter. Proceduren er en samling af individuelle offentliggjorte metoder til Prøvetilberedning, ægthedsbevis, rensning og identifikation efter højtryksvæskekromatografi (HPLC) og kvantificering af det derivatiserede ægthedsbevis ved absorbans eller Fluorescens detektering23,24,25. De afgørelser om ægthedsbeviser, der er opnået ved hjælp af denne procedure, har gjort det muligt at forstå ægthedsbeviset og udvikle en terapeutisk tilgang til behandling af ægthedsbeviser.

<table>
	<tbody>
		<tr>
			<td>2-(2-pyridyl)-ethyl silica gel SPE column</td>
			<td>Millipore-Sigma</td>
			<td>54127-U</td>
			<td></td>
		</tr>
		<tr>
			<td>coenzyme A</td>
			<td>Avanti Polar Lipids</td>
			<td>870700</td>
			<td></td>
		</tr>
		<tr>
			<td>Gemini C18 3 &#956;m 100 &#197; HPLC column</td>
			<td>Phenomenex</td>
			<td>00F-4439-E0</td>
			<td></td>
		</tr>
		<tr>
			<td>monobromobimane</td>
			<td>ThermoFisher Scientific</td>
			<td>M-1378</td>
			<td></td>
		</tr>
		<tr>
			<td>Omni-Tip probe tissue disrupter</td>
			<td>Omni International</td>
			<td>32750H</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisher</td>
			<td>S37440</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerGen 125 motorized rotor stator homogenizer</td>
			<td>ThermoFisher Scientific</td>
			<td>NC0530997</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin-X centrifuge tube filter</td>
			<td>CoStar</td>
			<td>8161</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma-HCl</td>
			<td>Fisher</td>
			<td>T395-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Waters 2475 fluorescence detector</td>
			<td>Waters</td>
			<td>2475</td>
			<td></td>
		</tr>
		<tr>
			<td>Waters 2489 UV-Vis detector</td>
			<td>Waters</td>
			<td>2489</td>
			<td></td>
		</tr>
		<tr>
			<td>Waters e2695 separations module</td>
			<td>Waters</td>
			<td>e2695</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of coenzyme a in cells and tissues

Ny forskning har afsløret, at den cellulære coenzym A (CoA) forsyning kan blive begrænsende med en skadelig indvirkning på vækst, metabolisme og overlevelse. Måling af cellulære CoA er en udfordring på grund af sin relativt lave overflod og den dynamiske omdannelse af gratis CoA til CoA thioesters at, til gengæld, deltage i talrige metaboliske reaktioner. En metode er beskrevet, der navigerer gennem potentielle faldgruber under prøveforberedelse til at give en analyse med et bredt lineært detekteringsområde, der er egnet til brug i mange biomedicinske laboratorier.

En relativt hurtig og pålidelig metode til påvisning af total CoA i dyrkede celler og væv er blevet udviklet ved at derivatizing thiol af CoA til et fluorescerende middel ved hjælp af mBBr, og derefter rense den derivatized CoA-Biman ved hjælp af reverse fase HPLC. Der genereres først en standardkurve, hvor kendte og stigende mængder af CoA-Biman-standarden injiceres enkeltvis, og arealerne under toppene i CoA-bimane-kromatogrammer afbildes som en funktion af input CoA-Biman (figur 4)...

Watch this Scientific Journal Video about Quantification of Coenzyme A in Cells and Tissues at JoVE.com

Quantification of Coenzyme A in Cells and Tissues

Denne metode beskriver prøveforberedelse fra dyrkede celler og animalsk væv, ekstraktion og derivatisering af coenzym A i prøverne, efterfulgt af højtryksvæskekromatografi til rensning og kvantificering af det derivatiserede coenzym A ved absorbans eller fluorescens detektering.

Kvantificering af coenzym A i celler og væv

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...

Coenzym A, kaldet CoA for korte, er en vigtig cofaktor i cellulære stofskifte. Kvantitative målinger afslører ændringer, der sker med ernæringsmæssige og hormonelle tilstande i dyremodeller. Denne metode benød den samlede mængde cellulære CoA, herunder CoA-derivater og gratis CoA, på en enkel, pålidelig metode.Flere typer af neurodegeneration med hjerne jern ophobning er forbundet med mutationer i generne af CoA biosyntetiske vej. Denne metode kan anvendes på ethvert biologisk system, da alle organismer kræver CoA for overlevelse, vækst og funktion. En første gang forsker kan kæmpe med de første skridt i prøve forberedelse, hvor det er vigtigt at holde prøverne helt koldt og derefter hurtigt overføre dem til kaliumhydroxid.Begræns antallet af prøver i et parti, før de overføres til kaliumhydroxid, og øv dig med overførslen, før der arbejdes med forsøgsprøverne. Mange forskere er ikke bekendt med fast fase ekstraktion i udarbejdelsen af biologiske prøver til senere biokemisk analyse. Til at begynde, inkubere tre eksemplarer kultur retter parallelt, to til biokemisk analyse og en til bestemmelse af levedygtige celletal.Når kulturen nærmer sig de sene underkonfluente tætheder, for eksempel, menneskelige HepG2/C3A celler vokset til en tæthed på seks til otte gange 10 til de seks eller HEK 293T celler dyrket til en tæthed på omkring 1,3 gange 10 til de syv pr 100-millimeter parabol, høste de klæbende celler i kulturen. Derefter tilsættes en milliliter iskoldt vand til den samme kulturskål. Skrab cellerne i det kolde vand i skålen, og overfør cellesuspensionen til et glasagensglas med 400 mikroliter på 0,25-molar kaliumhydroxid og 1,5 milliliter vand for at bringe pH-oli'en over 12.Bland suspensionen kraftigt ved at hvirvle på høj i 10 sekunder. Derefter dække stramt med paraffin film, og inkubere ved 55 grader Celsius i en time uden at ryste i et vandbad. Derefter tilsættes 160 mikroliter af en-molar Trizma-hydrochlorid opløsning og 10 mikroliter af 100-millimolar mBBr.Bland ved vortexing på høj i 10 sekunder. Dette bringer pH til ca otte til at støtte mBBr reaktion med gratis CoA. Dæk røret med paraffinfilm, og inkuber prøverne ved stuetemperatur i to timer i mørke, så mBBr reagerer med thiol af CoA.Efter to timer tilsættes 100 mikroliter eddikesyre, og blandes ved vortexing i 10 sekunder for at stoppe reaktionen. En nedbør former i røret. Dernæst centrifuge ved 2,000 gange g i 15 minutter.For at fjerne det udfældede celleaffald skal supernatanten overføres og gemmes i et glasagensglas til oprydning i den faste faseudvindingssøjle. Før analysen påbegyndes, tilsættes to milliliter kaliumhydroxid på en millimolar kalium til et glasreagensglas, der er beregnet til indføring af en sonde og vævsforstyrrelser med en rotor-statorhomogenisator. Hold røret på is, indtil det er brugt.De frosne vævsstykker vejes hurtigt, og den våde vægt registreres for hver prøve. Overfør vævet ind i glasrøret, og homogenisere vævet i 30 sekunder. Undgå fuldstændig optøning af vævet.Der tilsættes 500 mikroliter 0,25-molar kaliumhydroxid. Vortex på højt i 10 sekunder, og holde på is. Dette bringer pH-slutningen af prøven over 12 til hydrolysere CoA thioesters at give samlede CoA.Dæk røret med paraffinfilm. Fremstilling af dyrkede celler og fremstilling af væv er de mest kritiske trin i denne procedure. Det er vigtigt at tilsætte høj kaliumhydroxid hurtigt for at forhindre CoA nedbrydning.Inkuber prøverne ved 55 grader Celsius i to timer i et vandbad uden at ryste for at støtte hydrolyse. Derefter tilsættes 150 mikroliter af en-molar Trizma-hydrochlorid og 10 mikroliter af 100-millimolar mBBr at bringe pH til omkring otte for at støtte mBBr reaktion med gratis CoA. Vortex på højt i 10 sekunder.Dæk røret med paraffinfilm, og inkuber prøverne ved stuetemperatur i to timer i mørke for at sikre, at alle CoA'er er fraivatiseret med mBBr. Derefter tilsættes 100 mikroliter eddikesyre, og vortex på højt i 10 sekunder for at stoppe reaktionen. Centrifuge ved 2,000 gange g i 15 minutter til pellet udfældet cellerester, og overføre supernatanten i et glas reagensglas til fast fase ekstraktionssøjle oprydning.Ved stuetemperatur skal hver en milliliter engangs 2-2-pyridyl-ethylsiligel kolonne med en milliliter vaskebuffer bringes i ligevægt for at sikre, at pyridylfunktionsgruppen er protoneret og fungerer som anionbytter. Derefter pipette prøven supernatant på kolonnen, og indsamle eluat. Kolonnen vaskes to gange med en milliliter vaskepude og én gang med en milliliter vand for at fjerne eventuelle utilbetalte arter.Dernæst vaskes kolonnen to gange i et separat glasagagensglas med en milliliter elutionbuffer for at indsamle CoA-bimanen. CoA-bimaneprøven tørres af i røret under nitrogengas. Seal, helt dække røret, og opbevares på minus 20 grader Celsius.Når den er klar til HPLC-analyse, opslænkes prøven igen i 300 mikroliter vand, og blandes kraftigt ved at hvirvle højt i 10 sekunder. Den opsbrugte prøve overføres til et centrifugerørsfilter, og centrifugering ved 5.000 gange g i 10 minutter for at fjerne bundfald. Den filtrerede prøve overføres derefter til et glasglas, der er egnet til HPLC-injektion.I denne undersøgelse viser en repræsentativ HPLC-profil retentionstiden for CoA-bimane-standarden på C18 HPLC-kolonnen. Stigende mængder af CoA-bimane-standarden blev injiceret individuelt, og områderne under toppene i CoA-bimanekromatogrammet blev afbildet som en funktion af input CoA-bimane. Standardkurven afspejlede omfanget af absorbansen af CoA-bimane ved en bølgelængde på 393 nanometer.Fluorescensen af CoA-bimane blev også afspejlet ved excitation bølgelængden af 393 nanometer og emission bølgelængde på 470 nanometer. Efterfølgende HPLC-separation og typiske detektionsprofiler for muselever eller humant dyrkede C3A-celler er her angivet med røde toppe med en retentionstid på mellem 11 og 12 minutter ved hjælp af elution-programmet. Hvis reaktionen med mBBr ikke giver påviselige resultater, kan det være nødvendigt at justere mængden af Trizma-HCl for at sænke pH-oli'en til ca. otte.Det er muligt at opdage yderligere cellulære molekyler, der indeholder sulfhydryl eller thioester moieties som bimane derivater. For eksempel kan glutathion-bimane påvises med HPLC-metoden. Denne teknik vil give andre forskere til at undersøge den potentielle rolle CoA dysfunktion forbundet med metabolisk ubalance, såsom dyremodeller af type 2 diabetes.Forskere bør bruge personlige værnemidler, når de arbejder med organiske opløsningsmidler, der anvendes til fast fase ekstraktion.

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Research

JoVE Journal

Biochemistry

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m6A modifications have immense possibility to provide key insights into molecular physiology.

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

A modified northern blotting method for measuring N6-methyladenosine (m6A) modifications in RNA is described. The current method can detect modifications in diverse RNAs and controls under various experimental designs.

En metode til måling af RNA<em&gt; N</em<sup&gt; 6</sup&gt; -methyladenosine Ændringer i celler og væv

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their involvement in two-component signal transduction, a ubiquitous system through which bacteria sense and respond to environmental stimuli. Two-component signaling features autophosphorylation of a histidine kinase, followed by phosphotransfer to the receiver domain of a response regulator protein, which ultimately leads to an output response. Autophosphorylation of the histidine kinase is responsive to the presence of a cognate environmental stimulus, thereby giving bacteria a means to detect and respond to changes in the environment. Despite their importance in bacterial biology, histidine kinases remain poorly understood due to the inherent lability of phosphohistidine. Conventional methods for studying these proteins, such as SDS-PAGE autoradiography, have significant shortcomings. We have developed a nitrocellulose binding assay that can be used to characterize histidine kinases. The protocol for this assay is simple and easy to execute. Our method is higher throughput, less time-consuming, and offers a greater dynamic range than SDS-PAGE autoradiography.

We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.

Kvantificering af Bakteriel Histidin kinase Autophosphorylering Ved hjælp af en Nitrocellulose-bindingsassay

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

Kvantificering af overfloden og opladning niveauer af overførsel RNA'er i Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

Kvantificering af hypopigmentering aktivitet In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

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

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

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 ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

Kvantificering af protein interaktions netværk dynamik ved hjælp af Multiplexed Co-Immunopræcipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic factors affecting glucose uptake. The measurement of intracellular fluorescence is a widely used method to test the uptake of fluorescently-labeled glucose (FD-glucose) in cells in vitro, or for imaging glucose-consuming tissues in vivo. This assay assesses glucose uptake at a chosen time point. The intracellular analysis assumes that the metabolism of FD-glucose is slower than that of endogenous glucose, which participates in catabolic and anabolic reactions and signaling. However, dynamic glucose metabolism also alters uptake mechanisms, which would require kinetic measurements of glucose uptake in response to different factors. This article describes a method for measuring extracellular FD-glucose depletion and validates its correlation with intracellular FD-glucose uptake in cells and tissues ex vivo. Extracellular glucose depletion may be potentially applicable for high-throughput kinetic and dose-dependent studies, as well as identifying compounds with glycemic activity and their tissue-specific effects.

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

Extracellular depletion of fluorescently labeled glucose correlates with glucose uptake and could be used for high-throughput screening of glucose uptake in excised organs and cell cultures.

Ekstracellulær glukoseudtømning som et indirekte mål for glukoseoptagelse i celler og væv Ex Vivo

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic ...

Kvantificering af syntese og distribution af mtDNA med høj kapacitet

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

Forfatter Spotlight: Billedbaseret kvantificering af mitokondriel DNA-syntese og -fordeling med høj kapacitet  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

Billedbaseret kvantificering med høj kapacitet af mitokondrie-DNA-syntese og -distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...