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 erkjenner finansiering for sponset forskning levert av CoA legemiddel selskap, Inc., et datterselskap av BridgeBio LLC, National Institutes of Health Grant GM34496, og den amerikanske libanesiske syriske Associated velferdsorganisasjoner.

Dyret prosedyren referert til i denne protokollen ble utført i henhold til protokoller 323 og 556 og spesielt godkjent av St. Jude Children ' s Research Hospital institusjonelle Animal Care og use Committee.
1. utarbeidelse av løsninger
Merk: Bruk ultrarent vann for alle løsninger og når det fremgår av prosedyrene.
<ol><li>Forbered 1 mM kalium (KOH) i vann.</li>
	<li>Forbered 0,25 M KOH i vann.</li>
	<li>Forbered 1 M Trizma-HCl i vann og Juster til pH 8,0.</l...

<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 pålitelig, trinnvis fremgangsmåte for kvantifisere totale CoA i celler og animalsk vev med et bredt spekter av lineær deteksjon som er tilgjengelig i mange laboratorier som har en HPLC med enten en absorbansen eller fluorescens output detektor. Alternativt, masse massespektrometri er en vanlig teknikk for å evaluere CoA og CoA thioesters, men er ikke allment tilgjengelig på grunn av kostnaden av instrumentering og den spesialiserte kunnskapen som kreves for utvikling av metodikk og tolkning av data. ...

Koenzym A (CoA) er en essensiell kofaktor i alle levende organismer og er syntetisert fra Pantotensyre syre, også kalt pantothenate (salt av Pantotensyre syre) eller vitamin B5. CoA er den store intracellulære bærer av organiske syrer, inkludert kort-kjeden syrer som acetate og succinate, gren-kjeden syrer som propionate og methylmalonate, lang-kjeden fettsyrer som askorbylpalmitat og oleat, veldig lang kjede fettsyrer som flerumettede fettsyrer, og xenobiotics som Valproic syre. Den organiske syre danner en thioester sammenheng enzymatisk med CoA å muliggjøre bruken som et substrat i over 100 reaksjoner i mellomledd metabolisme1. CoA thioesters er også nukleosid regulatorer og transcriptional aktivator. Det er nå verdsatt2 at mobilnettet totale COA Supply er regulert3,4; Derfor kan CoA-tilgjengelighet begrenses, og at CoA-mangler kan være katastrofale, som et eksempel på arvet genetiske lidelser som påvirker CoA-biosyntesen5. Pantothenate kinase katalyserer det første trinnet i CoA-biosyntesen (figur 1) og pantothenate kinase Associated neurodegeneration, kalt PKAN, er forårsaket av mutasjoner i PANK2 Gene6. CoA-syntase, som er kodet av COASYN -genet, katalyserer de to siste trinnene i COA-biosyntesen (figur 1) og COASY-assosiert neurodegeneration, kalt CoPAN, forårsakes av en mutasjon i COASYN -genet7. Både PKAN og CoPAN er arvet nevrodegenerative sykdommer forbundet med jern akkumulering i hjernen og CoA mangler underliggende sykdommen patologi.
Cellulære nivåer av total CoA varierer mellom vev8 og total COA kan øke eller redusere under en rekke fysiologiske, patologiske og farmakologiske tilstander. Leveren CoA øker under drivstoff skifter fra matet til fastende stat9, og leveren COA nivåer er unormalt høy i leptin-mangelfull overvektige mus10. Liver CoA avtar som følge av kronisk etanol inntak11. Brain CoA nivåer i Pank2 knockout muse modellen er deprimert i løpet av Perinatal perioden, men senere i den voksne scenen hjernen COA innhold tilsvarer Wild-type nivåer, noe som indikerer en adaptiv COA respons under utbygging12. Manipulering av vev COA innhold ved transgenesis eller gen leveringsmetoder virkninger metabolske og nevrale funksjoner13,14,15. Prekliniske utvikling av potensielle terapi for PKAN eller CoPAN inkluderer celle eller vev COA målinger som indikatorer for effekt16,17,18,19,20 . Evaluering av alle disse forholdene og deres metabolske eller funksjonelle konsekvenser krever en kvantitativ metode for måling av total CoA.
En nøyaktig, pålitelig analysen for måling av CoA i biologiske prøver er en teknisk utfordring i mange laboratorier. Beklageligvis, der er nei sonder anvendelig å vurdere eller kvantifisere CoA eller CoA thioesters inne Behold celler, til tross for analogs av naturlig CoA thioesters ha blitt vidt anvendt idet mekanistisk sonder inne studier av CoA Ester bruker enzym21. Konverteringen av CoA, med en gratis sulfhydrylgruppen (-SH) moiety, til en CoA thioester (eller vice versa) er rask i celler eller animalsk vev under overføring til et annet miljø og under cellelyse. Tallrike acyl-CoA-synthetases og acyl-CoA-thioesterases i cellene megle i interconversions i ekthetsgarantien, og ytterligere enzymer som utnytter CoA-thioesters som underlag, forblir aktive i biologiske prøver til slukket av kjemiske eller fysiske Betyr. Off-lasting av acyl-grupper fra CoA til carnitine av acyl-transferases er ett eksempel innenfor nettverket av reaksjoner som kan endre CoA/CoA thioester fordelingen. Radioaktive Bevegelsesuskarphet kan brukes til å måle frekvensen av CoA syntese i celler. Gjeldende metoder for måling av CoA-og CoA-derivater i biologiske prøver har gjennomgått22 og inkluderer sammenkoblede enzymatisk Spektrofotometriske analyser, høytrykks væske kromatografi og masse massespektrometri prosedyrer. Men disse metodene er ofte fokusert på bestemte CoA molekyl arter og er blinde for variasjon av det totale CoA bassenget. De sammenkoblede enzymatisk analysene krever vanligvis større mengder inngangs materiale på grunn av lav deteksjons følsomhet og har et begrenset utvalg av linearitet.
Vårt laboratorium har utviklet en pålitelig prosedyre for kvantifisering av total CoA i kulturperler celler og animalsk vev. Strategien omfatter hydrolyse av alle CoA-thioesters for å gi bare gratis CoA under prøve forberedelser, i stedet for å gjøre arbeidet med å vedlikeholde og analysere hele spekteret av CoA-arter. Fremgangsmåten er en samling av individuelle, publiserte metoder for prøveklargjøring, CoA-derivatization, rensing og identifisering etter høytrykks kromatografi (HPLC), og kvantifisering av derivatized CoA ved absorbansen eller fluorescens Detection23,24,25. The CoA bestemmelser innhentet ved hjelp av denne prosedyren har aktivert vår forståelse av CoA regulering og utvikling av en terapeutisk tilnærming for behandling av CoA mangler.

<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

Emerging forskning har avdekket at mobilnettet koenzym A (CoA) forsyning kan bli begrensende med en ødeleggende innvirkning på vekst, metabolisme og overlevelse. Måler av Cellular CoA er en angripe på grunn av dens relativt lav overflod og det drivkraft omdanne av ledig CoA å CoA thioesters det, etter tur, delta inne tallrik metabolic reaksjonene. En metode er beskrevet som navigerer gjennom potensielle fallgruver under prøven forberedelse til å gi en analyse med et bredt lineær utvalg av deteksjon som er egnet for bruk i mange biomedisinsk laboratorier.

En relativt rask og pålitelig metode for påvisning av total CoA i kulturperler celler og vev har blitt utviklet av derivatizing tiolderivat av CoA til et fluorescerende middel ved hjelp mBBr, og deretter rense derivatized CoA-bimane bruker reverse fase HPLC. En standard kurve genereres først, der kjente og økende mengder av CoA-bimane-standarden injiseres individuelt, og områdene under toppene i CoA-bimane-kromatogrammene tegnes som en funksjon av inngangen CoA-bimane (Figur 4). CoA-bim...

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 metoden beskriver prøven forberedelse fra kultivert celler og animalsk vev, ekstraksjon og derivatization av koenzym A i prøvene, etterfulgt av høytrykks væske kromatografi for rensing og kvantifisering av derivatized koenzym A ved absorbansen eller fluorescens deteksjon.

Kvantifisering av koenzym A i celler og vev

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

Koenzym A, kalt CoA for kort, er en viktig kofaktor i cellulær metabolisme. Kvantitative målinger avslører endringer som skjer med ernæringsmessige og hormonelle tilstander i dyremodeller. Denne metoden kvanterer den totale mengden cellulær CoA, inkludert CoA-derivater og gratis CoA, i en enkel, pålitelig metode.Flere typer nevrodegenerasjon med hjernejernakkumulering er forbundet med mutasjoner i genene til CoA biosyntetisk vei. Denne metoden kan brukes på ethvert biologisk system siden alle organismer krever CoA for overlevelse, vekst og funksjon. En førstegangsforsker kan slite med de første trinnene i prøveforberedelsen, hvor det er viktig å holde prøvene helt kalde og deretter raskt overføre dem til kaliumhydroksid.Begrens antall prøver i en batch før overføring til kaliumhydroksid, og øv med overføringen før du arbeider med eksperimentelle prøver. Mange forskere er ikke kjent med solid faseutvinning i utarbeidelsen av biologiske prøver for senere biokjemisk analyse. Til å begynne med inkubere triplicate kulturretter parallelt, to for biokjemisk analyse og en for bestemmelse av levedyktig cellenummer.Når kulturen nærmer seg de sene subconfluent tettheter, for eksempel, menneskelige HepG2 / C3A celler vokst til en tetthet på seks til åtte ganger 10 til de seks eller HEK 293T celler vokst til en tetthet på ca 1,3 ganger 10 til syv per 100-millimeter parabolen, høste de vedleverske cellene i kulturen. Deretter legger du til en milliliter iskaldt vann til samme kulturrett. Skrap celler i det kalde vannet i fatet, og overfør cellesuspensjonen til et glassprøverør som inneholder 400 mikroliter på 0,25-molar kaliumhydroksid og 1,5 milliliter vann for å bringe pH over 12.Bland suspensjonen kraftig ved å virvle høyt i 10 sekunder. Dekk deretter tett med parafinfilm, og inkuber ved 55 grader Celsius i en time uten å riste i et vannbad. Deretter legger du til 160 mikroliter med en-molar Trizma-hydrokloridløsning og 10 mikroliter på 100 ml mBBr.Bland ved å virvle høyt i 10 sekunder. Dette bringer pH til ca åtte for å støtte mBBr-reaksjonen med gratis CoA. Dekk røret med parafinfilm, og inkuber prøvene ved romtemperatur i to timer i mørket for mBBr å reagere med thiol av CoA.Etter to timer, tilsett 100 mikroliter eddiksyre, og bland ved vortexing i 10 sekunder for å stoppe reaksjonen. En nedbør dannes i røret. Deretter sentrifuge på 2, 000 ganger g i 15 minutter.For å fjerne de utfelte celleavfallene, overfør og lagre supernatanten i et glassprøverør for opprydding av fastfaseekstraksjonskolonnen. Før analysen startet, tilsett to milliliter med en millimolar kaliumhydroksid til et glass reagensrør designet for innsetting av en sonde og vev forstyrrelse med en rotor-stator homogenisator. Hold røret på is til bruk.Vei de frosne vevsbitene raskt, og registrer den våte vekten for hver prøve. Overfør vevet inn i glassrøret, og homogeniser vevet i 30 sekunder. Unngå fullstendig tining av vevet.Tilsett 500 mikroliter med 0,25-molar kaliumhydroksid. Vortex på høy i 10 sekunder, og holde på isen. Dette bringer pH av prøven over 12 for å hydrolysere CoA-tørstene for å gi total CoA.Dekk røret med parafinfilm. Utarbeidelsen av kultiverte celler og fremstilling av vev er de mest kritiske trinnene i denne prosedyren. Det er viktig å legge til høy kaliumhydroksid raskt for å forhindre CoA-nedbrytning.Inkuber prøvene ved 55 grader Celsius i to timer i et vannbad uten å riste for å støtte hydrolyse. Deretter legger du til 150 mikroliter med en-molar Trizma-hydroklorid og 10 mikroliter på 100-millimolar mBBr for å bringe pH til omtrent åtte for å støtte mBBr-reaksjonen med gratis CoA. Vortex på høy i 10 sekunder.Dekk røret med parafinfilm, og inkuber prøvene ved romtemperatur i to timer i mørket for å sikre at all CoA er derivatisert med mBBr. Deretter legger du til 100 mikroliter eddiksyre, og vortex på høy i 10 sekunder for å stoppe reaksjonen. Sentrifuge ved 2000 ganger g i 15 minutter for å pellet utfelt celleavfall, og overføre supernatanten til et glassreagensrør for opprydding av massivfaseekstraksjonskolonnen.Ved romtemperatur, likevekt hver en-milliliter disponibel 2-2-pyridyl-etyl silika gel kolonne med en milliliter vaskebuffer for å sikre at pyridyl funksjonell gruppe er protonert og vil fungere som en anion veksler. Deretter pipette prøven supernatant på kolonnen, og samle eluate. Vask kolonnen to ganger med en milliliter vaskebuffer og en gang med en milliliter vann for å fjerne eventuelle uopphetede arter.Deretter, inn i et eget glassprøverør, vask kolonnen to ganger med en milliliter elutionbuffer for å samle CoA-bimane. Tørk CoA-bimane prøven i røret under nitrogengass. Forsegle, helt dekke røret, og lagre på minus 20 grader Celsius.Når du er klar for HPLC-analyse, bruker du prøven på 300 mikroliter vann, og bland kraftig ved å virvle høyt i 10 sekunder. Overfør den gjenbrukte prøven til et sentrifugerørfilter, og sentrifuge ved 5 000 ganger g i 10 minutter for å fjerne eventuelle bunnspapp. Deretter overfører du den filtrerte prøven til et hetteglass med glass som er egnet for HPLC-injeksjon.I denne studien viser en representativ HPLC-profil oppbevaringstiden til CoA-bimane-standarden i C18 HPLC-kolonnen. Økende mengder av CoA-bimane-standarden ble injisert individuelt, og områdene under toppene i CoA-bimanekromatrammer ble plottet som en funksjon av input CoA-bimane. Standardkurven reflekterte størrelsen på absorbansen av CoA-bimane ved en bølgelengde på 393 nanometer.Fluorescensen av CoA-bimane ble også reflektert ved eksitasjonsbølgelengden på 393 nanometer og utslippsbølgelengde på 470 nanometer. Påfølgende HPLC-separasjon og typiske deteksjonsprofiler for muselever- eller menneskekulturerte C3A-celler indikeres her med røde topper, med en oppbevaringstid mellom 11 og 12 minutter ved hjelp av elutionprogrammet. Hvis reaksjonen med mBBr ikke gir påviselige resultater, kan det hende at mengden Trizma-HCl må justeres for å senke pH-en til omtrent åtte.Det er mulig å oppdage flere cellulære molekyler som inneholder sulfhydryl eller tioester moieties som bimane derivater. Glutation-bimane kan for eksempel oppdages med HPLC-metoden. Denne teknikken vil tillate andre forskere å undersøke den potensielle rollen coa dysfunksjon forbundet med metabolsk ubalanse, som dyremodeller av type 2 diabetes.Forskere bør bruke personlig verneutstyr når de arbeider med organiske løsemidler som brukes til solidfaseutvinning.

quantification-of-coenzyme-a-in-cells-and-tissues

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 for måling av RNA<em&gt; N</em<sup&gt; 6</sup&gt; -methyladenosine Endringer i celler og vev

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.

Kvantifisering av bakteriell histidin kinase Autofosforylering Ved hjelp av en Nitrocellulose bindingsanalyse

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.

Kvantifisering av overflod og lading nivåer av overføring RNAs 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.

Kvantifisering av Hypopigmentation aktivitet i 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.

Dissipative Microgravimetry å studere Binding dynamikken i Phospholipid bindende Protein Annexin A2 til Solid-støttet Lipid Bilayers bruker 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.

Kvantifisering av protein samspill nettverk dynamikk ved hjelp av multiplekset co-Immunutfelling

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 glukoseuttømming som et indirekte mål på glukoseopptak i celler og vev ex vivo

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

Kvantifisering med høy gjennomstrømning av syntese og distribusjon av mtDNA

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: Bildebasert kvantifisering av mitokondriell DNA-syntese og distribusjon  

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.

High-throughput bildebasert kvantifisering av mitokondriell DNA-syntese og distribusjon

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