Detta arbete stöddes av NSF utmärkelse # 0920274 och South Carolina Experiment Station Project (SC-1.700.340) till KSS. Detta papper är tekniskt bidrag nr 5929 i Clemson University Experiment Station.

Det övergripande system av det här protokollet beskrivs i figur 1. 1. Beredning av lösningar för Standard Kurvor och analyser <ol><li> Bered 100 ml av en 2 mol / l lösning av hydroxylamin-HCl. Väg upp 13,9 g hydroxylamin hydroklorid (MW 69,49 g / mol) och lös upp i cirka 50 ml destillerat-avjoniserat vatten (DDH 2 O). Justera pH till 7,0 med hjälp av kaliumhydroxid pellets eller en koncentrerad lösning. Ta den slutliga volymen till 100 ml. Lösningen kan förvaras i rum...

<ol>
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Upptäckten av acetyl fosfat i denna analys är beroende av en tillräcklig koncentration av hydroxylamin hydroklorid och koncentration och syra av järnklorid lösningen. Ändring av analysen volymen kommer att kräva omprövning av båda dessa komponenter. Den enzymatiska reaktioner som beskrivs här utfördes vid 37 ° C med hydroxylamin uppsägning utföras vid 60 ° C i 5 minuter. Denna högre temperatur är avgörande för att möjliggöra snabb omställning av de återstående acetyl fosfat till acetyl hydroxamat...

<table border="1"><tr><td width="160"> Reagensets namn </td><td width="108"> Företag </td><td width="134"> Katalognummer </td><td width="103"> Kommentarer </td></tr><tr><td> Acetyl fosfat </td><td> Sigma-Aldrich </td><td> 01.409 </td><td> Litium Salt (97%) </td></tr><tr><td> Natrium monobasiskt (torka) </td><td> ThermoFisher </td><td> S381 </td><td></td></tr><tr><td> Dinatriumvätefosfat (vattenfritt) </td><td> ThermoFisher </td><td> S374 </td><td></td></tr><tr><td> Magnesiumklorid (hexahydrat) </td><td> ThermoFisher </td><td> M33 </td><td></td></tr><tr><td> Tris Base </td><td> ThermoFisher </td><td> B152 </td><td></td></tr><tr><td> Järnklorid (hexahydrat) </td><td> ThermoFisher </td><td> I88 </td><td></td></tr><tr><td> Triklorättiksyra </td><td> ThermoFisher </td><td> A324 </td><td></td></tr><tr><td> Hydroxylamin hydroklorid </td><td> ThermoFisher </td><td> H330 </td><td></td></tr><tr><td> Adenosin 5&#39;-difosfat natriumsalt </td><td> Sigma-Aldrich </td><td> A2754 </td><td></td></tr><tr><td> BIOMATE III Spektrofotometer </td><td> ThermoFisher </td><td> 142982082 </td><td> Standard UV / Vis spektrofotometer </td></tr></table>

direct detection of the acetate-forming activity  of the enzyme acetate kinase

Acetat kinas, en medlem av acetat och socker kinas-HSP70-aktin (ASKHA) enzym superfamiljen 1-5, ansvarar för den reversibla fosforyleringen av acetat till acetyl fosfat använda ATP som substrat. Acetat kinaser finns överallt i bakterier, som finns i ett släkte av arkéerna, och är också närvarande i mikrober i Eukarya 6. Den mest kännetecknas acetat kinas är att från metanbildande archaeon Methanosarcina thermophila 7-14. En acetat kinas som bara kan använda PP-I men inte ATP i acetyl fosfat-bildande riktning har isolerats från Entamoeba histolytica, vilka smittämnen av amöba dysenteri, och har hittills bara hittats i denna genus 15,16. I riktning mot acetyl fosfat bildas, är acetat kinasaktivitet normalt mätt med hydroxamate analys, som först beskrevs av Lipmann17-20, ett kombinerat test där omvandlingen av ATP till ADP är kopplad till oxidation av NADH till NAD + genom att enzymerna pyruvat kinas och laktatdehydrogenas 21,22 eller ett test som mäter utsläpp av oorganiskt fosfat efter reaktion acetyl fosfat produkt med hydroxylamin 23. Aktiviteten i den motsatta, är acetat-forming riktning mäts med koppling ATP bildas från ADP till en minskning av NADP + till NADPH av enzymer hexokinas och glukos-6-fosfatdehydrogenas 24. Här beskriver vi en metod för detektion av acetat kinasaktivitet i riktning mot acetat formation som inte kräver koppling enzymer, utan istället bygger på direkt bestämning av acetyl fosfat konsumtion. Efter den enzymatiska reaktionen är kvar acetyl fosfat omvandlas till en järnklorid hydroxamate komplex som kan mätas spektrofotometriskt, som för hydroxamate analysen. Till skillnad från S: tandard tillsammans assay för denna riktning som är beroende av produktionen av ATP från ADP, kan denna direkta analys användas för acetat kinaser som producerar ATP eller PP i.

Watch this Scientific Journal Video about Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase at JoVE.com

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

En metod för bestämning av acetat kinasaktivitet beskrivs. Denna analys använder en direkt reaktion för att bestämma enzymaktiviteten och kinetik för acetat kinas i acetat-bildande riktning med olika fosforylklorid som accepterar. Dessutom kan denna metod användas för testmetoder för andra acetyl fosfat eller Acetyl-CoA använder enzymer.

Direkt detektion av acetat-bildande aktivitet av enzymet acetat Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

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