Dette arbejde blev støttet af NSF pris # 0920274 og South Carolina Experiment Station Project (SC-1700340) til KSS. Dette papir er teknisk bidrag nr. 5929 af Clemson University Experiment Station.

Den overordnede opbygning af denne protokol er skitseret i figur 1. 1. Løsning Forberedelse til Standard Kurver og Analyser <ol><li> Forbered 100 ml af en 2 mol / L opløsning af hydroxylamin-HCl. Der afvejes 13,9 g hydroxylamin hydrochlorid (MW 69,49 g / mol) og opløses i 50 ml destilleret, demineraliseret vand (Hedeselskabet 2 O). Justér pH til 7,0 ved hjælp af kaliumhydroxid piller eller en koncentreret opløsning. Bring den endelige volumen til 100 ml. Løsningen kan opb...

<ol>
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Påvisning af acetyl fosfat i denne test er afhængig af en tilstrækkelig koncentration af hydroxylamin hydrochlorid og koncentrationen og surhedsgrad ferrichloridopløsning. Ændring af analysen volumen vil kræve en revurdering af begge disse komponenter. Den enzymatiske reaktioner beskrevet her blev udført ved 37 ° C med hydroxylamin opsigelse udføres ved 60 ° C i 5 minutter. Denne højere temperatur er afgørende for at muliggøre en hurtig konvertering af de resterende acetyl fosfat til acetyl hydroxamate. Tim...

<table border="1"><tr><td width="160"> Navn på reagenset </td><td width="108"> Firma </td><td width="134"> Katalog nummer </td><td width="103"> Kommentarer </td></tr><tr><td> Acetyl fosfat </td><td> Sigma Aldrich </td><td> 01409 </td><td> Lithium Salt (97%) </td></tr><tr><td> Sodium monobasisk (dehydrere) </td><td> ThermoFisher </td><td> S381 </td><td></td></tr><tr><td> Sodium dibasisk (vandfri) </td><td> ThermoFisher </td><td> S374 </td><td></td></tr><tr><td> Magnesiumchlorid (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> Ferrichlorid (hexahydrat) </td><td> ThermoFisher </td><td> I88 </td><td></td></tr><tr><td> Trichloreddikesyre </td><td> ThermoFisher </td><td> A324 </td><td></td></tr><tr><td> Hydroxylamin hydrochlorid </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 kinase, et medlem af acetat og sukker kinase-HSP70-actin (ASKHA) enzym superfamily 1-5, er ansvarlig for reversibel phosphorylering af acetat til acetyl fosfat udnytte ATP som substrat. Acetat kinaser er allestedsnærværende i de bakterier, der findes i en slægt af Archaea, og er også til stede i mikrober af Eukarya 6. Den mest velkarakteriserede acetat kinase er, at fra den metan-producerende archaeon Methanosarcina thermophila 7-14. En acetat kinase, der kun kan udnytte PP I, men ikke ATP i acetyl fosfat-dannende retning har været isoleret fra Entamoeba histolytica, det agens af dysenteri, og har hidtil kun fundet i denne slægt 15,16. I retning af acetyl fosfat dannelse, er acetat kinase aktivitet typisk måles efter den hydroxamate assay, første gang beskrevet af Lipmann17-20, en koblet assay, hvor omdannelsen af ATP til ADP, er koblet til oxidation af NADH til NAD + ved de enzymer pyruvat kinase og laktat dehydrogenase 21,22, eller en assay måler frigivelse af uorganisk fosfat efter reaktion af acetyl fosfat produktet med hydroxylamin 23. Aktiviteten i det modsatte, er acetat-dannende retning, målt ved at koble ATP dannelsen fra ADP til en reduktion af NADP + til NADPH af enzymer hexokinase og glucose 6-phosphat dehydrogenase-24. Her beskriver vi en metode til påvisning af acetat kinase aktivitet i retning af acetat formation, der ikke kræver kobling enzymer, men er i stedet baseret på direkte bestemmelse af acetyl fosfat forbrug. Efter den enzymatiske reaktion, der er tilbage acetyl fosfat omdannes til en ferri hydroxamate kompleks, der kan måles spektrofotometrisk, som for hydroxamate analysen. Således, i modsætning til standard koblet assay for denne retning, er afhængig af produktionen af ATP fra ADP, kan denne direkte analyse skal bruges til acetat kinaser, der producerer 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 metode til bestemmelse af acetat kinase aktivitet er beskrevet. Denne analyse anvender en direkte reaktion til bestemmelse af enzymaktivitet og kinetik af acetat kinase i acetat-dannende retning med forskellige phosphoryl receptorer. Desuden kan metoden anvendes til analysering andre acetyl fosfat eller acetyl-CoA udnytte enzymer.

Direkte påvisning af acetat-dannende aktivitet af 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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