Diese Arbeit wurde von der NSF award # 0920274 und South Carolina Experiment Station Project (SC-1700340), um KSS unterstützt. Dieses Papier ist technischen Beitrag Nr. 5929 von der Clemson University Experiment Station.

Das Gesamtkonzept dieses Protokolls ist in Abbildung 1 dargestellt. 1. Herstellung der Lösung für Standard-Kurven und Assays <ol><li> Bereiten Sie 100 mL einer 2 mol / L Lösung von Hydroxylamin-HCl. Abwiegen 13,9 g Hydroxylamin-Hydrochlorid (MW 69,49 g / mol) und lösen sich in ca. 50 mL destilliertem entionisiertem Wasser (ddH 2 O). Den pH-Wert auf 7,0 mit Kaliumhydroxid Pellets oder eine konzentrierte Lösung. Bringen Sie das Endvolumen von 100 mL. Die Lösung kann bei Raum...

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Der Nachweis von Acetyl-Phosphat in diesem Test ist abhängig von einer ausreichenden Konzentration von Hydroxylaminhydrochlorid und die Konzentration und Säuregehalt der Eisenchlorid-Lösung. Änderung der Assayvolumen erfordert Überprüfung dieser beiden Komponenten. Die enzymatischen Reaktionen hier beschriebenen wurden bei 37 ° C mit dem Hydroxylamin Kündigung bei 60 ° C für 5 Minuten. Diese höhere Temperatur ist entscheidend, um eine rasche Wandlung der restlichen Acetylphosphat zu Acetyl Hydroxamat ermögli...

<table border="1"><tr><td width="160"> Name des Reagenzes </td><td width="108"> Firma </td><td width="134"> Katalog-Nummer </td><td width="103"> Kommentare </td></tr><tr><td> Acetyl-Phosphat </td><td> Sigma Aldrich </td><td> 01409 </td><td> Lithium Salz (97%) </td></tr><tr><td> Mononatriumphosphat (dehydrieren) </td><td> ThermoFisher </td><td> S381 </td><td></td></tr><tr><td> Natriumdihydrogenphosphat (wasserfrei) </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> Eisenchlorid (Hexahydrat) </td><td> ThermoFisher </td><td> I88 </td><td></td></tr><tr><td> Trichloressigsäure </td><td> ThermoFisher </td><td> A324 </td><td></td></tr><tr><td> Hydroxylaminhydrochlorid </td><td> ThermoFisher </td><td> H330 </td><td></td></tr><tr><td> Adenosin-5&#39;-diphosphat Natriumsalz </td><td> Sigma Aldrich </td><td> A2754 </td><td></td></tr><tr><td> BioMate III-Spektralphotometer </td><td> ThermoFisher </td><td> 142982082 </td><td> Standard UV / Vis-Spektralphotometer </td></tr></table>

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

Acetat-Kinase, ein Mitglied der Acetat-und Zucker-Kinase-Hsp70-Aktin (ASKHA) Enzym-Superfamilie 1-5, ist verantwortlich für die reversible Phosphorylierung von Acetat zu Acetyl-Phosphat nutzen ATP als Substrat. Acetat-Kinasen sind ubiquitär in der Bakterien, in einer Gattung von Archaea gefunden und sind auch in Mikroben der Eukarya 6. Die am besten charakterisierten Acetat-Kinase ist, dass aus der Methan-produzierenden Archaeon Methanosarcina thermophila 7-14. Ein Acetat-Kinase, die nur nutzen können, PP i, aber nicht in die Acetyl-Phosphat-bildenden Richtung ATP ist von Entamoeba histolytica, der Erreger der Amöbenruhr isoliert worden, und bisher nur in dieser Gattung 15,16 gefunden. In Richtung der Acetyl-Phosphat-Bildung ist Acetat-Kinase-Aktivität in der Regel unter Verwendung der Hydroxamat Assay, die zuerst von Lipmann beschrieben17-20, eine gekoppelte Assay, bei dem Umsatz von ATP zu ADP um die Oxidation von NADH zu NAD + gekoppelt ist durch die Enzyme Pyruvatkinase und Lactat-Dehydrogenase 21,22 oder ein Assay Messung Freisetzung von anorganischem Phosphat nach der Reaktion der Acetyl-Phosphat-Produkts mit Hydroxylamin 23. Aktivität in das Gegenteil, ist Acetat-bildenden Richtung durch die Kopplung ATP-Bildung aus ADP auf die Reduktion von NADP + durch die Enzyme Hexokinase und Glucose-6-Phosphat-Dehydrogenase 24 NADPH gemessen. Hier beschreiben wir eine Methode zum Nachweis von Acetat-Kinase-Aktivität in Richtung der Acetat-Bildung, der keine Kopplung Enzyme, sondern ist auf die direkte Bestimmung von Acetyl-Phosphat-Verbrauch. Nach der enzymatischen Reaktion wird noch Acetylphosphat zu einem Eisen-III-Hydroxamat komplex, dass spektrophotometrisch gemessen werden kann, wie für die Hydroxamat Assay umgewandelt. So anders als die standard gekoppelte Assay für diese Richtung, dass abhängig von der Produktion von ATP aus ADP ist, kann diese direkt Assay für Acetat-Kinasen, ATP oder PP i zu produzieren verwendet werden.

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

Eine Methode zur Bestimmung von Acetat-Kinase-Aktivität beschrieben. Dieser Assay nutzt eine direkte Reaktion zur Bestimmung der Enzymaktivität und die Kinetik der Acetat-Kinase in die Acetat-bildenden Richtung mit unterschiedlichen Phosphoryl Akzeptoren. Darüber hinaus kann diese Methode für die Untersuchung auf andere Acetyl-Phosphat-oder Acetyl-CoA-Enzyme verwendet genutzt werden.

Direkter Nachweis der Acetat-bildenden Aktivität des Enzyms 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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