Bu çalışma, NSF ödül # 0920274 ve Güney Carolina Deney İstasyonu Projesi (SC-1.700.340) KSS tarafından desteklenen oldu. Bu makale, Clemson Üniversitesi Experiment Station 5929 Teknik Katkı No.

Bu protokolün genel şeması Şekil 1&#39;de özetlenmiştir. 1. Standart Eğriler ve Tahliller Çözüm Hazırlığı <ol><li> Hidroksilamin-HCl 2 mol / L çözelti 100 mL hazırlayın. Hidroksilamin hidroklorür 13,9 g (MW 69.49 g / mol) tartılır ve yaklaşık 50 ml distile deiyonize su (GKD 2 O) içinde çözülür. Potasyum hidroksit pelet veya konsantre bir çözüm kullanarak 7.0 pH ayarlayın. Son hacim 100 mL &#39;ye getirin. Çözüm 30 gün kadar ya da 4 ° C&#39;de...

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Bu testte asetil fosfat algılama hidroksilamin hidroklorür yeterli konsantrasyon ve ferrik klorür çözeltisi konsantrasyonu ve asit bağlıdır. Bu bileşenlerin her iki testin hacmi değişiklik yeniden gözden geçirilmesini gerektirebilir. Enzimatik reaksiyonları burada açıklanan 37 ° yapıldı ° C, 5 dakika 60 ° C hidroksilamin sonlandırma. Bu yüksek sıcaklık asetil hydroxamate kalan asetil fosfat hızlı dönüşüm için izin verecek kritik öneme sahiptir. Absorbans renk geliştirme adım ve ölçü...

<table border="1"><tr><td width="160"> Reaktifi Adı </td><td width="108"> Şirket </td><td width="134"> Katalog numarası </td><td width="103"> Yorumlar </td></tr><tr><td> Asetil fosfat </td><td> Sigma Aldrich </td><td> 01409 </td><td> Lityum tuz (% 97) </td></tr><tr><td> Sodyum fosfat tekbazlı (kurutmak) </td><td> ThermoFisher </td><td> S381 </td><td></td></tr><tr><td> Sodyum fosfat dibazik (susuz) </td><td> ThermoFisher </td><td> S374 </td><td></td></tr><tr><td> Magnezyum klorür (hekzahidrat) </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> Ferrik klorür (hekzahidrat) </td><td> ThermoFisher </td><td> I88 </td><td></td></tr><tr><td> Trikloroasetik asit </td><td> ThermoFisher </td><td> A324 </td><td></td></tr><tr><td> Hidroksilamin hidroklorür </td><td> ThermoFisher </td><td> H330 </td><td></td></tr><tr><td> Adenozin 5&#39;-difosfat sodyum tuzu </td><td> Sigma Aldrich </td><td> A2754 </td><td></td></tr><tr><td> Biomate III Spektrofotometre </td><td> ThermoFisher </td><td> 142982082 </td><td> Standart UV / Vis spektrofotometre </td></tr></table>

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

Asetat kinaz, asetat ve şeker kinaz-HSP70-aktin (ASKHA) enzim süperailesinin 1-5 bir üyesi, bir substrat olarak ATP kullanan asetil fosfat asetat geri dönüşümlü fosforilasyon için sorumludur. Asetat kinazlar Archaea bir cins bulundu, Bakteriler her yerde, ve aynı zamanda ökaryotlarının 6 mikroplar bulunmaktadır. En iyi karakterize asetat kinaz olduğunu metan üreten archaeon Methanosarcina thermophila 7-14. Sadece asetil fosfat oluşturan yönde ATP i PP yararlanmak ancak bir asetat kinaz Entamoeba histolytica, amip dizanteri etkeni izole edilmiş ve şimdiye kadar sadece bu cinsin 15,16 olmuştur. Asetil fosfat oluşum doğrultusunda, asetat kinaz aktivitesini genellikle ilk Lipmann tarafından açıklanan, hydroxamate tahlil kullanarak ölçülür.17-20, enzimler piruvat kinaz ve laktat dehidrogenaz 21,22 ADP ATP dönüşüm NAD NADH oksidasyonu birleştiğinde olduğu bir birleştiğinde tahlil + veya asetil fosfat ürün reaksiyon sonra inorganik fosfatın serbest ölçen bir test hidroksilamin 23. Etkinlik tam tersi, asetat oluşturan yönü ADP NADP azaltılması + enzimler heksokinaz ve glukoz 6-fosfat dehidrogenaz 24 NADPH, ATP oluşumu birleştirilmesi ile ölçülür . Burada kaplin enzimler gerektirmez asetat oluşumu yönünde asetat kinaz aktivitesinin tespiti için bir yöntem tarif, ama onun yerine asetil fosfat tüketimi doğrudan belirlenmesi üzerine dayanır. Enzimatik reaksiyon sonra, kalan asetil fosfat hydroxamate tayini için spektrofotometrik olarak ölçülmüş olabilir ferrik hydroxamate karmaşık dönüştürülür. Böylece, I. aksineADP ATP üretimine bağımlı bu yönde andard birleştiğinde tahlil, doğrudan bu testi ATP veya PP i üretmek asetat kinazlar için kullanılabilir.

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

Asetat kinaz aktivitesinin belirlenmesi için bir yöntem açıklanmıştır. Bu testte, asetat oluşturan yönde asetat kinaz enzim aktivitesi ve kinetiği farklı fosforilasyonunu alıcıları belirlemek için doğrudan bir tepki kullanır. Ayrıca, bu yöntem diğer asetil fosfat ya da asetil-CoA kullanan enzimler assaying için kullanılabilir.

Enzim Asetat Kinaz Asetat oluşturan Faaliyet Doğrudan Algılama

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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