醋酸形成醋酸酶激酶活性的直接检测
Summary
醋酸激酶活性测定方法的描述。本实验利用一个直接反应在醋酸形成的方向确定不同磷受体酶的活性和乙酸激酶动力学。此外，这种方法可以用于化验等乙酰磷酸盐或乙酰CoA利用酶。
Abstract
醋酸激酶，醋酸盐和糖激酶- HSP70 -肌动蛋白（ASKHA）1-5酶超家族成员，负责对醋酸的可逆磷酸化乙酰磷酸盐作为基质利用ATP 。醋酸激酶是无处不在的细菌 ，发现一个古属的，而且也存在于 真核生物 6微生物。最特征的醋酸激酶是由产甲烷古菌甲烷嗜 热 7-14 。 阿米巴 ，阿米巴痢疾的病原体已被隔离醋酸激酶只能利用聚丙烯 ，但不乙酰磷酸形成的方向ATP，并迄今只发现该属15,16。
在乙酰磷酸形成的方向，醋酸激酶活性通常是测量使用的氧肟检测，首先由李普曼描述17-20日 ，耦合检测的ATP转化为ADP耦合NADH氧化为NAD +的酶丙酮酸激酶和乳酸脱氢酶21,22，或乙酰磷酸盐产品的反应后释放的无机磷的实验测量羟胺23。醋酸形成方向相反的活动，是衡量耦合ATP的形成，从ADP 减少NADP +，NADPH酶己糖激酶和葡萄糖-6 – 磷酸脱氢酶24。
在这里，我们描述了醋酸醋酸形成，不需要耦合酶的方向激酶活性检测的方法，而是被直接测定乙酰磷酸消费。酶反应后，剩余的乙酰磷酸转化为羟肟检测，可以测量分光光度计的一个铁氧肟复杂。因此，不像STandard加上这个方向是依赖于从ADP ATP的生产检测，这种直接的检测可用于醋酸激酶产生ATP或PP 我 。
Protocol
此协议的总体方案概述图1。 1。标准曲线和检测解决方案的准备工作准备100毫升2 mol / L的羟胺盐酸溶液。称取13.9克盐酸羟胺（兆瓦69.49克/摩尔），溶于约50毫升蒸馏水，去离子水（DDH 2 O）的。使用氢氧化钾颗粒或浓缩液，调整pH至7.0。带来的最终体积至100 mL。该解决方案可在常温下保存长达30天，或在4 ° C下90天。 准备一个三氯化铁/盐酸溶液100毫升。?…
Discussion
在这个实验中的乙酰磷酸的检测是根据足够浓度的盐酸羟胺和氯化铁溶液的浓度和酸度依赖。更改的检测量将需要重新考虑这两个组件。酶促反应这里描述的是37 °与5分钟，在60 ° C时，羟胺终止。这较高的温度是至关重要的，以便其余乙酰磷酸迅速转化为乙酰氧肟。颜色一步发展和吸光度的测量时间是不太重要的，羟胺溶液此外，可以在短短的5分钟至15分钟进行。样品应该离心读取吸光度之前，…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作是由国家科学基金会奖＃0920274和南卡罗来纳州试验站项目（SC – 1700340）幼稚园资助计划的支持。本文是技术贡献的克莱姆森大学实验站5929号。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Acetyl phosphate | Sigma Aldrich | 01409 | Lithium Salt (97% ) |
| Sodium phosphate monobasic (dehydrate) | ThermoFisher | S381 | |
| Sodium phosphate dibasic (anhydrous) | ThermoFisher | S374 | |
| Magnesium chloride (hexahydrate) | ThermoFisher | M33 | |
| Tris Base | ThermoFisher | B152 | |
| Ferric chloride (hexahydrate) | ThermoFisher | I88 | |
| Trichloroacetic acid | ThermoFisher | A324 | |
| Hydroxylamine hydrochloride | ThermoFisher | H330 | |
Adenosine 5’-diphosphate sodium salt |
Sigma Aldrich | A2754 | |
| Biomate III Spectrophotometer | ThermoFisher | 142982082 | Standard UV/Vis spectrophotometer |
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Tags
Acetate KinaseAcetate-forming ActivityEnzymeEnzyme SuperfamilyPhosphorylationATPAcetyl PhosphateBacteriaArchaeaEukaryaMethanosarcina ThermophilaEntamoeba HistolyticaAmoebic DysenteryHydroxamate AssayNADHNAD+Pyruvate KinaseLactate DehydrogenaseInorganic PhosphateHydroxylamineATP FormationADPNADP+NADPHHexokinaseGlucose 6-phosphate Dehydrogenase
Citar este artículo
Fowler, M. L., Ingram-Smith, C. J., Smith, K. S. Direct Detection of the Acetate-forming Activity of the Enzyme Acetate Kinase. J. Vis. Exp. (58), e3474, doi:10.3791/3474 (2011).