Determination of the H+-ATP Synthase and Hydrolytic Activities
测定 H+-ATP 合成酶的合成和水解活性
(*contributed equally to this work) 发布: 2016年08月20日第6卷第16期 DOI: 10.21769/BioProtoc.1905 浏览次数: 9497
评审: Masahiro MoritaDaniel KrausAntoine de Morree
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参见作者原研究论文
The authors used this protocol in:
Sep 2015
Abstract
The H+-ATP synthase of the inner mitochondrial membrane utilizes the proton gradient generated by the respiratory chain to synthesize ATP. Under depolarizing conditions, it can function in reverse by hydrolyzing ATP to generate a proton gradient. The protocols presented here allow the facile determination of both the synthetic and hydrolytic activities of the H+-ATP synthase in isolated mitochondria and in permeabilized mammalian cells. Since the protocol requires the isolation of polarized and well-coupled mitochondria, first we describe the protocol for mitochondrial isolation from mouse tissues. Second, we describe the protocol for measuring the ATP synthetic activity as end-point and kinetic modes in isolated mitochondria and in permeabilized cells. Finally, we describe the protocol for the determination of the ATP hydrolytic activity of the enzyme in isolated mitochondria.
Keywords: Mitochondria (线粒体)
Oxidative phosphorylation (氧化磷酸化)
Enzyme activity (酶的活性)
ATP synthase (ATP合成酶)
ATP hydrolase (ATP水解酶)
Part I. The isolation of mitochondria from mouse tissues
Materials and Reagents
- 10 ml tubes
- 1.5 ml Eppendorf tubes
- Sucrose (Merck Millipore Corporation, catalog number: 107651 )
- Ethylenediaminetetraacetic acid disodium salt dehydrate (Sigma-Aldrich, catalog number: ED2SS )
- D-Sorbitol (Sigma-Aldrich, catalog number: S1876 )
- Ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (Sigma-Aldrich, catalog number: E4378 )
- Trizma® base (Sigma-Aldrich, catalog number: T1503 )
- Bovine serum albumin (Sigma-Aldrich, catalog number: A7906 )
- Protein assay dye reagent concentrate (Bio-Rad Laboratories, catalog number: 5000006 )
- Medium A (see Recipes)
- Medium H (see Recipes)
Equipment
- 15 ml Dounce All-Glass tissue grinder (Kimble, catalog number: 885303-0015 )
- Beckman Avanti J-25 centrifuge (Beckman Coulter, model: Avanti J-25 )
- JA-25.50 rotor, fixed angle, aluminum, single lock lid, 8 x 50 ml, 25,000 rpm, 75,600 x g (Beckman Coulter, catalog number: 363055 )
- Bio-VialTM tube, PP, 4 ml, 14 x 55 mm, 0.56 x 2.2 in (Beckman Coulter, catalog number: 566353 )
- Centrifuge 5415R (Sigma-Aldrich, Eppendorf®, model: 5415R )
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
García-Bermúdez, J., Nuevo-Tapioles, C. and Cuezva, J. M. (2016). Determination of the H+-ATP Synthase and Hydrolytic Activities. Bio-protocol 6(16): e1905. DOI: 10.21769/BioProtoc.1905.
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分类
生物化学 > 蛋白质 > 活性
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