Isolation of Tomato Fruit Chromoplasts and Determination of ATP Levels
番茄果实有色体的分离和ATP含量的测定
发布: 2014年08月05日第4卷第15期 DOI: 10.21769/BioProtoc.1192 浏览次数: 10624
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参见作者原研究论文
The authors used this protocol in:
Apr 2013
Abstract
It has recently been reported that tomato fruit chromoplasts can synthesize ATP de novo using an ATP synthase complex harboring an atypical γ-subunit which is also present in a variety of plant species. However many aspects related with the biochemical processes underlying this process remain largely unknown. Here we describe detailed protocols for the isolation of tomato fruit chromoplasts and the determination of ATP levels (end-point measurements) and ATP synthesis rates (kinetic measurements) in these organelles using bioluminescent luciferin/luciferase based assays.
Keywords: Chromoplast (成色素细胞)
Tomato (番茄)
Fruit (水果)
ATP levels (ATP水平)
Materials and Reagents
- Red tomato fruits (MicroTom or Ailsa Craig, harvested 5-7 and 7-9 days after breaker stage, respectively)
- Ultrapure water
- NaCl (Sigma-Aldrich, catalog number: S7653 )
- Trizma® base (Tris) (Sigma-Aldrich, catalog number: T6066 )
- HCl (Merck KGaA, catalog number: 100317 )
- Sorbitol (Sigma-Aldrich, catalog number: S1878 )
- MgCl2 (Sigma-Aldrich, catalog number: M2670 )
- KCl (Sigma-Aldrich, catalog number: P9541 )
- L-Ascorbic acid (Sigma-Aldrich, catalog number: 255564 )
- L-Cysteine (Sigma-Aldrich, catalog number: C7352 )
- DL-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43819 )
- Sucrose (Sigma-Aldrich, catalog number: S8501 )
- Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
- MnCl2 (Sigma-Aldrich, catalog number: M3634 )
- KH2PO4 (Merck KGaA, catalog number: 104873 )
- β-Nicotinamide adenine dinucleotide 2’-phosphate reduced tetrasodium salt hydrate (NADPH) (Sigma-Aldrich, catalog number: N7505 )
- β-Nicotinamide adenine dinucleotide phosphate hydrate (NADP+) (Sigma-Aldrich, catalog number: N5755 )
- β-Nicotinamide adenine dinucleotide reduced disodium salt hydrate (NADH) (Sigma-Aldrich, catalog number: N8129 )
- β-Nicotinamide adenine dinucleotide hydrate (NAD+) (Sigma-Aldrich, catalog number: N1636 )
- Flavin adenine dinucleotide disodium salt hydrate (FAD) (Sigma-Aldrich, catalog number: F6625 )
- Sodium pyruvate (Sigma-Aldrich, catalog number: P2256 )
- ATP Bioluminescence Assay Kit HS II (Roche Diagnostics, catalog number: 11699709001 )
- ENLITEN® rLuciferase/Luciferin Reagent (Promega Corporation, catalog number: FF2021 )
- RC DC Protein Assay kit (Bio-Rad Laboratories, catalog number: 500-0122 )
- Adenosine 5’-diphosphate sodium salt (ADP) (Sigma-Aldrich, catalog number: A2754 )
- Adenosine 5’-triphosphate disodium salt hydrate (ATP) (Sigma-Aldrich, catalog number: A2383 )
- P1, P5-di(adenosine-5’) pentaphosphate pentasodium salt (DAPP) (Sigma-Aldrich, catalog number: D4022 )
- 2-[(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid, N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES) (Sigma-Aldrich, catalog number: T1375 )
- 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) (HEPES) (Sigma-Aldrich, catalog number: H4034 )
- Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
- Tween 20 (Sigma-Aldrich, catalog number: P1379 )
- Miracloth (pore size of 22-25 µm) (Calbiochem®, catalog number: 475855 )
- 1 mM DAPP (stored at -20 °C)
- 10 mM FAD (stored at -80 °C)
- 100 mM NADH (stored at -80 °C)
- 100 mM NAD+ (stored at -80 °C)
- 100 mM NADPH (stored at -80 °C)
- 100 mM NADP+ (stored at -80 °C)
- 10 µM ATP standard solutions (stored at -80 °C)
- 0.1 µM ATP standard solutions (stored at -80 °C)
- 0.001 µM ATP standard solutions (stored at -80 °C)
- 1 M DTT (stored at -20 °C)
- 0.5 mM ADP
- Washing solution (see Recipes)
- 0.5 M Tris-HCl buffer (see Recipes)
- 15% sucrose solution (see Recipes)
- 30% sucrose solution (see Recipes)
- 40% sucrose solution (see Recipes)
- 50% sucrose (see Recipes)
- Buffer A (see Recipes)
- Buffer B (see Recipes)
- Buffer C (see Recipes)
- Buffer D (see Recipes)
- Buffer E (see Recipes)
Equipment
- Glomax® 96 Microplate Luminometer w/Dual Injectors (Promega Corporation, catalog number: E6521 )
- Centrifuge Avanti J-E (Beckman Coulter)
- Ultracentrifuge Optima L-100xPI (Beckman Coulter)
- SW 28 swinging-bucket rotor (Beckman Coulter) or equivalent
- JA-14 rotor (Beckman Coulter) or equivalent
- JA-20 rotor (Beckman Coulter) or equivalent
- Polypropylene centrifuge tubes (250 ml) (Beckman Coulter)
- Polypropylene centrifuge tubes (30 ml) (Beckman Coulter)
- Polyallomer centrifuge tubes for SW 28 rotor (Beckman Coulter)
- Waring blender 8011S (Dynamics Corporation of America)
- Support stand, clamp holder and extension clamp
- Glass beakers of different volume capacity
- 96-well microplates white (Porvair Sciences, catalog number: 204003 )
- Bench orbital shaker
- Micropipettes
- Heat block
- Benchtop microcentrifuge
- Gauze (1 m x 1 m)
Software
- Microsoft Excel
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Hernández-Gras, F., Petrizzo, R., Pateraki, I., Renato, M., Angaman, M., Azcón-Bieto, J. and Boronat, A. (2014). Isolation of Tomato Fruit Chromoplasts and Determination of ATP Levels. Bio-protocol 4(15): e1192. DOI: 10.21769/BioProtoc.1192.
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分类
植物科学 > 植物细胞生物学 > 细胞器分离
植物科学 > 植物生物化学 > 蛋白质
细胞生物学 > 细胞器分离 > 分级分离
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