番茄胞质中的磷酸烯醇丙酮酸羧激酶（PEPCK）和质体中的NADP依赖性苹果酸酶（ME）的提取和活性测定

Sonia  Osorio; José G.  Vallarino; Marek  Szecowka; Shai  Ufaz; Vered  Tzin; Ruthie  Angelovici; Gad  Galili; Alisdair R.  Fernie

doi:10.21769/BioProtoc.1122

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Peer-reviewed

Extraction and Measurement the Activities of Cytosolic Phosphoenolpyruvate Carboxykinase (PEPCK) and Plastidic NADP-dependent Malic Enzyme (ME) on Tomato (Solanum lycopersicum)

番茄胞质中的磷酸烯醇丙酮酸羧激酶（PEPCK）和质体中的NADP依赖性苹果酸酶（ME）的提取和活性测定

SO Sonia Osorio email

AF Alisdair R. Fernie

发布: 2014年05月05日第4卷第9期 DOI: 10.21769/BioProtoc.1122 浏览次数: 10743

评审: Tie Liu

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参见作者原研究论文

The authors used this protocol in:

Cover of Plant Physiology, featuring study using the protocol.

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Abstract

A recent study demonstrated that cytosolic phosphoenolpyruvate carboxykinase (PEPCK) and NADP-malic enzyme (NADP-ME) have an important role in malate metabolism during fruit ripening (Osorio et al., 2013). PEPCK catalyze the ATP-dependent decarboxylation of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) and NADP-ME, the reversible conversion of malate and pyruvate. Here, we present the detailed protocols to measure PEPCK activity in carboxylation direction by following oxidation of NADH and to measure NADP-ME activity based upon the reduction of NADP⁺.

Materials and Reagents

PEPCK activity
1. Un-harvested plant tissues (how to freeze the tissue is explained in the procedure section)
2. Liquid N₂
3. Ice
4. Bicine (Sigma-Aldrich, catalog number: B8660 )
5. KOH
6. EDTA (Sigma-Aldrich, catalog number: EDS-100G )
7. Poly (ethylene glycol)-4000 (Sigma-Aldrich, catalog number: 81240 )
8. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43815 )
9. β-nicotinamide adenine dinucleotide reduced form (NADH) (Roche Diagnostics, catalog number: 10128015001 )
10. 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) (Sigma-Aldrich, catalog number: H3375 )
11. KCl
12. MnCl₂
13. Phosphoelnolpyryvate (PEP) (Bio Vectra, catalog number: 2552)
14. Adenosine 5′-diphosphate sodium salt (ADP) (Sigma-Aldrich, catalog number: A2754 )
15. KHCO₃
16. L-Malate dehydrogenase (Roche Diagnostics, catalog number: 10127248001 )
17. Bradford stock solution (Bio-Rad Laboratories, catalog number, 500-0006 )
18. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2058 )
19. Extraction buffer 1 (see Recipes)
20. Extraction buffer 2 (see Recipes)
21. Buffer 3 (see Recipes)
22. PEPCK assay mix 1 (see Recipes)
NADP-ME activity
1. Un-harvested plant tissues (how to freeze the tissue is explained in the procedure section)
2. Liquid N₂
3. Ice
4. Tris-Base (United State Biological, catalog number: T8600 )
5. MnCl₂
6. EDTA (Sigma-Aldrich, catalog number: EDS-100G)
7. Glycerol (Sigma-Aldrich, catalog number: G5516 )
8. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
9. β-nicotinamide adenine dinucleotide phosphate (NADP⁺) (Roche Diagnostics, catalog number: 10128058001 )
10. L-Malate (Sigma-Aldrich, catalog number: M1000 )
11. Bradford stock solution (Bio-Rad Laboratories, catalog number, 500-0006)
12. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2058)
13. Extraction buffer 4 (see Recipes)
14. NADP-ME assay mix 1 (see Recipes)

Equipment

Small mortar and pestle
2 ml and 1.5 ml microfuge tubes
Pipettes
Balance
2 ml centrifuge (Hettich Mikro 22R)
96 well polystyrene microplate (flat bottom) (Corning, catalog number: 3300 )
A computer supported microplate spectrophotometer for kinetic (time-course) measurement mode (Elisa microplate-spetrophotometer) (BioTek Instruments, model: EL808 )

Procedure

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Osorio, S., Vallarino, J. G., Szecowka, M., Ufaz, S., Tzin, V., Angelovici, R., Galili, G. and Fernie, A. R. (2014). Extraction and Measurement the Activities of Cytosolic Phosphoenolpyruvate Carboxykinase (PEPCK) and Plastidic NADP-dependent Malic Enzyme (ME) on Tomato (Solanum lycopersicum). Bio-protocol 4(9): e1122. DOI: 10.21769/BioProtoc.1122.
Osorio, S., Vallarino, J. G., Szecowka, M., Ufaz, S., Tzin, V., Angelovici, R., Galili, G. and Fernie, A. R. (2013). Alteration of the interconversion of pyruvate and malate in the plastid or cytosol of ripening tomato fruit invokes diverse consequences on sugar but similar effects on cellular organic acid, metabolism, and transitory starch accumulation. Plant Physiol 161(2): 628-643.