体外比色法测定植物谷氨酸脱氢酶的活性

Asier Sarasketa; Izargi Vega-Mas; Daniel Marino

doi:10.21769/BioProtoc.1560

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Peer-reviewed

In vitro Colorimetric Method to Measure Plant Glutamate Dehydrogenase Enzyme Activity

体外比色法测定植物谷氨酸脱氢酶的活性

Asier Sarasketa

Izargi Vega-Mas

DM Daniel Marino email

发布: 2015年08月20日第5卷第16期 DOI: 10.21769/BioProtoc.1560 浏览次数: 11378

评审: Arsalan DaudiAnonymous reviewer(s)

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Cover of Journal of Experimental Botany, featuring study using the protocol.

Nov 2014

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Abstract

Glutamate dehydrogenase (GDH) is an NAD(H) dependent enzyme that catalyzes, in vitro, the reversible amination of glutamate. Here we describe how to determine spectrophotometrically GDH activity monitoring NADH evolution. This protocol is described here for Arabidopsis thaliana (A. thaliana) although it is also valid for other plant species. GDH protein is a hexamer composed, in the case of Arabidopsis, of a combination of GDHα, GDHβ and GDHγ subunits. Every combination of subunits is possible; however, it is still barely known whether different combinations affect the enzymatic properties of the hexamers. In other species, hexamers are a combination of GDHα and GDHβ but it cannot be discarded the existence of other genes since for instance GDHγ subunit in Arabidopsis was described in Fontaine et al. (2012).

Glutamate + NAD⁺ + H⁺ → 2-Oxoglutarate + NADH + NH4⁺

Keywords: Glutamate dehydrogenase (谷氨酸脱氢酶)

Enzyme activity (酶的活性)

Spectrophotometry (分光光度法)

Materials and Reagents

4-weak old Arabidopsis thaliana leaves and roots
Ultrapure water
Liquid N₂
Ice
Flat bottom microplates (Deltalab, catalog number: 900011.1 )
4 mm diameter glass beads (Glaswarenfabrik Karl Hecht) (VWR International, catalog number: 201-0278 )
1.2 ml deep well storage plate (Thermo Fisher Scientific, catalog number: AB-0564 )
EVA Capband for capping 8 tubes (Micronic, catalog number: MP227B1 )
Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, AbD Serotec^®, catalog number: 500-006 )
NADH (Sigma-Aldrich, catalog number: 43420 )
PVPP (Sigma-Aldrich, catalog number: 77627 )
Glycerol (EMD Millipore Corporation, catalog number: 104092 )
Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
Triton X-100 (Sigma-Aldrich, catalog number: T-9284 )
HEPES (Sigma-Aldrich, catalog number: H3375 )
KOH (EMD Millipore Corporation, catalog number: 105029 )
MnCl₂ (Sigma-Aldrich, catalog number: M8266 )
EDTA (Sigma-Aldrich, catalog number: ED255 )
EGTA (Sigma-Aldrich, catalog number: E4378 )
Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
PMSF (Sigma-Aldrich, catalog number: P7626 )
Delta-aminocaproic acid (Sigma-Aldrich, catalog number: A2504 )
Leupeptin (Sigma-Aldrich, catalog number: L2884 )
Tricine (Sigma-Aldrich, catalog number: T0377 )
CaCl₂ (Panreac Applichem, catalog number: 131232 )
2-Oxogutarate (Merck KGaA, catalog number: 5194 )
(NH₄)₂SO₄ (Panreac Applichem, catalog number: 131140 )
Extraction buffer (see Recipes)
Reaction buffer (see Recipes)
Bradford solution (see Recipes)

Equipment

TissueLyser (RETSCH, model: MM400 )
Plate centrifuge (Sigma, model: 2-16K )
Absorbance microplate reader (Biotek Powerwave X 384 Microplate spectrophotometer)
TissueLyser Adapter Set 2 x 96 (QIAGEN, catalog number: 69984 )

Procedure

English

中文翻译

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如何引用

Sarasketa, A., Vega-Mas, I. and Marino, D. (2015). In vitro Colorimetric Method to Measure Plant Glutamate Dehydrogenase Enzyme Activity. Bio-protocol 5(16): e1560. DOI: 10.21769/BioProtoc.1560.