莱茵衣藻中二磷酸核酮糖羧化酶活性的测定

Hemanth P. K. Sudhani; María Jesús  García-Murria; Julia Marín-Navarro; Carlos García-Ferris; Lola Peñarrubia; Joaquín Moreno

doi:10.21769/BioProtoc.1672

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Peer-reviewed

Assay of the Carboxylase Activity of Rubisco from Chlamydomonas reinhardtii

莱茵衣藻中二磷酸核酮糖羧化酶活性的测定

Hemanth P. K. Sudhani

María Jesús García-Murria

Julia Marín-Navarro

Carlos García-Ferris

Lola Peñarrubia

Joaquín Moreno email

发布: 2015年12月05日第5卷第23期 DOI: 10.21769/BioProtoc.1672 浏览次数: 9706

评审: Maria SinetovaAgnieszka ZienkiewiczAnonymous reviewer(s)

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Abstract

The performance of the carbon-fixing enzyme, ribulose 1, 5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39, Rubisco), controls biomass accumulation in green plants, algae and most autotrophic bacteria. In particular, the carboxylase activity of Rubisco incorporates carbon from CO₂ to ribulose 1, 5-bisphosphate (RuBP) producing two molecules of 3-phosphoglycerate. Here a detailed protocol is given for the assay of the carboxylase activity of Rubisco from Chlamydomonas reinhardtii, a model organism for chloroplast studies and a fitting host for biotechnologically oriented genetic manipulation of the enzyme. Rubisco has to be pre-incubated with Mg²⁺ ions and bicarbonate to induce the catalytically competent active center (Laing and Christeller, 1976). Once Rubisco is activated, the assay of its carboxylase activity described here is based on the fixation of ¹⁴C-carbon dioxide/bicarbonate into acid-resistant radioactivity (Lorimer et al., 1977). Although a spectrophotometric assay is also available (Lilley and Walker, 1974), the method based on fixation of a radioactive substrate is irreplaceable when processing a large number of samples, and it is still the technique most often used for the determination of Rubisco activity.

Keywords: Ribulose 1,5-bisphosphate carboxylase/oxygenase (二磷酸核酮糖的羧化酶/加氧酶)

Carbon dioxide fixation (二氧化碳的固定)

Radioisotopic assay (radioisotopic试验)

Chlamydomonas reinhardtii (莱茵衣藻)

Rubisco (Rubisco)

Materials and Reagents

Sephadex G-25 columns (PD-10) (GE Healthcare, catalog number: 17-0851-01 )
Snap cap plastic tubes of 4 ml (Bio-Vials) (Beckman Coulter, catalog number: 566353 ) and tube racks
Laboratory film (Parafilm M) (Thomas Scientific, Pechiney Plastic Packaging, catalog number: PM-996 )
Chlamydomonas reinhardtii cell extract containing Rubisco
Tris [2-amino, 2-hydroxymethyl, 1, 3-propanediol] (Trizma base) (Sigma-Aldrich, catalog number: T-1503 )
Magnesium chloride hexahydrate (MgCl₂^.6H₂O) (Merck Millipore Corporation, catalog number: 1.05833 )
Sodium bicarbonate (NaHCO₃) (Merck Millipore Corporation, catalog number: 1.06329 )
D-Ribulose 1, 5-bisphosphate sodium salt hydrate (Sigma-Aldrich, catalog number: R-0878 )
Note: this is presented as a sodium salt hydrate, containing 4 Na⁺ and 3 H₂O per molecule, with a global weight of 452.1 g/mol.
Sodium ¹⁴C-bicarbonate (52 mCi/mmol) (PerkinElmer, catalog number: NEC086H )
2, 5-diphenyl oxazol (PPO) (Sigma-Aldrich, Fluka, catalog number: 43140 )
1, 4-bis-(5-phenyl-2-oxazolyl) benzene (POPOP) (Sigma-Aldrich, Fluka, catalog number: 15150 )
Note: Product 15150 has been discontinued.
2-Phenylethylamine (Merck Millipore Corporation, catalog number: 8.07334 )
Toluene (Panreac, catalog number: 131745-1611 )
Methanol (Scharlau S.L., catalog number: ME-0301 )
Scintillation cocktail (Cocktail 22 Normascint) (Scharlau S.L., catalog number: CO-0135 )
dH₂O (deionized water) (processed by the Milli-Q system from Merck Millipore Corporation)
Activation buffer (AB) (see Recipes)
Reaction buffer (RB) (see Recipes)
RuBP stock (see Recipes)
Alkaline scintillation cocktail (see Recipes)
Radioactive stock (see Recipes)
2 M Hydrochloric Acid (HCl) (VWR International, J.T. Baker^®, catalog number: 6081 ) (see Recipes)

Equipment

Radioactivity licensed laboratory equipped with a security extraction hood
Thermostatic water bath (SBS, model: TI-03 )
Stopwatch chronometer (Oregon Scientific Inc., model: TR118 )
Vacuum oven (Thermo Fisher Scientific, Heraeus, model: Vacutherm VT6025 ) connected to an alkaline trap
Radioactivity scintillation counter (PerkinElmer, model: Tri-Carb 2810 TR )

Procedure

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Sudhani, H. P. K., García-Murria, M. J., Marín-Navarro, J., García-Ferris, C., Peñarrubia, L. and Moreno, J. (2015). Assay of the Carboxylase Activity of Rubisco from Chlamydomonas reinhardtii. Bio-protocol 5(23): e1672. DOI: 10.21769/BioProtoc.1672.