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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   

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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

  1. Sephadex G-25 columns (PD-10) (GE Healthcare, catalog number: 17-0851-01 )
  2. Snap cap plastic tubes of 4 ml (Bio-Vials) (Beckman Coulter, catalog number: 566353 ) and tube racks
  3. Laboratory film (Parafilm M) (Thomas Scientific, Pechiney Plastic Packaging, catalog number: PM-996 )
  4. Chlamydomonas reinhardtii cell extract containing Rubisco
  5. Tris [2-amino, 2-hydroxymethyl, 1, 3-propanediol] (Trizma base) (Sigma-Aldrich, catalog number: T-1503 )
  6. Magnesium chloride hexahydrate (MgCl2.6H2O) (Merck Millipore Corporation, catalog number: 1.05833 )
  7. Sodium bicarbonate (NaHCO3) (Merck Millipore Corporation, catalog number: 1.06329 )
  8. 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.
  9. Sodium 14C-bicarbonate (52 mCi/mmol) (PerkinElmer, catalog number: NEC086H )
  10. 2, 5-diphenyl oxazol (PPO) (Sigma-Aldrich, Fluka, catalog number: 43140 )
  11. 1, 4-bis-(5-phenyl-2-oxazolyl) benzene (POPOP) (Sigma-Aldrich, Fluka, catalog number: 15150 )
    Note: Product 15150 has been discontinued.
  12. 2-Phenylethylamine (Merck Millipore Corporation, catalog number: 8.07334 )
  13. Toluene (Panreac, catalog number: 131745-1611 )
  14. Methanol (Scharlau S.L., catalog number: ME-0301 )
  15. Scintillation cocktail (Cocktail 22 Normascint) (Scharlau S.L., catalog number: CO-0135 )
  16. dH2O (deionized water) (processed by the Milli-Q system from Merck Millipore Corporation)
  17. Activation buffer (AB) (see Recipes)
  18. Reaction buffer (RB) (see Recipes)
  19. RuBP stock (see Recipes)
  20. Alkaline scintillation cocktail (see Recipes)
  21. Radioactive stock (see Recipes)
  22. 2 M Hydrochloric Acid (HCl) (VWR International, J.T. Baker®, catalog number: 6081 ) (see Recipes)

Equipment

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

Procedure

  1. Activation of Rubisco
    1. Rubisco extracted from C. reinhardtii cultures can be assayed at any level of purity. As a first step, the Rubisco should be transferred to a medium containing Mg2+ and dissolved CO2 (introduced as bicarbonate) to get the catalytically active form of the enzyme (Laing and Christeller, 1976). Therefore, desalt the starting extract loading 2.5 ml onto a Sephadex G-25 (PD-10) column equilibrated with activation buffer (AB), and elute with 3.5 ml of the same buffer.
      Note: This section is equivalent to section D in the protocol on “Purification of Rubisco from Chlamydomonas reinhardtii” (Sudhani et al., 2015) and might be omitted if the Rubisco has been already transferred to AB after purification.

  2. Preparation of the substrate mixture and specific radioactivity controls
    1. Working in a security extraction hood, mix 1 ml of reaction buffer (RB), 0.1 ml of RuBP stock and 15 μl of the radioactive stock into a 1.5 ml Eppendorf tube. This will be the substrate reaction mixture (SR mix). In parallel, prepare an identical mixture but replace the 0.1 ml of RuBP stock by 0.1 ml of dH2O. This will be the no-RuBP reaction mixture (NR mix). Table 1 summarizes the composition of both reaction mixtures.

      Table 1. Components and volumes of the reaction mixtures
      Reaction mixture
      RB
      RuBP stock
      dH2O
      Radioactive stock
      SR mix
      1 ml
      0.1 ml
      0 ml
      15 μl
      NR mix
      1 ml
      0 ml
      0.1 ml
      15 μl

      Note: Volumes given above are for the preparation of 1.115 ml of mixture, which is enough for assaying ca 21 vials (i.e., triplicates of seven independent determinations, including samples and controls). In the case that the number of vials intended for assay is higher than 21, the volumes should be scaled up accordingly.
    2. Immediately place three 50 μl aliquots of the SR mix and three 50 μl aliquots of the NR mix in snap-cap plastic tubes (Bio-vial) used for radioactive counting. Add 3 ml of alkaline scintillation cocktail to each vial, mix, and count for radioactivity. These measurements are the SR and NR controls, and they will be used for determining the exact specific radioactivity of the fixed CO2.
      Note: Commercial scintillation cocktails are usually acidic and, therefore, cannot be used for these controls because the radioactivity would be lost in the gas phase as 14CO2.
    3. Meanwhile, the Eppendorf tube containing the substrate mixture should remain tightly closed at room temperature for at least 10 min in order to let the 14C-bicarbonate equilibrate with the non-radioactive CO2/bicarbonate pool.

  3. Preparation of the blank, non-RuBP fixation control and sample vials
    1. Place open vials in a rack for the samples and remaining controls. The latter include the blanks (measuring the radioactivity retained in the absence of sample) and the non-RuBP fixation control (monitoring potential CO2 fixation by the sample due to carboxylases other than Rubisco). You may prepare triplicates for each determination.
    2. Mix 180 μl of AB and 20 μl of sample extract (containing 0.2 to 10 μg of Rubisco) in each tube, except for the blank vials in which 200 μl of AB should be delivered. Submerge the tube rack in a thermostatic water bath at 30 °C inside the extraction hood and let stand for a few minutes to homogenize the temperature before starting the assay.
      Note: Instead of a water bath, tubes may be alternatively placed in a thermoblock at 30 °C inside the hood.

  4. Assay of the activity
    1. Place a stopwatch chronometer in a place easily readable while manipulating the samples, and start the assay at time 0 by delivering 50 μl of radioactive SR mixture to the first tube. Proceed subsequently by adding 50 μl to additional tubes every 20 sec. Switch from the SR to the NR mixture (delivering also 50 μl of it) when starting the set of samples for determining the non-RuBP carbon fixation. Tubes should be kept at 30 °C throughout the assay.
    2. Stop the enzymatic reaction by adding 50 μl of 2 M HCl to each tube exactly 5 min after the radioactive mixture was delivered. Table 2 summarizes the additions to each tube hitherto.

      Table 2. Components and volumes added to each vial throughout the assay
      Type of vial
      AB
      Extract
      SR mix
      NR mix
      2 M HCl
      Blank
      200 μl
      0 μl
      50 μl
      0 μl
      50 μl
      No-RuBP (NR)
      180 μl
      20 μl
      0 μl
      50 μl
      50 μl
      + RuBP (SR)
      180 μl
      20 μl
      50 μl
      0 μl
      50 μl

    3. Place the rack of vials (without cap) in a vacuum oven connected to a water jet pump through an alkaline trap. This oven should also be placed inside the security extraction hood. Evacuate at 90 °C until total dryness of the content of the tubes in order to remove the unfixed radioactivity. This may take less than one hour, but the actual duration is highly dependent on the vacuum level achieved by the pump.
    4. Redissolve the dry content of each tube in 200 μl of dH2O and evacuate again at 90 °C until dryness.
    5. Redissolve again the dry content of each tube in 200 μl of dH2O and add 3 ml of a commercial scintillation cocktail (admitting a 7% of water in a single phase). Mix thoroughly before counting.
    6. Count all samples in a scintillation counter equipped with calibrated efficiency correction facilities. Sample vials should be counted long enough to accumulate more than 10,000 total counts (e.g., 5 min if more than 2,000 cpm are detected).

  5. Assessment of the results and calculations
    1. After efficiency correction by the scintillation counter, radioactivity data will be in (or will be easily converted to) disintegrations per minute (dpm). Begin by calculating the average from triplicates, and subtracting the average of the blanks from all other means.
    2. Once the blanks have been deducted, divide the average radioactivity of the SR and NR controls by the number of μmoles of CO2/bicarbonate present in the reaction (0.2 ml of 10 mM bicarbonate in AB + 0.05 ml of 50 mM bicarbonate in the reaction mix which equals 4.5 μmoles) to obtain the specific radioactivity of the SR and NR assays. Specific radioactivity should be between 2.0.105 and 3.5.105 dpm/μmol.
    3. Divide the mean radioactivity of each sample (with blanks deducted) by the specific radioactivity of the corresponding control to obtain the number of μmoles of CO2 fixed in each assay.
    4. Subtract the μmoles of CO2 fixed in the no-RuBP assay from the μmoles of CO2 fixed in the corresponding assay with RuBP to get the net amount of RuBP-dependent CO2 fixation due to Rubisco.
    5. Divide the net μmoles of CO2 fixed by 5 min and by 0.02 ml to get the final activity (μmoles of CO2 fixed per min and ml of enzyme extract). If the concentration of Rubisco in the assays is known, results can be expressed as specific activities (e.g., μmoles of CO2 fixed per min and mg of enzyme). Typical specific activity of Rubisco from a wild type strain of C. reinhardtii is about 2 μmoles of CO2 fixed per min per mg of enzyme.

Representative data

Table 3 illustrates the calculation of the enzymatic activity from raw data with a numerical example. Note that the radioactivity of the SR and NR controls is not exactly the same probably due to limited reproducibility at pipetting small volumes of the radioactive stock. This leads to slightly different specific radioactivities for the assays in presence (SR series) and absence (NR series) of RuBP.

Table 3. Calculation of the Rubisco carboxylase activity from experimental triplicate determination of radioactivity (dpm)

aAverages of the 3 replicas
bAverages with mean blank deduced (net radioactivity)
cSpecific radioactivity (radioactivity of controls divided by 4.5 μmol of CO2/bicarbonate)
dCO2 fixation (net radioactivity divided by the corresponding specific radioactivity)
eNet CO2 fixation due specifically to Rubisco (NR fixation subtracted)
fNormalized Rubisco activity = net Rubisco fixation divided by assay duration (5 min) and volume of extract (0.02 ml)

Notes

  1. This assay might be performed by a single person if the number of vials does not exceed 15 (typically 5 triplicates). For assaying more vials, it is convenient to engage 2 persons, one initiating the reactions by adding the substrate mixture every 20 sec and the other stopping them with HCl after a 5 min delay.
  2. When assaying moderately purified Rubisco, the RuBP-independent fixation is usually negligible. Consequently, the set of no-RuBP vials (NR series) can be suppressed, thereby reducing the assay length considerably.
  3. The carbon-fixation activity of eukaryotic Rubiscos decays slowly when assayed in vitro (in the absence of the auxiliary enzyme Rubisco activase and ATP) due to blocking of the catalytic site in an inactive form by the RuBP substrate, a process termed “fallover” (Edmondson et al., 1990). Fallover is negligible in the case of the C. reinhardtii enzyme for a 5 min-fixation assay (but it will become increasingly noticeable for prolonged incubations). Moreover, the Rubiscos of higher plants experience a much stronger fallover, and the fixation time should be shortened accordingly (typically to 1 min) when adapting this procedure to these enzymes. In any case, when facing the assay of a Rubisco from a new species, the interval of linearity should be checked.

Recipes

  1. Activation buffer (AB) (100 mM Tris-HCl, 10 mM MgCl2, 10 mM NaHCO3, pH 8.2)
    For 100 ml:
    Dissolve 1.210 g of Tris and 0.203 g of MgCl2.6H2O in some 80 ml of dH2O
    Adjust the pH to 8.19 using diluted (e.g., 1 M) HCl
    Add 0.084 g of NaHCO3 (previously weighed on aluminum foil) and dissolve. The final pH should be very nearly 8.2 (but do not readjust it further).
    Bring the final volume to 100 ml and transfer to a 100 ml bottle leaving little headspace. Prepared this buffer shortly before use to prevent a significant loss of bicarbonate as CO2.
  2. Reaction buffer (RB) (100 mM Tris-HCl, 10 mM MgCl2, 55 mM NaHCO3, pH 8.2)
    For 100 ml:
    Dissolve 1.210 g of Tris and 0.203 g of MgCl2.6H2O in some 80 ml of dH2O
    Adjust the pH to 8.18 using diluted (e.g., 1 M) HCl
    Add 0.462 g of NaHCO3 (previously weighed on aluminum foil) and dissolve. The final pH should be very nearly 8.2 (but do not readjust it further).
    Bring the final volume to 100 ml
    This buffer should be prepared immediately before preparing the reaction mixtures with it.
  3. RuBP stock
    RuBP comes as a dry powder, but it is very hygroscopic and should not be weighed. Therefore, once the sealed bottle is opened, immediately dilute the powder inside the original container with dH2O to a 10 mg/ml concentration (about 22 mM) according to the amount declared by the manufacturer. You may verify afterwards the actual amount by comparing the weight of the filled and the empty (dry) container, readjusting the nominal concentration if necessary.
    Aliquot the RuBP solution in 150 μl portions, and freeze them in liquid nitrogen. Aliquots are stored at -80 °C and thawed as needed immediately before preparing the reaction mixture in order to minimize the spontaneous oxidation of the sugar, which results in an impurity that inhibits Rubisco activity (Kane et al., 1998).
  4. Alkaline scintillation cocktail
    For 115 ml:
    Dissolve 0.980 g of PPO and 0.020 g of POPOP in 57 ml of toluene
    Add 50 ml of 2-phenylethylamine and mix
    Add 3 ml of methanol and 5 ml of dH2O
    The final mixture should remain as a single phase.
    Stored at 4 °C protected from light
  5. Radioactive stock
    14C-bicarbonate is usually delivered by the manufacturers as an aqueous solution at high pH (e.g., 9.5) inside a sealed glass ampoule. Once opened, the remaining solution should be transferred into a container leaving little headspace (e.g., an Eppendorf tube of an adequate volume) and stored tightly closed (e.g., wrapped in several layers of Parafilm) at room temperature inside the security hood. There will be nonetheless some unavoidable exchange with atmospheric CO2 resulting in a very slow decline of the specific radioactivity. This is the reason why the actual specific radioactivity should be measured for each assay.
  6. 2 M HCl
    For 10 ml:
    Mix 2 ml of concentrated (36-38%, about 10 M) HCl with 8 ml of dH2O

Acknowledgments

This work was supported by a grant (UV-INV-AE14-269247) of the University of Valencia.

References

  1. Edmondson, D. L., Badger, M. R. and Andrews, T. J. (1990). Slow inactivation of ribulosebisphosphate carboxylase during catalysis is caused by accumulation of a slow, tight-binding inhibitor at the catalytic site. Plant Physiol 93(4): 1390-1397.
  2. Kane, H. J., Wilkin, J. M., Portis, A. R. and John Andrews, T. (1998). Potent inhibition of ribulose-bisphosphate carboxylase by an oxidized impurity in ribulose-1, 5-bisphosphate. Plant Physiol 117(3): 1059-1069.
  3. Laing, W. A. and Christeller, J. T. (1976). A model for the kinetics of activation and catalysis of ribulose 1,5-bisphosphate carboxylase. Biochem J 159(3): 563-570.
  4. Lilley, R. M. and Walker, D. A. (1974). An improved spectrophotometric assay for ribulosebisphosphate carboxylase. Biochim Biophys Acta 358(1): 226-229.
  5. Lorimer, G. H., Badger, M. R. and Andrews, T. J. (1977). D-Ribulose-1,5-bisphosphate carboxylase-oxygenase. Improved methods for the activation and assay of catalytic activities. Anal Biochem 78(1): 66-75.
  6. Sudhani, H. P. K., García-Murria, M. J., Marín-Navarro, J., García-Ferris, C., Peñarrubia, L. and Moreno, J. (2015). Purification of Rubisco from Chlamydomonas reinhardtii. Bio-protocol 5(23): e1673.

简介

碳固定酶,核酮糖1,5-二磷酸羧化酶/加氧酶(EC 4.1.1.39,Rubisco)的性能控制绿色植物,藻类和大多数自养细菌中的生物量积累。特别地,Rubisco的羧化酶活性掺入来自CO 2的碳到产生两分子3-磷酸甘油酸的核酮糖1,5-二磷酸(RuBP)。这里给出了用于来自莱茵衣藻的Rubisco的羧化酶活性的测定的详细方案,其是用于叶绿体研究的模式生物体和用于生物技术定向的酶的遗传操作的拟合宿主。 Rubisco必须与Mg 2+离子和碳酸氢盐预孵育以诱导催化活性中心(Laing和Christeller,1976)。一旦Rubisco被活化,本文所述的其羧化酶活性的测定基于将14 C-二氧化碳/碳酸氢盐固定在耐酸放射性中(Lorimer等人, ,1977)。虽然也可以使用分光光度测定法(Lilley和Walker,1974),但是当处理大量样品时,基于放射性底物固定的方法是不可替代的,并且它仍然是最常用于测定Rubisco活性的技术。

关键字:二磷酸核酮糖的羧化酶/加氧酶, 二氧化碳的固定, radioisotopic试验, 莱茵衣藻, Rubisco

材料和试剂

  1. Sephadex G-25柱(PD-10)(GE Healthcare,目录号:17-0851-01)
  2. 4毫升(Bio-Vials)(Beckman Coulter,目录号:566353)的快速盖塑料管和管架
  3. 实验室膜(Parafilm M)(Thomas Scientific,Pechiney Plastic Packaging,目录号:PM-996)
  4. 含有Rubisco的莱茵衣藻细胞提取物
  5. 三[2-氨基-2-羟基甲基-1,3-丙二醇](Trizma碱)(Sigma-Aldrich,目录号:T-1503)
  6. 氯化镁六水合物(MgCl 2·6H 2 O·6H 2 O)(Merck Millipore Corporation,目录号:1.05833)
  7. 碳酸氢钠(NaHCO 3)(Merck Millipore Corporation,目录号:1.06329)
  8. D-核酮糖1,5-二磷酸钠盐水合物(Sigma-Aldrich,目录号:R-0878)
    注意:这表示为钠盐水合物,含有4 Na + 和3 H em> 2 O,总重量为452.1 g/mol。
  9. 钠14 C碳酸氢盐(52mCi/mmol)(PerkinElmer,目录号:NEC086H)
  10. 2,5-二苯基恶唑(PPO)(Sigma-Aldrich,Fluka,目录号:43140)
  11. 1,4-双 - (5-苯基-2-恶唑基)苯(POPOP)(Sigma-Aldrich,Fluka,目录号:15150)
    注意:产品15150已停产。
  12. 2-苯乙胺(Merck Millipore Corporation,目录号:8.07334)
  13. 甲苯(Panreac,目录号:131745-1611)
  14. 甲醇(Scharlau S.L.,目录号:ME-0301)
  15. 闪烁混合物(Coktail 22 Normascint)(Scharlau S.L.,目录号:CO-0135)
  16. dH 2 O(去离子水)(由来自Merck Millipore Corporation的Milli-Q系统处理)
  17. 激活缓冲区(AB)(参见配方)
  18. 反应缓冲液(RB)(参见配方)
  19. RuBP储备(见配方)
  20. 碱性闪烁鸡尾酒(见配方)
  21. 放射性物质(见配方)
  22. 2M HCl盐(VWR International,J.T.Baker ,目录号:6081)(参见配方)

设备

  1. 配有安全抽气罩的放射性执照实验室
  2. 恒温水浴(SBS,型号:TI-03)
  3. 秒表计时器(Oregon Scientific Inc.,型号:TR118)
  4. 连接到碱性阱的真空烘箱(Thermo Fisher Scientific,Heraeus,型号:Vacutherm VT6025)
  5. 放射性闪烁计数器(PerkinElmer,型号:Tri-Carb 2810TR)

程序

  1. 激活Rubisco
    1. Rubisco从C提取。可以在任何地方测定培养物 纯度水平。作为第一步,Rubisco应该转移到 ?含有Mg 2+和溶解的CO 2(作为碳酸氢盐引入)的培养基 ?得到催化活性形式的酶(Laing和Christeller, ?1976)。因此,将起始萃取物脱盐至2.5ml上 用激活缓冲液(AB)平衡的Sephadex G-25(PD-10)柱, 并用3.5ml相同的缓冲液洗脱 注意:本节为 相当于"Rubisco的纯化"的方案中的D节 ?衣藻(Chlamydomonas reinhardtii)"(Sudhani等人,2015),并且可以省略 如果Rubisco在纯化后已经转移到AB。

  2. 底物混合物和比放射性对照的制备
    1. 在安全提取罩中工作,混合1毫升反应缓冲液(RB), ?0.1ml RuBP储液和15μl放射性储备液放入1.5ml Eppendorf管。这将是底物反应混合物(SR mix)。在 ?平行,制备相同的混合物,但代替0.1ml的RuBP 储备液通过0.1ml dH 2 O。这将是无RuBP反应混合物(NR 混合)。表1总结了两种反应混合物的组成
      表1.反应混合物的组分和体积
      反应混合物
      RB
      RuBP股票
      dH 2 2 O 放射性物品
      SR mix
      1 ml
      0.1 ml
      0 ml
      15微升
      NR mix
      1 ml
      0 ml
      0.1 ml
      15微升

      注意:上面给出的体积用于制备1.115ml 混合物,其足以分析约21个小瓶(即一式三份 七个独立测定,包括样品和对照)。在 用于测定的小瓶数目高于21的情况, 卷应相应地按比例放大。
    2. 立即放置 三个50μl等分的SR混合物和三个50μl等分的NR 混合在用于放射性计数的卡口塑料管(Bio-vial)中。 向每个小瓶中加入3ml碱性闪烁剂,混合并计数 ?用于放射性。这些测量是SR和NR控制,和 它们将用于确定的确切比放射性 固定CO 2。
      注意:商业闪烁鸡尾酒通常是 ?酸性,因此,不能用于这些控制,因为 放射性将在气相中丢失,如 2
    3. 与此同时, ?应保留含有底物混合物的微量离心管 在室温下密封至少10分钟以使其通过 14 C碳酸氢盐与非放射性CO 2 2 /碳酸氢盐平衡 ?池。

  3. 制备空白,非RuBP固定对照和样品瓶
    1. 将开放的小瓶放在样品和其余对照的架子上。的 后者包括空白(测量保留在中的放射性 缺少样品)和非RuBP固定对照(由于除了Rubisco以外的羧化酶引起的样品的监测潜在CO 2固定)。您可以为每次测定准备三次。
    2. 混合180微升的AB和20微升的样品提取物(含有0.2到10微克 ?Rubisco),除了其中加入200μlAB的空白小瓶 ?应交付。将管架浸入恒温水中 在30℃在抽提罩内浴中,并静置几分钟 ?以在开始测定之前均化温度 注意:代替水浴,管可以替代地放置在罩内的30℃的加热块中。

  4. 活动测验
    1. 将秒表计时器放在一个方便阅读的地方 操作样品,并在时间0通过递送50开始测定 ?μl的放射性SR混合物加入第一管中。随后由 ?每20秒向另外的管中加入50μl。从SR切换到 开始时的NR混合物(也提供50微升) 样品用于测定非RuBP碳固定。管应该 在整个测定中保持在30℃
    2. 停止酶反应 通过在每个管中恰好5分钟后加入50μl的2M HCl 放射性混合物。表2总结了对的添加 到目前为止
      表2.在整个测定中添加到每个小瓶中的组分和体积
      小瓶类型
      AB
      解压缩
      SR mix
      NR mix
      2 M HCl
      空白
      200μl
      0微升
      50微升
      0微升
      50微升
      No-RuBP(NR)
      180微升
      20微升
      0微升
      50微升
      50微升
      + RuBP(SR)
      180微升
      20微升
      50微升
      0微升
      50微升

    3. 将样品瓶架(无盖)放在与a连接的真空烘箱中 ?水喷射泵通过碱性阱。这个烤箱也应该 放置在安全抽吸罩内。在90°C抽真空 总干燥度的管内容物以除去未固定的 ?放射性。这可能需要不到一个小时,但实际 持续时间高度取决于泵所达到的真空度
    4. 将每个管的干燥内容物再溶解在200μldH 2 O中,并在90℃下再次抽空直至干燥。
    5. 再次将每管的干燥内容物再溶解在200μldH 2 O中, ?加入3ml市售闪烁混合物(允许7% 水单相)。计数前充分混匀。
    6. 计数 所有样品在装备有校准的闪烁计数器中 效率校正设施。样品瓶应计数长 足以累积超过10,000个总计数(例如,如果更多,则为5分钟) 检测到2,000cpm)。

  5. 评估结果和计算
    1. 经闪烁计数器效率校正后,放射性 数据将在(或将容易转换成)崩解 分钟(dpm)。首先计算一式三份的平均值, 从所有其他平均值中减去空白的平均值
    2. 一旦 ?空白被扣除,除以平均放射性 SR和NR通过存在于中的CO 2/NaHCO 3的微摩尔数控制 ?反应(0.2ml的10mM碳酸氢盐在AB + 0.05ml的50mM 碳酸氢盐在反应混合物中,其等于4.5μmol)以获得 SR和NR测定的特异性放射性。比放射性 应该在2.0 10 之间。 10 5 dpm /μmol。
    3. 除以平均值 每个样品的放射性(扣除空白) 放射性的相应控制得到的数量 在每个测定中固定的微摩尔CO 2
    4. 从固定在无RuBP测定中的CO 2的微摩尔减去固定在CO 2中的CO 2的微摩尔 用RuBP进行相应的测定,得到RuBP的净量 CO 2 固定。
    5. 除以固定的CO 2的净微摩尔数 5分钟和0.02ml以获得最终活性(μmolCO 2固定 每分钟和ml的酶提取物)。如果Rubisco的浓度 测定是已知的,结果可以表示为比活性 (例如,每分钟固定的CO 2微摩尔数和mg酶)。典型具体 来自野生型菌株的Rubisco的活性。 reinhardtii 约为2 ?μmol的每分钟每mg酶固定的CO 2。

代表数据

表3说明了用数值实施例从原始数据计算酶活性。注意,SR和NR对照的放射性不完全相同,可能是由于在移取小体积的放射性原料时的有限的再现性。这导致在RuBP存在(SR系列)和不存在(NR系列)的测定中略微不同的比放射性。

表3.从实验一式三份测定放射性(dpm)计算Rubisco羧化酶活性

a 3个副本的平均值
b 平均空白推算(净放射性)的平均值
比放射性(对照的放射性除以4.5μmolCO 2 /碳酸氢盐)
d CO 固定(净放射性除以相应的比放射性)
e Net CO <2> 固定,特别是Rubisco(减去NR固定)
f 标准化Rubisco活性=净Rubisco固定除以测定持续时间(5分钟)和提取物体积(0.02ml)

笔记

  1. 如果小瓶数量不超过15(通常为5个三次),则该测定可由单个人进行。为了测定更多的小瓶,方便地吸入2人,一次通过每20秒加入底物混合物引发反应,另一次在延迟5分钟后用HCl停止反应。
  2. 当测定中等纯化的Rubisco时,RuBP非依赖性固定通常可忽略。因此,可以抑制一组无RuBP小瓶(NR系列),从而大大降低测定时间
  3. 真核Rubiscos的碳固定活性在体外测定时(在不存在辅助酶Rubisco活化酶和ATP的情况下)由于RuBP底物阻断无活性形式的催化位点而缓慢衰减,称为"失败"的过程(Edmondson等人,1990)。在 C的情况下,Fallover是可忽略的。用于5分钟固定测定(但其对于延长的孵育将变得越来越明显)。此外,高等植物的Rubiscos经历更强的失败,并且当将该程序适应这些酶时,固定时间应相应地缩短(通常至1分钟)。在任何情况下,当面对来自新物种的Rubisco的测定时,应检查线性的间隔

食谱

  1. 活化缓冲液(AB)(100mM Tris-HCl,10mM MgCl 2,10mM NaHCO 3,pH8.2)
    对于100 ml:
    在约80ml dH 2 SO 4溶液中溶解1.210g Tris和0.203g MgCl 2。 O
    使用稀释的(例如,,1M)HCl
    将pH调节至8.19 加入0.084g NaHCO 3(先前在铝箔上称重)并溶解。最终的pH值应该非常接近8.2(但不要再调整它)。
    使最终体积为100毫升,并转移到一个100毫升的瓶子,留下很少的顶空。在使用前不久制备该缓冲液以防止碳酸氢盐作为CO 2的显着损失。
  2. 反应缓冲液(RB)(100mM Tris-HCl,10mM MgCl 2,55mM NaHCO 3,pH8.2)
    对于100 ml:
    在约80ml的dH 2中溶解1.210g的Tris和0.203g的MgCl 2·6H 2 O·6H 2 O。 O
    使用稀释的(例如,,1M)HCl
    将pH调节至8.18 加入0.462g NaHCO 3(预先称量在铝箔上)并溶解。最终的pH值应该非常接近8.2(但不要再调整它)。
    使最终体积为100 ml
    该缓冲液应在用其制备反应混合物之前立即制备
  3. RuBP股票
    RuBP是干粉末,但它是非常吸湿,不应称重。因此,一旦打开密封瓶,立即用dH 2 O将原容器内的粉末稀释至10mg/ml浓度(约22mM),根据制造商声明的量。您可以通过比较填充和空(干燥)容器的重量,然后根据需要重新调整标称浓度来验证实际量。
    等分RuBP溶液150μl,并在液氮中冷冻。将等分试样储存在-80℃,并在制备反应混合物之前立即解冻,以使糖的自发氧化最小化,这导致抑制Rubisco活性的杂质(Kane等人, ,1998)。
  4. 碱性闪烁鸡尾酒
    对于115毫升:
    将0.980g PPO和0.020g POPOP溶于57ml甲苯中 加入50ml 2-苯乙胺并混合
    加入3ml甲醇和5ml dH 2 O 2 / 最终混合物应保持为单相。
    储存在4°C避光
  5. 放射性物品
    14 C-碳酸氢盐通常由制造商在密封的玻璃安瓿内在高pH(例如9.5)下作为水溶液递送。一旦打开,剩余的溶液应该被转移到容器内,留下很少的顶部空间(例如足够体积的Eppendorf管),并且紧密封闭(例如包裹在几层石蜡膜)在室温下在安全罩内。但是与大气CO 2存在一些不可避免的交换,导致比放射性的非常缓慢的下降。这就是为什么应该测量每次测定的实际比放射性的原因
  6. 2 M HCl
    对于10ml:
    将2ml浓缩的(36-38%,约10M)HCl与8ml dH 2 O混合。

致谢

这项工作是由瓦伦西亚大学的赠款(UV-INV-AE14-269247)支持的。

参考文献

  1. Edmondson,D.L.,Badger,M.R。和Andrews,T.J。(1990)。 催化期间核酮糖二磷酸羧化酶的缓慢失活是由缓慢,紧密结合的抑制剂在催化位点。 植物生理学 93(4):1390-1397
  2. Kane,H.J.,Wilkin,J.M.,Portis,A.R.and John Andrews,T。(1998)。 通过核酮糖-1,5-二磷酸中的氧化杂质有效抑制核酮糖 - 二磷酸羧化酶。/a> Plant Physiol 117(3):1059-1069。
  3. Laing,W.A。和Christeller,J.T。(1976)。 核酮糖1,5-二磷酸羧化酶的活化和催化动力学模型。 Biochem J 159(3):563-570。
  4. Lilley,R.M。和Walker,D.A。(1974)。 改进的核酮糖二磷酸羧化酶的分光光度测定法。生物化学生物物理学> 358(1):226-229。
  5. Lorimer,G.H.,Badger,M.R。和Andrews,T.J。(1977)。 D-核酮糖-1,5-二磷酸羧化酶 - 加氧酶。改进的用于活化和测定催化活性的方法。 Anal Biochem 78(1):66-75。
  6. Sudhani,H.P.K.,García-Murria,M.J.,Marín-Navarro,J.,García-Ferris,C.,Pe?arrubia,L.and Moreno,J.(2015)。 从莱茵衣藻中纯化Rubisco 生物协议 5(23):e1673。
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引用:Sudhani, H. P., 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.
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