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Measurement of Glucose-6-phosphate Dehydrogenase Activity in Bacterial Cell-free Extracts
无细菌细胞的提取物总葡萄糖-6-磷酸活性的测量   

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Abstract

Glucose-6-phosphate dehydrogenase (G6PDH) (EC 1.1.1.49) is the first enzyme of the oxidative pentose phosphate cycle and catalyses the conversion of glucose-6-phosphate (G6P) to 6-phosphoglucono-δ-lactone and transfers one electron to NADP+ producing one NADPH. Conversion of G6P to 6-phosphoglucono-δ-lactone is proportional to the production of NADPH. The increase in NADPH concentration results in an increase in absorbance at 340 nm. To assay G6PDH activity, therefore, production of NADPH is determined by measuring increase in absorbance at 340 nm spectrophotometrically. This increase rate is then converted to unit of activity and specific activity of G6PDH. In this procedure, a generalized method is given for bacterial G6PDH assays emphasizing on a cyanobacterium Synechocystis sp. PCC6803 (Schaeffer and Stanier, 1978; Karakaya et al., 2008, 2012) and a heterotrophic bacterium E.coli (Hylemon and Phibbs, 1972; Barnel et al., 1990).

Keywords: Cyanobacteria(蓝藻), Glucose-6-phosphate dehydrogenase(6-磷酸葡萄糖脱氢酶), Specific activity(具体活动), Cell-free extract(无细胞提取物)

Materials and Reagents

  1. 1.5 ml Eppendorf tubes (Eppendorf, catalog number: 022363204 )
  2. Micropipette tips (200 μl) (Sigma-Aldrich, catalog number: CLS4866 )
  3. Micropipette tips (1,000 μl) (Sigma-Aldrich, catalog number: CLS4868 )
  4. Glass beads (unwashed) (212-300 μm) (Sigma-Aldrich, catalog number: G9143 )
  5. 1.5 ml polystyrene spectrophotometer cuvettes with 10 mm path length (Sigma-Aldrich, catalog number: C5416 )
  6. Parafilm (Sigma-Aldrich, catalog number: P7793 )
  7. Bacterial cells
    Note: Amount of the cell depends on how many assays will be carried out. Supernatant yielded from a cell pellet of 50 ml well-grown cyanobacterial culture (OD750 ≥ 1.0) and 10 ml overnight grown Escherichia coli will be sufficient for about 20 assays depending on what volume is used for each assay.
  8. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
  9. β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  10. Glucose-6-phosphate disodium salt (G6P) (Sigma-Aldrich, catalog number: G7250 )
  11. Potassium phosphate dibasic trihydrate (K2HPO4·3H2O) (Sigma-Aldrich, catalog number: P5504 )
  12. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P0662 )
  13. Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: M7506 )
  14. Hydrochloric acid (HCl) (36.5-38.0%) (Sigma-Aldrich, catalog number: H1758 )
  15. Maleic acid (Sigma-Aldrich, catalog number: M0375 )
  16. β-nicotinamide adenine dinucleotide phosphate hydrate (NADP+) (Sigma-Aldrich, catalog number: N5755 )
  17. Paraffin
  18. 50 mM Tris-maleate solution (see Recipes)
  19. Extraction buffers
    1. Tris-maleate buffer, pH 6.8 for Synechocystis sp. PCC6803 (see Recipes)
    2. Potassium phosphate buffer, pH 6.8 for E.coli (see Recipes)
  20. Assay buffers
    a. Tris-maleate buffer, pH 7.4 for Synechocystis sp. PCC6803 (see Recipes)
    b. Tris-HCl buffer, pH 8.0 for E.coli (see Recipes)
  21. 500 mM Tris-HCl stock solution (pH 8.0) (see Recipes)
  22. 500 mM G6P solution (see Recipes)
  23. 100 mM NADP+ solution (see Recipes)

Equipment

  1. Micropipettes (20-200 μl capacity) (Nichiryo, catalog number: 00-NPX2-200 )
  2. Micropipettes (100-1,000 μl capacity) (Nichiryo, catalog number: 00-NPX2-1000 )
  3. Microcentrifuge, 1.5 ml Eppendorf tube rotor and at least 10,000 x g force (Sigma-Aldrich, Hettich®, model: MIKRO120 )
  4. FastPrep FP120 (BioSurplus, Thermo-Savant, model: FP120 )
  5. Light microscope
  6. Vortex mixer (Bibby Scientific, Stuart, model: SA8 )
  7. Balance (Precision Weighing Balances, Salter Brecknell, model: ESA-150 )
  8. pH meter (Hanna Instruments, model: HI5221 )
  9. Vis-Spectrophotometer with 1.5 ml cuvette holder (Shimadzu Scientific Instruments, model: UV-1800 )

Procedure

  1. Extraction of G6PDH from bacterial cells
    Note: Bacterial cells and cell-free extracts must be kept on ice or at 4 °C throughout all steps of extraction.
    1. Resuspend the cell pellets in 500 μl extraction buffer (Tris-maleate buffer pH 6.8 for cyanobacterial cells or potassium phosphate buffer pH 6.8 for E.coli cells). The extraction buffers keep G6PDH enzyme in an active state.
      Note: G6PDH of the photosynthetic prokaryotes like cyanobacteria is a redox modulated oligomeric enzyme. In case, redox modulation properties are planned to be tested, β-mercaptoethanol must be omitted from the buffers.
    2. To wash glass beads, add 500 μg glass beads and 1 ml extraction buffer to two separate Eppendorf tubes, mix with a whirly mixer well, centrifuge at 10,000 x g for 1 min, and discard the supernatant.
    3. Add 500 μl cell suspension to each glass beads containing tubes and mix the tube to resuspend the glass beads.
    4. Put the tubes in fast prep FP120 and run the device twice at 5.5 m/sec for 40 sec.
      Note: This step may be done by mixing the tubes vigorously on a whirly mixer for 1-2 min until most of the cells are disrupted. This may be confirmed by examining a small amount of the crude extract for unbroken cells under a light microscope.
    5. Centrifuge the extract at 10,000 x g for 10 min.
    6. Transfer the supernatant to a new Eppendorf tube and keep at 4 °C. Use this cell-free extract as enzyme solution.
      Note: It is better to use the extract immediately. However, the extract may be stored at 4 °C for a week albeit some degree of activity loss.

  2. Assay of G6PDH activity in cell-free extract
    1. Set the wavelength of spectrophotometer to 340 nm. Select absorbance function.
    2. Prepare a blank and a test mixture as follows (Table1).

      Table 1. Regent contents of test and blank mixtures


    3. Add assay buffer, NADP+ and enzyme solutions first to a 1 ml spectrophotometer cuvette, then, start the enzyme reaction by adding G6P and mix the content by flipping the paraffin sealed cuvette.
      Note: Assay buffer is Tris-maleate (pH 7.4) for cyanobacteria and Tris-HCl (pH 8.0) for E.coli. A blank reaction mixture is needed to check any endogenous NADP+ reduction in enzyme solution. A minimum amount of 200 mg protein per assay is necessary albeit it may vary depending on factors such as growth phase of the culture used.
    4. Place the cuvette in the cell holder of the spectrophotometer, close the lid and follow the absorbance for 3-5 min at room temperature by recording absorbance value in 15-30 sec intervals. Temperature of the reaction mixture should be kept at a certain point. Some enzymes need specific temperature like 37 °C. In this case reaction cuvette must be treated with a thermostatic apparatus integrated to the spectrophotometer. However, some enzymes work well at room temperature.
    5. Estimate ∆A340 (rate) dividing the total absorbance change to the total assay time in min. For example, if the absorbance change in a reaction mixture at 340 nm is 0.450 after 3 min, the rate is 0.150.
      Note: Some spectrophotometers have rate measurement function. Using such a device, the rate may be determined directly.
    6. Repeat steps B4 to B6 for the blank tube to test whether any detectable endogenous NADP+ is present in the enzyme solution. If so, subtract this value from the test value to find net ∆A340 value.

  3. Estimation of unit of activity and specific activity of G6PDH
    One of the standard expression ways of enzyme activity is the unit of activity. Unit of G6PDH activity is defined here as the formation of 1 μmol NADPH in one min. NADP+ reduction (G6PDH activity) determined as rate (∆A340) is needed to be converted to concentration in μmol. Once the unit of activity is determined, it is often converted to specific activity. Specific activity is expressed as units per mg protein.
    1. Estimate G6PDH activity in units by using the formula as follows:


      In this protocol the dilution factor is 20 (50 μl extract in 1,000 μl test or blank mixture). For example, if a rate of 0.85 is yielded in the test assay and 0.05 in the blank assay, the net rate will be 0.8. Then the units of activity will be estimated as 0.8 x 20/6.22 = 2.572 μmol min-1 ml-1.
    2. To estimate specific activity, the protein amount in the extract must be determined by a standard method such as Bradford assay. Specific activity is then estimated as the amount of units per mg protein by using the formula below:


      For example, if the protein content of the extract is 0.5 mg ml-1, the specific activity will be 2.572/0.5 = 5.44 units/mg protein.

Notes

  1. One important point is to keep the enzyme in the active state in the supernatant especially by protecting it from oxidation damage. To avoid oxidation damage and keep the enzyme in the active state, the reducing agent β-mercaptoethanol may be added to the supernatant. However, this agent also reduces disulphide bonds in the enzyme and should be omitted when studying the redox properties of the enzyme.
  2. It is known that higher amounts of G6PDH, G6P and NADP positively affect the enzyme’s activity. If the effects of some agents on G6PDH activity are studied, minimal amounts of enzyme solution and substrates should be used. It is difficult to define standard minimal concentrations for enzyme and substrates since several factors may affect these values. Therefore, the researchers must determine minimal concentrations themselves.

Recipes

  1. 50 mM Tris-maleate solution
    Dissolve 6.05 g Trizma-base in 800 ml water and adjust pH to 6.8 (for extraction buffer) or 7.4 (for assay buffer) by adding maleic acid.
    Keep the solution at room temperature for a few months.
  2. Tris-maleate extraction buffer
    50 mM Tris-maleate (pH 6.8)
    0.1% β-mercaptoethanol
    10 mM G6P
    Mix the reagents just before use.
  3. Tris-maleate assay buffer
    50 mM Tris-maleate (pH 7.4)
    0.1% β-mercaptoethanol
    10 mM MgSO4
  4. Potassium phosphate extraction buffer (pH 6.8) (Barnell et al., 1990)
    50 mM K2HPO4
    50 mM KH2PO4
    Titrate against each other to adjust pH to 6.8.
  5. 500 mM Tris-HCl stock solution pH 8.0
    Dissolve 60.57 g Trizma base in 800 ml water and adjust pH to 8.0 with 2 N HCl solution.
    Keep the solution at room temperature for a few months.
  6. Tris-HCl assay buffer for E.coli (Hylemon and Phibbs,1972)
    50 mM Tris-HCl (pH 8.0)
    0.1% β-mercaptoethanol
    10 mM MgSO4
  7. 500 mM G6P solution
    Prepare 500 mM G6P solution (152.05 mg G6P in 1 ml ultrapure water), separate in aliquots and store at -20 °C for a few months.
  8. 100 mM NADP+ solution
    Prepare 100 mM NADP+ solution (74.34 mg NADP+ in 1 ml ultrapure water), separate in aliquots and store at -20 °C for a few months.

Acknowledgments

This protocol was adapted and modified from previously published studies by Schaeffer and Stanier (1978), Hylemon and Phibbs (1972) and Barnell et al. (1990) and applied in the studies of Karakaya et al. (2008 and 2012) which were supported by TUBITAK (Project TBAG 1985 100T100) and Ondokuz Mayıs Üniversity (Projects F261 and PYO 1904 09 21).

References

  1. Barnell, W. O., Yi, K. C. and Conway, T. (1990). Sequence and genetic organization of a Zymomonas mobilis gene cluster that encodes several enzymes of glucose metabolism. J Bacteriol 172(12): 7227-7240.
  2. Hylemon, P. B. and Phibbs, P. V., Jr. (1972). Independent regulation of hexose catabolizing enzymes and glucose transport activity in Pseudomonas aeruginosa. Biochem Biophys Res Commun 48(5): 1041-1048.
  3. Karakaya, H., Ay, M. T., Ozkul, K. and Mann, N. H. (2008). A Delta zwf (glucose-6-phosphate dehydrogenase) mutant of the cyanobacterium Synechocystis sp PCC 6803 exhibits unimpaired dark viability. Annals of Microbiology 58(2): 281-286.
  4. Karakaya, H., Erdem, F., Özkul, K. and Yilmaz, A. (2012). Analysis of glucose-6-phosphate dehydrogenase of the cyanobacterium Synechococcus sp. PCC7942 in the zwf mutant Escherichia coli DF214 cells. Annals of Microbiology 63: 1319-1325.
  5. Schaeffer, F. and Stanier, R. Y. (1978). Glucose-6-phosphate dehydrogenase of Anabaena sp. kinetic and molecular properties. Arch Microbiol 116: 9-19.

简介

葡萄糖-6-磷酸脱氢酶(G6PDH)(EC 1.1.1.49)是氧化戊糖磷酸循环的第一个酶,并催化葡萄糖-6-磷酸(G6P)转化为6-磷酸葡萄糖酸-δ-内酯并将一个电子 到NADP +产生一种NADPH。 G6P向6-磷酸葡萄糖酸-δ-内酯的转化与NADPH的产生成比例。 NADPH浓度的增加导致340nm处吸光度的增加。 因此,为了测定G6PDH活性,通过分光光度法测量340nm处吸光度的增加来确定NADPH的产生。 然后将该增加速率转化为G6PDH的活性和比活性的单位。 在该程序中,给出了强调蓝细菌集胞藻的细菌G6PDH测定的一般方法。 PCC6803(Schaeffer和Stanier,1978; Karakaya等人,2008,2012)和异养细菌大肠杆菌(Hylemon和Phibbs,1972; Barnel等人 ,1990)。

关键字:蓝藻, 6-磷酸葡萄糖脱氢酶, 具体活动, 无细胞提取物

材料和试剂

  1. 1.5ml Eppendorf管(Eppendorf,目录号:022363204)
  2. 微量吸头(200μl)(Sigma-Aldrich,目录号:CLS4866)
  3. 微量吸头(1000μl)(Sigma-Aldrich,目录号:CLS4868)
  4. 玻璃珠(未洗涤)(212-300μm)(Sigma-Aldrich,目录号:G9143)
  5. 1.5ml具有10mm路径长度的聚苯乙烯分光光度计比色皿(Sigma-Aldrich,目录号:C5416)
  6. 石蜡膜(Sigma-Aldrich,目录号:P7793)
  7. 细菌细胞
    注意:细胞的量取决于进行多少测定。从50ml充分生长的蓝细菌培养物(OD 750 = 1.0)的细胞沉淀物产生的上清液和10ml过夜生长的大肠杆菌将足以用于约20次测定,这取决于用于每次测定的体积。
  8. (Sigma-Aldrich,目录号:T1503)
  9. β-巯基乙醇(Sigma-Aldrich,目录号:M3148)
  10. 葡萄糖-6-磷酸二钠盐(G6P)(Sigma-Aldrich,目录号:G7250)
  11. 磷酸氢二钾三水合物(K 2 HPO 4·3H 2 O)(Sigma-Aldrich,目录号:P5504)
  12. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P0662)
  13. 硫酸镁(MgSO 4)(Sigma-Aldrich,目录号:M7506)
  14. 盐酸(HCl)(36.5-38.0%)(Sigma-Aldrich,目录号:H1758)
  15. 马来酸(Sigma-Aldrich,目录号:M0375)
  16. β-烟酰胺腺嘌呤二核苷酸磷酸盐水合物(NADP )(Sigma-Aldrich,目录号:N5755)
  17. 石蜡
  18. 50mM Tris-马来酸盐溶液(参见配方)
  19. 提取缓冲区
    1. Tris-马来酸盐缓冲液,pH6.8,用于集胞藻属(Synechocystis) PCC6803(请参阅配方)
    2. 磷酸钾缓冲液,pH 6.8用于大肠杆菌(参见配方)
  20. 测定缓冲区
    一个。 Tris-马来酸盐缓冲液,pH 7.4,用于集胞藻。 PCC6803(请参阅配方)
    b。 Tris-HCl缓冲液,pH 8.0用于大肠杆菌(参见Recipes)
  21. 500 mM Tris-HCl储备液(pH 8.0)(见配方)
  22. 500 mM G6P溶液(参见配方)
  23. 100mM NADP溶液(参见Recipes)

设备

  1. 微量移液管(20-200μl容量)(Nichiryo,目录号:00-NPX2-200)
  2. 微量移液器(100-1000μl容量)(Nichiryo,目录号:00-NPX2-1000)
  3. 微量离心机,1.5ml Eppendorf管式转子和至少10,000×g的力(Sigma-Aldrich,Hettich ,型号:MIKRO120)
  4. FastPrep FP120(BioSurplus,Thermo-Savant,型号:FP120)
  5. 光学显微镜
  6. 涡旋混合器(Bibby Scientific,Stuart,型号:SA8)
  7. Balance(精密称重天平,Salter Brecknell,型号:ESA-150)
  8. pH计(Hanna Instruments,型号:HI5221)
  9. 具有1.5ml比色杯架(Shimadzu Scientific Instruments,型号:UV-1800)的Vis分光光度计

程序

  1. 从细菌细胞中提取G6PDH
    注意:在提取的所有步骤中,细菌细胞和无细胞提取物必须保存在冰上或4℃。
    1. 重悬细胞沉淀在500微升提取缓冲液(Tris-马来酸盐缓冲液pH6.8对于蓝藻细胞或磷酸钾缓冲液pH6.8用于大肠杆菌细胞)。提取缓冲液保持G6PDH酶处于活性状态 注意:光合原核生物如蓝细菌的G6PDH是氧化还原调节的寡聚酶。在计划测试氧化还原调节性质的情况下,必须从缓冲液中省略β-巯基乙醇。
    2. 为了洗涤玻璃珠,向两个单独的Eppendorf管中加入500μg玻璃珠和1ml提取缓冲液,用涡旋混合器孔混合,以10,000xg离心1分钟,弃去上清液。
    3. 向每个含玻璃珠的管中加入500μl细胞悬浮液,并混合管以重悬玻璃珠
    4. 将管置于快速制备FP120中,并以5.5m/sec运行该装置两次,持续40秒。
      注意:该步骤可以通过在旋转混合器上剧烈混合管1-2分钟直到大部分细胞被破坏来完成。这可以通过在光学显微镜下检查少量未破裂细胞的粗提取物来确认。
    5. 以10,000×g离心提取物10分钟。
    6. 转移上清液到新的Eppendorf管,并保持在4°C。使用这种无细胞提取物作为酶溶液 注意:最好立即使用提取。然而,提取物可以在4℃下储存一周,虽然一定程度的活性损失
  2. 在无细胞提取物中测定G6PDH活性
    1. 将分光光度计的波长设置为340nm。选择吸光度函数。
    2. 准备空白和测试混合物如下(表1)。

      表1.测试和空白混合物的摄影内容


    3. 将测定缓冲液,NADP +和酶溶液首先加入1ml分光光度计比色皿,然后通过加入G6P开始酶反应,并通过翻转石蜡密封的比色皿混合内容物。
      注意:测定缓冲液是用于蓝细菌的Tris-马来酸盐(pH 7.4)和用于大肠杆菌的Tris-HCl(pH 8.0)。需要空白反应混合物来检查酶溶液中的任何内源性NADP还原。每次测定的最小量为200mg蛋白质是必要的,尽管其可以取决于诸如所用培养物的生长期的因素而变化。
    4. 将比色杯放置在分光光度计的细胞保持器中,关闭盖子,并在室温下通过以15-30秒的间隔记录吸光度值,遵循吸光度3-5分钟。反应混合物的温度应保持在某一点。一些酶需要特定的温度,如37℃。在这种情况下,反应容器必须用集成到分光光度计的恒温装置处理。然而,一些酶在室温下工作良好
    5. 估计将总吸光度变化除以总测定时间(分钟)的ΔA 340(速率)。例如,如果反应混合物在340nm处的吸光度变化在3分钟后为0.450,则比率为0.150。
      注意:一些分光光度计有速率测量功能。使用这种设备,可以直接确定速率。
    6. 对空白试管重复步骤B4至B6,以测试酶溶液中是否存在任何可检测的内源性NADP sup + +。如果是,从测试值中减去该值,以找到净△A <340> 值
  3. G6PDH的活性单位和比活性的估计
    酶活性的标准表达方式之一是活性单位。 G6PDH活性的单位在本文中定义为在1分钟内形成1μmolNADPH。需要将测定为速率(ΔA340)的NADP +还原(G6PDH活性)转化为以μmol计的浓度。一旦确定了活动单位,通常将其转换为特定活动。比活性表示为每mg蛋白质的单位
    1. 使用以下公式估算单位G6PDH活性:

      在这个协议中,稀释因子是20(在1000μl测试或空白混合物中的50μl提取物)。例如,如果在测试测定中产生0.85的比率并且在空白测定中产生0.05,则净速率将为0.8。然后,活性单位将估计为0.8×20/6.22 =2.572μmolmin -1 - ml -1。
    2. 为了估计比活性,必须通过标准方法如Bradford测定法测定提取物中的蛋白质量。然后通过使用以下公式将比活性估计为每mg蛋白质的单位量:

      例如,如果提取物的蛋白质含量为0.5mg ml -1 -1,则比活性为2.572/0.5 = 5.44单位/mg蛋白质。

笔记

  1. 一个重要的点是将酶保持在上清液中的活性状态,特别是通过保护其免受氧化损伤。为了避免氧化损伤并保持酶处于活性状态,可以将还原剂β-巯基乙醇加入到上清液中。然而,该试剂还减少酶中的二硫键,并且当研究酶的氧化还原性质时应当省略。
  2. 已知较高量的G6PDH,G6P和NADP正面影响酶的活性。如果研究一些试剂对G6PDH活性的影响,应使用最小量的酶溶液和底物。很难定义酶和底物的标准最低浓度,因为几个因素可能影响这些值。因此,研究人员必须自己确定最低浓度。

食谱

  1. 50mM Tris-马来酸盐溶液 将6.05g Trizma碱溶解在800ml水中,并通过加入马来酸将pH调节至6.8(用于提取缓冲液)或7.4(用于测定缓冲液)。 将溶液在室温下保存几个月。
  2. Tris-马来酸盐提取缓冲液
    50mM Tris-马来酸盐(pH 6.8)
    0.1%β-巯基乙醇 10 mM G6P
    在使用前混合试剂。
  3. Tris-马来酸盐测定缓冲液
    50mM Tris-马来酸盐(pH7.4) 0.1%β-巯基乙醇 10mM MgSO 4
  4. 磷酸钾提取缓冲液(pH 6.8)(Barnell等人,1990)
    50mM K 2 HPO 4
    50mM KH 2 PO 4 sub/
    相互滴定以将pH调节至6.8
  5. 500mM Tris-HCl储备溶液pH 8.0
    将60.57g Trizma碱溶于800ml水中,并用2N HCl溶液调节pH至8.0。 将溶液在室温下保存几个月。
  6. 用于大肠杆菌的Tris-HCl测定缓冲液(Hylemon和Phibbs,1972)
    50mM Tris-HCl(pH8.0)
    0.1%β-巯基乙醇 10mM MgSO 4
  7. 500 mM G6P溶液
    准备500 mM G6P溶液(152.05 mg G6P在1 ml超纯水中),等分分装,在-20°C保存几个月。
  8. 100mM NADP +溶液
    制备100mM NADP +溶液(74.34mg NADP +在1ml超纯水中),分成等份,在-20℃下储存几个月。 >

致谢

该协议根据Schaeffer和Stanier(1978),Hylemon和Phibbs(1972)和Barnell等人以前发表的研究进行了修改和修改。 (1990)并应用于Karakaya等人的研究中。 (2008年和2012年),得到TUBITAK(项目TBAG 1985 100T100)和OndokuzMay?s大学(项目F261和PYO 1904 09 21)的支持。

参考文献

  1. Barnell,WO,Yi,KC and Conway,T。(1990)。  编码几种葡萄糖代谢酶的运动发酵单胞菌(Zymomonas mobilis)基因簇的序列和遗传组织。细菌 172(12): 7227-7240。
  2. Hylemon,PB和Phibbs,PV,Jr.(1972)。  58(2):281-286。
  3. Karakaya,H.,Erdem,F.,?zkul,K.和Yilmaz,A。(2012)。  分析蓝细菌聚球藻属的葡萄糖-6-磷酸脱氢酶。 PCC7942在 zwf 突变大肠杆菌 DF214细胞中。 微生物学年鉴 63:1319-1325。
  4. Schaeffer,F。和Stanier,RY(1978)。  鱼腥藻的葡萄糖-6-磷酸脱氢酶。动力学和分子性质。 Arch Microbiol 116:9-19。
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引用:Karakaya, H. and Özkul, K. (2016). Measurement of Glucose-6-phosphate Dehydrogenase Activity in Bacterial Cell-free Extracts. Bio-protocol 6(19): e1949. DOI: 10.21769/BioProtoc.1949.
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