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Stem Microsome Preparation and Mannan Synthase Activity Assay
干细胞微粒体的制备和甘露聚糖合成酶活性检测   

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Abstract

Mannans are hemicellulosic polysaccharides and are present in cell walls of all land plants. Mannan polysaccharides are synthesized by two enzymes, mannan synthase (ManS) for backbone (mannan or glucomannan) synthesis and galactomannan galactosyl transferase for side-chain (galactosyl) addition. Here, a method for ManS activity assay using microsomes freshly isolated from Arabidopsis stems is described. This method can be applied to isolation of microsomes from any tissues of Arabidopsis or any other plants.

Keywords: Arabidopsis(拟南芥), Cell wall(细胞壁), Microsomes(微粒体), Galactomannan(半乳甘露聚糖), Mannan synthase(甘露聚糖合成酶)

Materials and Reagents

  1. cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets (Roche, catalog number: 11836170001 )
  2. GDP-[14C]-Man (9.694 GBq/mmol, 262.0 mCi/mmol, 76.34 μM) (PerkinElmer, catalog number: NEC536050UC )
  3. Bicinchoninic acid (BCA) Protein Assay Kit (Pierce Antibodies, catalog number: 23225 or 23227 )
  4. OptiPhase Supermix Cocktail (PerkinElmer, catalog number: 1200-439 )
  5. GDP-Man (Sigma-Aldrich, catalog number: G-7377 )
  6. Carob galactomannan (Megazyme International, catalogue number: P-GALML )
  7. Sucrose
  8. MgCl2
  9. Extraction buffer (EB) (see Recipes)
  10. 4x ManS assay buffer (see Recipes)
  11. 1 mM GDP-Man (see Recipes)
  12. 1% carob galactomannan (see Recipes)

Equipment

  1. Mortar and pestle
  2. 13 ml Sarstedt tube (Sarstedt, catalog number: 60.540.500 )
  3. Centrifuge
  4. 4 ml plastic liquid scintillation counting vial (PerkinElmer, catalog number: 1200-421 )
  5. 1450 MicroBeta TriLux Microplate Scintillation and Luminescence Counter (PerkinElmer, catalog number: 1450-024 )

Procedure

  1. Grow Arabidopsis plants in a growth chamber under standard growth conditions (20 °C, 100 μmol/m2/s, 16 h light/8 h dark, 60% humidity) for 6-7 weeks.
  2. Harvest the whole stem (including secondary stems) from a single plant, and remove leaves, flowers and siliques. The stem should be big enough (at least 200 mg of fresh weight) but not very old or hard (without any yellow siliques).
  3. Weigh the stem and cut it into approximately 1 cm segments.
  4. Immediately transfer the stem segments into a mortar pre-chilled on ice, and add ice-cold EB (~1 ml EB/100 mg stem).
  5. Grind the stem segments in EB on ice with a mortar and pestle until the tissue is homogenized well (~5 min).
    I usually grind the tissue in the cold room.
  6. Transfer stem homogenate to a pre-chilled 13 ml Sarstedt tube using a 1 ml wide-bore pipetman tip (with the sharp end cut off), and centrifuge it at 3,000 x g at 4 °C for 10 min.
  7. Transfer the supernatant to a new pre-chilled 13 ml Sarstedt tube, and centrifuge it at 17,000 x g at 4 °C for 20 min.
  8. Centrifuge the supernatant from step 7 at 100,000 x g at 4 °C for 90 min to pellet microsome membranes.
  9. Resuspend the membrane pellet in EB (0.5 μl EB/mg stem) by pipetting up and down using a 200 μl tip. The membranes are pelleted tightly, so it takes some time to resuspend the pellet. I usually do it in the cold room.
    Aliquot a small volume of the microsome membranes, diluted it by 10 fold with EB, and store it at –20 °C. The diluted sample will be used for quantifying protein concentration using the BCA Protein Assay Kit.
  10. Transfer microsome suspension to a 1.5 ml Eppendorf tube, vortex briefly, and incubate on ice for 5 min to let large particulates settle down.
  11. Aliquot 20 μl of microsomes (do not pipet the precipitate on the bottom) into 1.5 ml Eppendorf tubes pre-chilled on ice. Boil 2-3 tubes of microsomes at 100 °C for 10 min, and the boiled samples will be used as a boiled control.
  12. Prepare reaction cocktail by adding and mixing the following reagents.
    4x ManS assay buffer
    10 μl
    (final concentration in reaction: 1x)
    1 mM GDP-Man
    0.85 μl
    (final concentration in reaction: 21.18 μM)
    GDP-[14C]-Man (76.34 μM)
    2 μl
    (final concentration in reaction: 3.82 μM)
    Deionized H2O
    7.15 μl

    Make master mix by scaling up the volumes based on the numbers of reaction tubes.
  13. Pipet 20 μl of the reaction cocktail into each tube containing 20 μl of microsomes, and briefly vortex to mix. Allow ~5 sec staggering time between two samples.
  14. Conduct reactions at room temperature for 1 h.
  15. Stop the reactions by adding 1 ml of 70% ethanol containing 2 mM EDTA and 10 μl of 1% carob galactomannan. Precipitate assay products at –20 °C for at least 1 h.
  16. Pellet the assay products by centrifuging at 16,000 x g at 4 °C for 10 min.
  17. Wash the pellets with 70% ethanol containing 2 mM EDTA four times, and repeat centrifugation after each wash.
  18. Resuspend each washed pellet in 300 μl of water.
  19. Transfer suspension to a 4 ml plastic scintillation vial, and then add 3 ml OptiPhase Supermix Cocktail to the vial. Cap the vial and mix the sample well by vortexing briefly.
  20. Perform liquid scintillation counting using a 1450 MicroBeta TriLux Microplate Scintillation and Luminescence Counter.
  21. Calculate the in vitro ManS enzymatic activities (shown as picomoles of GDP-Man incorporation per hour per mg protein) based on radioactivity of the assay products using the following formula.

    Specific ManS activity = (CPM ÷ counting efficiency) ÷ 2,220,000 ÷ (GDP-[14C]-Man radioactivity shown as μCi/μmol) x 1,000,000 x [(cold GDP-Man concentration + hot GDP-Man concentration) ÷ hot GDP-Man concentration] ÷ 1 h ÷ [20 x (protein concentration as μg/μl) ÷ 1,000]

    Detailed explanations are as follows:

    DPM (decaying per minute) = CPM (counting per minute) ÷ counting efficiency
    (This formula is used to convert the detected radioactivity (cpm) of the assay products to the actual radioactivity (dpm) based on the detection efficiency of the of the liquid scintillation counting machine)

    Hot GDP-Man incorporation (pmol) = DPM ÷ 2,220,000 dpm/μCi ÷ (GDP-[14C]-Man radioactivity shown as μCi/μmol) x 1,000,000 pmol/μmol
    (This formula is used to convert the radioactivity (dpm) of the assay products to pmol radio-labeled (hot) GDP-Man incorporation. Note the unit conversions: 1 μCi = 2,220,000 dpm; 1 μmol = 1,000,000 pmol.)

    Total GDP-Man incorporation (pmol) = hot GDP-Man incorporation (pmol) x [(cold GDP-Man concentration + hot GDP-Man concentration) ÷ hot GDP-Man concentration]
    [The total GDP-Man incorporation (pmol) is calculated by dividing hot GDP-Man incorporation (pmol) by the ratio of the hot GDP-Man concentration to the total (hot + cold) GDP-Man concentration]

    Protein mass (mg) of 20 μl microsomes = 20 μl x (protein concentration as μg/μl) ÷ 1,000 μg/mg

    Specific ManS activity (pmol GDP-Man incorporation/h/mg protein) = total GDP-Man incorporation (pmol) ÷ 1 h ÷ protein mass (mg)

    For cold and hot GDP-Man reagents used in this protocol:
    Specific ManS activity = (CPM ÷ counting efficiency) ÷ 2,220,000 ÷ 262 x 1,000,000 x [(21.18 + 3.82) ÷ 3.82] ÷ 1 h ÷ [20 x (protein concentration as μg/μl) ÷ 1,000]

Recipes

  1. Extraction Buffer (EB)
    50 mM HEPES-KOH, pH 7.5
    0.4 M Sucrose
    10 mM MgCl2
    Filter sterilize and store at 4 °C
    Before use, add 1 cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail Tablet per 10 ml buffer.
  2. 4x ManS Assay Buffer
    200 mM HEPES-KOH (pH 7.5)
    10 mM DTT
    10 mM MgCl2
    20 mM MnCl2
    24% glycerol
    Store at –20 °C
  3. 1 mM GDP-Man
    Dissolve 5 mg GDP-Man in 770 μl of 10 mM HEPES-KOH (pH 7.5) to a final concentration of 10 mM. Dilute a small volume of 10 mM GDP-Man to 1 mM with H2O
  4. 1% carob galactomannan
    Dissolve 100 mg carob galactomannan in H2O with a final volume of 10 ml. Heat to facilitate dissolution of galactomannan

Acknowledgments

This protocol is adapted from Wang et al. (2013).

References

  1. Wang, Y., Mortimer, J. C., Davis, J., Dupree, P. and Keegstra, K. (2013). Identification of an additional protein involved in mannan biosynthesis. Plant J 73(1): 105–117.

简介

甘露聚糖是半纤维素多糖,并且存在于所有陆地植物的细胞壁中。 甘露聚糖由两种酶合成:用于主链(甘露聚糖或葡甘露聚糖)合成的甘露聚糖合酶(ManS)和用于侧链(半乳糖基)加成的半乳甘露聚糖半乳糖基转移酶。 这里,描述了使用新鲜分离自拟南芥茎的微粒体进行ManS活性测定的方法。 该方法可以应用于从拟南芥或任何其它植物的任何组织分离微粒体。

关键字:拟南芥, 细胞壁, 微粒体, 半乳甘露聚糖, 甘露聚糖合成酶

材料和试剂

  1. cOmplete,Mini,无EDTA的蛋白酶抑制剂混合片(Roche,目录号:11836170001)
  2. GDP(9.694GBq/mmol,262.0mCi/mmol,76.34μM)(PerkinElmer,目录号:NEC536050UC)。
  3. 双金鸡宁酸(BCA)蛋白测定试剂盒(Pierce Antibodies,目录号:23225或23227)
  4. OptiPhase Supermix Cocktail(PerkinElmer,目录号:1200-439)
  5. GDP-Man(Sigma-Aldrich,目录号:G-7377)
  6. 角豆半乳甘露聚糖(Megazyme International,目录号:P-GALML)
  7. 蔗糖
  8. MgCl 2
  9. 提取缓冲液(EB)(参见配方)
  10. 4x ManS测定缓冲液(参见配方)
  11. 1 mM GDP-Man(见配方)
  12. 1%角豆半乳甘露聚糖(参见食谱)

设备

  1. 砂浆和杵
  2. 13ml Sarstedt管(Sarstedt,目录号:60.540.500)
  3. 离心机
  4. 4ml塑料液体闪烁计数小瓶(PerkinElmer,目录号:1200-421)
  5. 1450 MicroBeta TriLux微板闪烁和发光计数器(PerkinElmer,目录号:1450-024)

程序

  1. 在生长室中在标准生长条件(20℃,100μmol/m 2/s,16h光/8h黑暗,60%湿度)下生长拟南芥植物6-7周。
  2. 从单个植物收获整个茎(包括次级茎),并删除叶,花和长角果。 茎应该足够大(至少200毫克的鲜重),但不是很老或硬(没有任何黄色长角)。
  3. 称重茎并将其切成约1厘米的区段
  4. 立即将茎段转移到在冰上预冷的研钵中,加入冰冷的EB(〜1ml EB/100mg茎)。
  5. 在冰上用研钵和杵研磨EB中的茎段,直到组织匀浆(〜5分钟)。
    我通常在冷室里擦拭纸巾。
  6. 使用1毫升大口径移液器尖端(尖锐端切除)将茎匀浆转移至预冷的13ml Sarstedt管,并在3,000xg在4℃离心10分钟。
  7. 将上清液转移到新的预冷的13ml Sarstedt管中,并在4℃下以17,000×g离心20分钟。
  8. 在4℃下将来自步骤7的上清液以100,000xg离心90分钟以沉淀微粒体膜。
  9. 重悬细胞沉淀在EB(0.5微升EB /毫克茎)通过吸取上下使用200微升提示。膜紧密地沉淀,因此需要一些时间来重悬沉淀物。我通常在寒冷的房间里做 等分小体积的微粒体膜,用EB稀释10倍,并将其储存在-20℃。稀释的样品将用于使用BCA蛋白测定试剂盒定量蛋白浓度
  10. 将微粒体悬浮液转移到1.5ml Eppendorf管中,短暂涡旋,并在冰上孵育5分钟,以使大颗粒沉降。
  11. 等分20微升的微粒体(不移取底部的沉淀)到1.5毫升在冰上预冷冻的Eppendorf管。 在100℃煮沸2-3管微粒体10分钟,煮沸的样品将用作煮沸的对照。
  12. 通过加入和混合以下试剂制备反应混合物
    4x ManS测定缓冲液
    10微升
    (反应终浓度:1x)
    1 mM GDP-Man
    0.85μl
    (反应终浓度:21.18μM)
    GDP- [14 C] -Man(76.34μM)
    2微升
    (反应终浓度:3.82μM)
    去离子H 2 O 2 / 7.15微升

    根据反应管的数量放大体积,使主混合
  13. 吸取20μl反应混合物到每个管含有20微升的微粒体,并简要涡旋混合。 允许两个样品之间约5秒的交错时间。
  14. 在室温下反应1小时。
  15. 通过加入1ml含有2mM EDTA和10μl1%角豆荚半乳甘露聚糖的70%乙醇停止反应。 沉淀测定产物在-20°C至少1小时
  16. 通过在4℃下以16,000×g离心10分钟来沉淀测定产物。
  17. 用含有2mM EDTA的70%乙醇洗涤沉淀4次,每次洗涤后重复离心
  18. 将每个洗涤的沉淀重悬在300μl水中
  19. 转移悬浮液到4毫升塑料闪烁瓶,然后加入3毫升OptiPhase Supermix鸡尾酒到小瓶。盖上小瓶,通过短暂涡旋混合样品
  20. 使用1450 MicroBeta TriLux微孔板闪烁和发光计数器进行液体闪烁计数
  21. 基于测定产物的放射性,使用下式计算体外ManS酶活性(显示为每小时每mg蛋白的GDP-Man掺入的皮摩尔)。

    特定ManS活动=(CPM > ÷计数效率)÷2,220,000÷(GDP- [ 14 C] - 放射性显示为μCi/μmol)×1,000,000 ÷1 h÷[20×(蛋白质浓度以μg/μl计)÷1,000] x>>>>>>>>>>>>>>>>>>>>>>>>>>
    详细说明如下:

    DPM(每分钟衰减)= CPM(每分钟计数)÷计数效率
    (该公式用于根据液体闪烁计数机的检测效率将检测产物的检测放射性(cpm)转换为实际放射性(dpm))

    热GDP-人掺入(pmol)= DPM÷2,220,000dpm /μCi÷(GDP- [14 C] - 人放射性,以μCi/μmol表示)×1,000,000pmol /μmol
    (该公式用于将测定产物的放射性(dpm)转换为pmol放射性标记(热)GDP-Man掺入。注意单位转换:1μCi= 2,220,000dpm;1μmol= 1,000,000pmol) >
    总GDP-人结合(pmol)=热GDP-人结合(pmol)x [(冷GDP - 人浓度+热GDP-人浓度)÷热GDP-人浓度]
    [通过将热GDP-Man掺入(pmol)除以热GDP​​-Man浓度与总(热+冷)GDP-Man浓度的比率来计算总GDP-人掺入(pmol)]

    20μl微粒体的蛋白质质量(mg)=20μlx(蛋白质浓度以μg/μl计)÷1,000μg/mg

    特异性ManS活性(pmol GDP-Man掺入/h/mg蛋白)=总GDP-人掺入(pmol)÷1h÷蛋白质量(mg)

    对于本协议中使用的冷和热GDP-Man试剂:
    特异性ManS活性=(CPM÷计数效率)÷2,220,000÷262×1,000,000×[(21.18 + 3.82)÷3.82]÷1h÷[20×(蛋白质浓度,μg/μl)÷

食谱

  1. 提取缓冲液(EB)
    50mM HEPES-KOH,pH 7.5
    0.4 M蔗糖
    10mM MgCl 2/
    过滤灭菌并在4℃下保存
    使用前,每10ml缓冲液中加入1cOmplete,Mini,无EDTA的蛋白酶抑制剂混合物片剂。
  2. 4x ManS测定缓冲液
    200mM HEPES-KOH(pH7.5) 10 mM DTT
    10mM MgCl 2/
    20mM MnCl 2
    24%甘油 储存于-20°C
  3. 1 mM GDP-Man
    将5mg GDP-Man溶解在770μl的10mM HEPES-KOH(pH7.5)中至终浓度为10mM。 用H 2 O稀释小体积的10mM GDP-Man至1mM
  4. 1%角豆半乳甘露聚糖
    将100mg角豆半乳甘露聚糖溶解在H 2 O中,最终体积为10ml。 加热以促进半乳甘露聚糖的溶解

致谢

该协议改编自Wang等人(2013)。

参考文献

  1. Wang,Y.,Mortimer,J.C.,Davis,J.,Dupree,P。和Keegstra,K。(2013)。 鉴定涉及甘露聚糖生物合成的其他蛋白。植物J em> 73(1):105-117。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wang, Y. (2013). Stem Microsome Preparation and Mannan Synthase Activity Assay. Bio-protocol 3(10): e569. DOI: 10.21769/BioProtoc.569.
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