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Lysosomal Amino Acid Efflux Assay
溶酶体氨基酸外流检测试验   

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Abstract

As the cellular “recycling” organelle, lysosomes break down proteins into amino acids, which are then transported into cytosol for reuse by various amino acid transporters. The transport rate of an amino acid is presumably regulated by cellular conditions such as organelle pH, membrane potential and metabolic states. Because of their intracellular localization and the relative inaccessibility, lysosomal amino acid transporters have been studied largely via indirect measurements. Using lysosome purification and 14C-labeled amino acids, this protocol provides a method to measure the efficiency of specific amino acid transporters on lysosomes.

Materials and Reagents

  1. Mouse liver
  2. 14C-labeled amino acids (PerkinElmer) (select the amino acid of your interest)
  3. 3 N methanolic HCl (Sigma-Aldrich, catalog number: 33051 )
  4. Anhydrous methanol (Sigma-Aldrich, catalog number: 322415 )
  5. Sucrose
  6. HEPES
  7. MOPS
  8. Na2EDTA
  9. KCl
  10. MgCl2
  11. ATP-Mg
  12. Phosphate buffered saline (PBS)
  13. Scintillation cocktail (Research Products International, catalog number: 111175 )
  14. Protein assay kit (Bio-Rad Laboratories, catalog number: 500-0112 )
  15. Homogenization buffer (HB) (see Recipes)
  16. High sucrose buffer (see Recipes)
  17. Uptake buffer (see Recipes)
  18. Efflux buffer (see Recipes)

Equipment

  1. Nitrogen gas tank (with regulator and connecting tube)
  2. 25-ml glass conical flask
  3. 1.5 ml and 15 ml tubes
  4. 30 G needles
  5. Dounce homogenizer
  6. High speed centrifuge (up to 25,000 x g) with temperature control
  7. Glassfiber filters (Grade GF/F) (Whatman, catalog number: 1825-025 )
  8. Vacuum filtration manifold (EMD Millipore, model: 1225 )
  9. Liquid scintillation counter (Beckman Coulter, model: LS6500 )
  10. Personal protective equipment for working with radioactive materials

Procedure

Note: This protocol involves 14C-labeled amino acids. The uses of radioactive isotopes and the necessary safety precautions to be taken should follow institutional regulations.

  1. Preparation of amino acid methyl esters
    1. Transfer 12.5 µCi 14C-labeled amino acid into a 25-ml glass conical flask and let it dry under stream of nitrogen.
      Note: The drying time depends on the speed of nitrogen flow and is about 10 min.
    2. Add 3 ml 3 N methanolic HCl into the flask and incubate for 24 h at room temperature. This will transform amino acid into amino acid methyl ester.
      Note: Seal the opening of the flask with parafilm to prevent evaporation of methanolic HCl. The transformation efficiency can be measured with chromatography and high voltage electrophoresis (Reeves, 1979; Steinherz et al., 1982).
    3. Dry the resulted amino acid methyl ester under stream of nitrogen. Add 1 ml anhydrous methanol to dissolve the pellet and dry it again under stream of nitrogen.
      Note: The drying time depends on the speed of nitrogen flow.
    4. Add 500 μl anhydrous methanol to dissolve the methyl ester. Bring down the volume to 250 μl through evaporation under stream of nitrogen.
    5. Shortly before efflux assay, transfer appropriate amount into a 1.5 ml tube. Dry under stream of nitrogen. Dissolve in uptake buffer (final concentration 0.005 μCi/μl).
      Note: Amino acid methyl ester is not stable in aqueous solution.

  2. Crude lysosome preparation
    1. To prepare crude lysosome preparation from mouse liver, follow animal use protocols and dissect out the liver after the animal is euthanized. Rinse in ice-cold HB.
      Note: Liver from one adult mouse is enough for one assay. If you need to test multiple amino acids, calculate the total protein concentration you can get from one mouse liver and scale up the animal number.
    2. Transfer the liver into a Dounce homogenizer, add 8 ml ice-cold HB.
    3. Homogenize the liver on ice to break liver cells (20 strokes).
    4. Centrifuge the homogenate at 1,000 x g for 10 min at 4 °C; transfer the supernatant into a new centrifuge tube and keep on ice.
    5. Homogenize the pellet again in 7 ml HB (15 strokes); centrifuge at 1,000 x g for 10 min at 4 °C.
    6. Combine the supernatant from steps B4-5; centrifuge at 20,000 x g for 20 min at 4 °C.
    7. Resuspend the pellet in 10 ml 250 mM KCl (pH 7.4, buffered by 1 mM MOPS) and centrifuge at 1,600 x g for 10 min at 4 °C. Transfer the supernatant to a fresh tube. This step pellets most of the mitochondria.
    8. Dilute the supernatant by 2.5x volume high sucrose buffer and centrifuge at 25,000 x g for 10 min to spin down the crude lysosomes.
    9. Resuspend the crude lysosome pellet in 200 μl uptake buffer. Centrifuge at 600 x g for 2 min.
    10. Transfer the supernatant to a new tube and keep it on ice. Resuspend the pellet in 100 μl uptake buffer by passing through a 30 G needle for 30 times. Combine it with the supernatant. This will be the crude lysosome sample.
    11. Use a small aliquot of crude lysosome to measure total protein concentration with protein assay. Adjust the concentration to 2 mg protein-equivalent amount of lysosomes/ml. Keep the rest of the preparation on ice and do the efflux assay within 12 h. Longer storage is not recommended.
      Note: If needed, lysosomes can be further purified from the above preparation using, for example, OptiPrep density gradient centrifugation (see References 4 and 5).

  3. Amino acid efflux assay
    1. For each reaction, dispense 50 μl aliquot (containing 100 μg of protein-equivalent amount of lysosomes) into a 1.5 ml tube.
    2. To initiate amino acid ester loading, add 50 μl of amino acid methyl ester (0.25 μCi, in uptake buffer) into each lysosome aliquot tube. Gently mix by pipetting. Incubate at 37 °C for 30 min.
      Note: The optimum loading time and temperature need to be experimentally determined for the amino acid and the type of lysososomes used.
    3. During the loading time, assemble the Whatman filters (GF/F) to vacuum filtration manifold. Rinse the filters with 5 ml PBS. Adjust the filtering speed to ~10 ml/min.
    4. At the end of loading, dilute the loading reaction 50x by transferring the 100 μl loading reaction into a 15 ml tube containing 5 ml pre-warmed (37 °C) efflux buffer; mix well and incubate at 37 °C. This will start the efflux.
    5. At the end of each efflux time point, combine a tube of efflux reaction (containing 5.1 ml) with 5 ml ice-cold PBS. Filter immediately through Whatman filter under vacuum; wash twice with 5 ml ice-cold PBS.
    6. For background count, incubate lysosomes and amino acid methyl ester separately (without loading). Mix and filter immediately after incubation.
    7. Soak the filter in scintillation cocktail overnight. Count using a scintillation counter.
    8. Subtract background from the isotope counts. Normalize the background-subtracted counts to that obtained at efflux time point 0. The normalized count at each time point indicates the portion of amino acid remaining in the lysosome and reflects the activity of the amino acid transporter. (See Figure 1 for an example.)


      Figure 1. A representative amino acid efflux result using mouse crude lysosomes and 14C-labeled arginine. Data are presented as mean ± SEM.

Recipes

  1. Homogenization buffer (HB)
    250 mM sucrose
    1 mM Na2EDTA
    10 mM HEPES (pH 7.0)
  2. High sucrose buffer
    450 mM sucrose
    0.5 mM Na2EDTA
    5 mM HEPES (pH 7.2)
  3. Uptake buffer
    250 mM sucrose
    5 mM MgCl2
    20 mM HEPES (pH 7.0)
  4. Efflux buffer
    250 mM sucrose
    5 mM MgCl2
    20 mM HEPES
    2 mM ATP-Mg (pH 7.0)

Acknowledgments

This protocol was adapted from previously published papers (Cang et al., 2013; Reeves, 1979; Steinherz et al., 1982; Graham et al., 1982) and the technical bulletin of the lysosome isolation kit from Sigma-Alderich (Reference 5). We thank Dr. Yandong Zhou and Dr. Youngjun Seo for discussion and conducting the experiments, and Dr. Denia Ramirez-Montealegre for advice on the protocol. The development of this protocol was supported, in part, by funding from American Heart Association, NIH and the University of Pennsylvania Research Foundation.

References

  1. Cang, C., Zhou, Y., Navarro, B., Seo, Y. J., Aranda, K., Shi, L., Battaglia-Hsu, S., Nissim, I., Clapham, D. E. and Ren, D. (2013). mTOR regulates lysosomal ATP-sensitive two-pore Na(+) channels to adapt to metabolic state. Cell 152(4): 778-790.
  2. Graham, J., Ford, T. and Rickwood, D. (1994). The preparation of subcellular organelles from mouse liver in self-generated gradients of iodixanol. Anal Biochem 220(2): 367-373. 
  3. Reeves, J. P. (1979). Accumulation of amino acids by lysosomes incubated with amino acid methyl esters. J Biol Chem 254(18): 8914-8921.
  4. Steinherz, R., Tietze, F., Raiford, D., Gahl, W. A. and Schulman, J. D. (1982). Patterns of amino acid efflux from isolated normal and cystinotic human leucocyte lysosomes. J Biol Chem 257(11): 6041-6049. 
  5. Technical Bulletin of Lysosome Isolation Kit (LYSISO1). (Sigma-Aldrich)

简介

作为细胞"再循环"细胞器,溶酶体将蛋白质分解成氨基酸,然后将其转运到细胞质中以供各种氨基酸转运蛋白重复使用。 氨基酸的转运速率可能通过细胞条件如细胞器pH,膜电位和代谢状态来调节。 由于它们的细胞内定位和相对不可接近性,溶酶体氨基酸转运蛋白主要通过间接测量进行研究。 使用溶酶体纯化和14 C标记的氨基酸,该协议提供了一种测量特定氨基酸转运蛋白在溶酶体上的效率的方法。

材料和试剂

  1. 小鼠肝脏
  2. 14 C标记的氨基酸(PerkinElmer)(选择您感兴趣的氨基酸)
  3. 3N甲醇HCl(Sigma-Aldrich,目录号:33051)
  4. 无水甲醇(Sigma-Aldrich,目录号:322415)
  5. 蔗糖
  6. HEPES
  7. MOPS
  8. Na 2 EDTA
  9. KCl
  10. MgCl 2
  11. ATP-Mg
  12. 磷酸盐缓冲盐水(PBS)
  13. 闪烁鸡尾酒(Research Products International,目录号:111175)
  14. 蛋白质测定试剂盒(Bio-Rad Laboratories,目录号:500-0112)
  15. 均质缓冲液(HB)(参见配方)
  16. 高蔗糖缓冲液(见配方)
  17. 摄取缓冲区(请参阅配方)
  18. 流出缓冲区(参见配方)

设备

  1. 氮气罐(带调节器和连接管)
  2. 25毫升玻璃锥形瓶
  3. 1.5 ml和15 ml试管
  4. 30 G针
  5. Dounce匀浆器
  6. 具有温度控制的高速离心机(最高25,000 x g)
  7. 玻璃纤维过滤器(GF/F级)(Whatman,目录号:1825-025)
  8. 真空过滤歧管(EMD Millipore,型号:1225)
  9. 液体闪烁计数器(Beckman Coulter,型号:LS6500)
  10. 用于使用放射性物质的个人防护设备

程序

注意:此协议涉及 14 C标记的氨基酸。放射性同位素的使用和必要的安全预防措施应遵循制度规定。

  1. 氨基酸甲酯的制备
    1. 将12.5μCi14 C标记的氨基酸转移到25ml玻璃锥形瓶中,并在氮气流下干燥。
      注意:干燥时间取决于氮气流的速度,约为10分钟。
    2. 加入3ml 3N甲醇HCl到烧瓶中,并在室温下孵育24小时。这将将氨基酸转化为氨基酸甲酯。
      注意:用石蜡膜密封烧瓶的开口以防止甲醇HCl蒸发。转化效率可以用色谱法和高压电泳测定(Reeves,1979; Steinherz等,1982)。
    3. 在氮气流下干燥所得的氨基酸甲酯。加入1ml无水甲醇溶解沉淀,并在氮气流下再次干燥 注意:干燥时间取决于氮气流的速度。
    4. 加入500μl无水甲醇溶解甲酯。通过在氮气流下蒸发使体积降至250μl。
    5. 在流出测定之前,将适量转移到1.5ml管中。在氮气流下干燥。溶解于摄取缓冲液(终浓度0.005μCi/μl) 注意:氨基酸甲酯在水溶液中不稳定。

  2. 粗溶酶体制备
    1. 为了从小鼠肝制备粗制溶酶体制剂,遵循动物使用方案并在动物安乐死后切除肝脏。在冰冷的HB中冲洗 注意:来自一只成年小鼠的肝脏足以进行一次测定。如果你需要测试多个氨基酸,计算一个小鼠肝脏可以得到的总蛋白浓度,并放大动物数量。
    2. 将肝脏转移到Dounce匀浆器中,加入8ml冰冷的HB
    3. 在冰上均质肝脏以破碎肝细胞(20次)
    4. 在4℃下以1,000×g离心匀浆10分钟; 将上清液转移到新的离心管中并保持在冰上。
    5. 在7ml HB(15次)中再次匀化沉淀; 在4℃下以1,000×g离心10分钟
    6. 合并步骤B4-5的上清液; 在4℃下以20,000×g离心20分钟
    7. 将沉淀重悬在10ml 250mM KCl(pH 7.4,1mM MOPS缓冲)中,并在4℃下以1,600×g离心10分钟。 转移上清液到一个新鲜的管。 此步骤使大多数线粒体沉淀。
    8. 用2.5倍体积的高蔗糖缓冲液稀释上清液,并以25,000×g离心10分钟,以离心沉淀粗溶酶体。
    9. 重悬在200μl吸收缓冲液中的粗溶酶体沉淀。以600×g离心2分钟。
    10. 转移上清液到一个新的管,并保持在冰上。通过通过30 G针30次,重悬浮在100微升摄取缓冲液中的沉淀。将其与上清液合并。这将是粗溶酶体样品。
    11. 使用一小部分粗溶酶体测量蛋白质测定的总蛋白浓度。调整浓度至2mg蛋白质当量的溶酶体/ml。保持其余的制剂在冰上,并进行外渗测定在12小时内。不建议更长的存储。
      注意:如果需要,可以使用例如OptiPrep密度梯度离心从上述制备中进一步纯化溶酶体(参见参考文献4和5)。

  3. 氨基酸流出测定
    1. 对于每个反应,将50μl等分试样(含有100μg蛋白质等量的溶酶体)分配到1.5ml管中。
    2. 为了引发氨基酸酯负载,向每个溶酶体等分试管中加入50μl氨基酸甲酯(0.25μCi,在吸收缓冲液中)。轻轻混匀通过吸移。在37℃孵育30分钟。
      注意:最佳加载时间和温度需要通过实验确定氨基酸和所用溶酶体的类型。
    3. 在装载时间期间,组装Whatman过滤器(GF/F)到真空过滤歧管。用5ml PBS冲洗过滤器。调整过滤速度〜10 ml/min
    4. 在加载结束时,通过将100μl加载反应转移到含有5ml预热(37℃)外排缓冲液的15ml管中,将加载反应稀释50倍;混合均匀并在37℃下孵育。这将开始流出。
    5. 在每个流出时间点结束时,结合一个流出反应管(含5.1ml)与5ml冰冷PBS。在真空下立即通过Whatman过滤器过滤;用5ml冰冷洗涤两次 PBS。
    6. 对于背景计数,分别孵育溶酶体和氨基酸甲酯(不加载)。 孵育后立即混合和过滤。
    7. 将滤膜在闪烁鸡尾酒中浸泡过夜。 使用闪烁计数器计数。
    8. 从同位素计数中减去背景。 将背景扣除计数归一化为在流出时间点0获得的计数。在每个时间点的归一化计数表示保留在溶酶体中的氨基酸部分并反映氨基酸转运蛋白的活性。 (参见图1的示例。)


      图1.使用小鼠粗制溶酶体和14 C-标记的精氨酸的代表性氨基酸流出结果。数据以平均值±SEM表示。

食谱

  1. 均质缓冲液(HB)
    250mM蔗糖 1mM Na 2 EDTA 10mM HEPES(pH 7.0)
  2. 高蔗糖缓冲液
    450mM蔗糖 0.5mM Na 2 EDTA 5mM HEPES(pH 7.2)
  3. 吸收缓冲区
    250mM蔗糖 5mM MgCl 2/
    20mM HEPES(pH 7.0)
  4. 流出缓冲区
    250mM蔗糖 5mM MgCl 2/
    20 mM HEPES
    2mM ATP-Mg(pH7.0)

致谢

该方案改编自以前发表的论文(Cang等人,2013; Reeves,1979; Steinherz等人,1982; Graham等人, 1982)和来自Sigma-Alderich的溶酶体分离试剂盒的技术公报(参考文献5)。 我们感谢Yandong Zhou博士和Dr. Youngjun Seo讨论和进行实验,并感谢Denia Ramirez-Montealegre博士就方案提供咨询。 该协议的开发部分得到美国心脏协会,NIH和宾夕法尼亚大学研究基金会的资助。

参考文献

  1. Cang,C.,Zhou,Y.,Navarro,B.,Seo,YJ,Aranda,K.,Shi,L.,Battaglia-Hsu,S.,Nissim,I.,Clapham,DE和Ren,D。 2013)。 mTOR调节溶酶体ATP敏感性两孔Na(+)通道以适应代谢状态。/a> 152(4):778-790。
  2. Graham,J.,Ford,T。和Rickwood,D。(1994)。 在自发生成的碘克沙醇梯度中从小鼠肝脏制备亚细胞器官。 em> Anal Biochem 220(2):367-373。 
  3. Reeves,J.P。(1979)。 与氨基酸甲酯孵育的溶酶体积累氨基酸。 Biol Chem 254(18):8914-8921。
  4. Steinherz,R.,Tietze,F.,Raiford,D.,Gahl,W.A。和Schulman,J.D。(1982)。 氨基酸从分离的正常和胱氨酸的人白细胞溶酶体流出的模式。 J Biol Chem 257(11):6041-6049。
  5. 溶酶体分离技术公告 Kit(LYSISO1)。(Sigma-Aldrich)
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Cang, C. and Ren, D. (2014). Lysosomal Amino Acid Efflux Assay. Bio-protocol 4(4): e1048. DOI: 10.21769/BioProtoc.1048.
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