搜索

Amino Acid Transport Assays in Resting Cells of Lactococcus lactis
乳酸乳球菌静息细胞中的氨基酸转运分析   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Many bacteria are auxotrophic for at least a number of amino acids for which they lack the biosynthetic pathways. The organisms are still able to grow in media containing free amino acids as the sole source of amino acid when transport systems for the free amino acids are present in the cytoplasmic membrane. A range of transport systems for essential as well as non-essential amino acids has been described that use the proton motive force (by proton symport) or ATP hydrolysis as driving force to allow for the accumulation of the amino acid in the cells. The most widely used assay for uptake of amino acids (or any other substrate) is the rapid filtration assay using radiolabelled substrates. Here we describe the assay for uptake in resting cells of Lactococcus lactis (L. lactis) that are energized by glucose.

Keywords: Lactococcus lactis(乳酸乳球菌), Amino acid(氨基酸), Transport(运输), Resting cells(静息细胞), Radiolabelled(放射性标记)

Materials and Reagents

  1. L. lactis cells
  2. Whatman, Protran® BA85 Nitrocellulose Membranes (2.5 cm diameter, 0.45-μm-pore-size nitrocellulose filter ) (Sigma-Aldrich, catalog number: Z670634 )
  3. LiCl
  4. K2HPO4
  5. KH2PO
  6. 14C-labelled amino acids (PerkinElmer)
    Note: stock solutions typically contain 50-100 mCi/ml corresponding to 100 to 500 μM concentrations.
  7. Scintillation fluid (e.g. Emulsifier Scintillator Plus, Packard Bioscience)
  8. 100 mM potassium phosphate (KPi) pH 5.8 buffer at 4 °C (see Recipes)
  9. KPi containing 0.2% glucose at 4 °C (see Recipes)

Equipment

  1. Filtration setup (e.g. Ten-place filtration manifold, catalog number: FH225V )
  2. Vacuum pump
  3. Thermostat water bath with magnetic stirrer
  4. Liquid scintillation analyser (e.g. Tri-Carb 2000CA, Packard Instruments)
  5. Glass tubes (10 x 75 mm)
  6. Tweezers
  7. Magnetic stirring bars
  8. Timer
  9. Vortex

Procedure

  1. L. lactis cells harvested in the late exponential growth phase between OD660 of 0.6 and 1.0, washed and resuspended to OD660 of 2 in potassium phosphate (KPi) buffer, kept on ice.
  2. Homogenize cell suspension by briefly vortexing.
  3. Distribute alliquotes of 100 μl of the cell suspension over 5 glass tubes. Keep on ice.
  4. Incubate one of the tubes for 5 min at 30 °C under continuous stirring.
  5. Mount filter in setup and apply vacuum.
  6. At t = 0, add 1 μl of the 14C-labelled amino acid stock solution to the cells in each of the five tubes giving 1-5 μM concentrations.
  7. At each of the time points t = 0.5, 1, 2, 4 min, add 2 ml of ice cold 0.1 M LiCl  and filtrate cells over the nitrocellulose filter. For most amino acids these four time points cover a complete uptake curve. Occasionally, other time points may have to be selected.
  8. Immediately rinse tube with another 2 ml of 0.1 M LiCl and filtrate over the same filter.
  9. Remove vacuum, and transfer filter with tweezer to a 2 ml Eppendorf tube and put a new filter back to the filtration setup for the next sample.
  10. For the zero time point (final tube), skip the incubation at 30 °C (step 3); add the radioactivity to the cell suspension on ice , immediately followed by 2 ml LiCl and filtration plus rinsing (steps 6 and 7). Keeping the sample on ice prevents uptake during processing of the sample.
  11. Add 1 μl of the 14C-labelled amino acid stock solution to a 2 ml Eppendorf tube to determine the total amount of radioactivity used per time point.
  12. Add 1.8 ml of scintillation fluid to the Eppendorf tubes.
  13. Measure radioactivity retained on the filters by scintillation counting. Best results are obtained after allowing the filter plus bacteria to dissolve in the scintillation fluid.
  14. Uptake (rate) can be calculated from retained activity and total added activity and expressed in nmol amino acid/(mg protein x minute). The amount of amino acid is calculated from the data provided by the manufacturer and the amount of protein determined by standard assays for cell protein.

Recipes

  1. 0.1 M KPi pH 5.8 buffer (1,000 ml)
    Mix 1 M K2HPO4 and 1 M KH2PO4 to a final pH of 5.8 and dilute 10x with dH2O.
    Mix 10 ml KPi pH 5.8 and 0.1 ml of a 20% glucose solution. Store at 4 °C.

Acknowledgments

This protocol was adapted from and recently used in Trip et al. (2013).

References

  1. Trip, H., Mulder, N. L. and Lolkema, J. S. (2013). Cloning, expression, and functional characterization of secondary amino acid transporters of Lactococcus lactis. J Bacteriol 195(2): 340-350.

简介

许多细菌是至少一些氨基酸的营养缺陷型,它们缺少生物合成途径。 当游离氨基酸的运输系统存在于细胞质膜中时,生物体仍然能够在含有游离氨基酸作为唯一氨基酸来源的培养基中生长。 已经描述了使用质子动力(通过质子对数)或ATP水解作为驱动力以允许氨基酸在细胞中积累的必需氨基酸和非必需氨基酸的一系列运输系统。 用于摄取氨基酸(或任何其它底物)的最广泛使用的测定法是使用放射性标记底物的快速过滤测定法。 在这里,我们描述了由葡萄糖激发的乳酸乳球菌(乳酸乳球菌)的静息细胞中摄取的测定。

关键字:乳酸乳球菌, 氨基酸, 运输, 静息细胞, 放射性标记

材料和试剂

  1. L。 乳酸细胞
  2. Whatman,Protran?BA85硝化纤维素膜(2.5cm直径,0.45μm孔径硝酸纤维素滤膜)(Sigma-Aldrich,目录号:Z670634)
  3. LiCl
  4. K 2 HPO 4
  5. KH 2 PO 4
  6. < sup> 14 C-标记的氨基酸(PerkinElmer)
    注意:储备溶液通常含有50-100mCi/ml,相当于100至500μM浓度。
  7. 闪烁液(例如Emulsifier Scintillator Plus,Packard Bioscience)
  8. 100mM磷酸钾(KPi)pH5.8缓冲液(4℃)(见配方)
  9. KPi在4℃含有0.2%葡萄糖(参见配方)

设备

  1. 过滤设置(如十位过滤歧管,目录号:FH225V)
  2. 真空泵
  3. 恒温水浴用磁力搅拌器
  4. 液体闪烁分析仪(例如Tri-Carb 2000CA,Packard Instruments)
  5. 玻璃管(10×75mm)
  6. 镊子
  7. 磁力搅拌棒
  8. 计时器
  9. 涡流

程序

  1. L。 乳酸菌在后期指数生长期收获 OD 660为0.6和1.0,洗涤并在钾中重悬浮至OD 660为2 磷酸盐(KPi)缓冲液,保存在冰上
  2. 通过短暂涡旋匀浆细胞悬浮液。
  3. 分布100微升的细胞悬液的5微升玻璃管的alliquotes。 保持冰上。
  4. 在连续搅拌下,在30℃下孵育其中一个管5分钟
  5. 在安装过程中安装过滤器并应用真空
  6. 在t = 0时,向5个管中的每个中的细胞中加入1μl的14 C标记的氨基酸储备溶液,得到1-5μM浓度。
  7. 在每个时间点t = 0.5,1,2,4分钟,加入2ml冰冷的0.1M LiCl 并在硝酸纤维素滤膜上过滤细胞。 对于大多数氨基酸,这四个时间点覆盖完整的摄取曲线。 有时,可能必须选择其他时间点。
  8. 立即用另外2ml的0.1M LiCl洗涤管,并在同一过滤器上过滤
  9. 取出真空,用镊子将过滤器转移到2ml Eppendorf管中,并将新的过滤器放回下一个样品的过滤装置。
  10. 对于零时间点(最终管),在30℃下跳过孵育(步骤3);在冰上向细胞悬浮液中加入放射性,随后立即加入2ml LiCl并过滤加冲洗(步骤6和7)。将样品保持在冰上防止样品处理过程中的吸收
  11. 将1μl的14 C标记的氨基酸储备溶液加入到2ml Eppendorf管中,以确定每个时间点使用的放射性的总量。
  12. 向Eppendorf管中加入1.8 ml闪烁液
  13. 通过闪烁计数测量保留在过滤器上的放射性。在使滤器加细菌溶解在闪烁液中之后获得最佳结果
  14. 摄取(速率)可以从保留的活性和总添加活性计算,并以nmol氨基酸/(mg蛋白x分钟)表示。氨基酸的量由制造商提供的数据计算 通过细胞蛋白的标准测定法测定蛋白质的量

食谱

  1. 0.1M KPi pH5.8缓冲液(1,000ml) 将1×MK 2 HPO 4和1M KH 2 PO 4 4混合至最终pH为5.8,稀释10× 与dH <2> O。
    混合10ml KPi pH5.8和0.1ml 20%葡萄糖溶液。 储存于4°C。

致谢

这个协议改编自Trip 等人(2013)中最近使用的协议。

参考文献

  1. Trip,H.,Mulder,N.L。和Lolkema,J.S。(2013)。 乳酸乳球菌的次级氨基酸转运蛋白的克隆,表达和功能表征 > 。 J Bacteriol 195(2):340-350。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Trip, H. and Lolkema, J. S. (2013). Amino Acid Transport Assays in Resting Cells of Lactococcus lactis. Bio-protocol 3(12): e793. DOI: 10.21769/BioProtoc.793.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。