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LDH-A Enzyme Assay
乳糖脱氢酶(LDH-A)酶活分析   

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Abstract

LDH (Lactate dehydrogenase) enzyme catalyzes the reversible conversion of pyruvate to lactate using NAD+ as a cofactor. Although the physiological significance of lactate accumulation in tumor cells, a dead-end product in cellular metabolism, is currently a topic of debate, it has long been known that many tumor cells express a high level of LDH-A (Koukourakis et al., 2003; Koukourakis et al., 2006; Koukourakis et al., 2009). So detection of its enzyme activity in vitro is important for researching on LDH-A. Recently, it has been reported that Lys-5 acetylation could decrease LDH-A enzyme activity (Zhao et al., 2013).

Keywords: LDH(LDH), Enzyme activity(酶的活性), Lactate(乳酸), NAD+(NAD +)

Materials and Reagents

  1. 293T cells
  2. DMEM + 10% NCS
  3. Aprotinin (BBI Solutions, catalog number: AD0153-50mg )
  4. Leupeptin (AMRESCO, catalog number: J580-25MG )
  5. Pepstatin (AMRESCO, catalog number: J583 )
  6. PMSF (Sangon Biotech, catalog number: P0754-5g )
  7. Tris-HCl (pH 7.3) (Sangon Biotech)
  8. 250 μg/ml Flag peptide (in PBS buffer) (GL Biochem, sequence: DYKDDDDK)
  9. Pyruvate (Sigma-Aldrich, catalog number: 80443 )
  10. NADH (Sigma-Aldrich, catalog number: N8129 )
  11. Flag-beads (Sigma-Aldrich, catalog number: M8823 )
  12. Lipofectamine 2000 (Invitrogen)
  13. Reaction buffer (see Recipes)
  14. 0.3% NP-40 buffer (Lysis buffer) (see Recipes)

Equipment

  1. F-4600 Fluorescence Spectrophotometer
  2. 37 °C, 5% CO2 incubator
  3. 90 mm cell culture plate

Procedure

  1. Prepare LDH-A protein. You could ectopically overexpress and purify it from E. coli, or ectopically express Flag-LDH-A plasmid in 293T cells, followed by immunoprecipitation by Flag-beads and eluted using Flag peptide.
  2. 293T cells were cultured in DMEM + 10% NCS, in 5% CO2 incubator at 37 °C. Cell transfection was performed using Lipofectamine 2000 or calcium phosphate methods. 2 μg plasmids was transfected into 90 mm plate of 293T cells. And cells were cultured for 30 hours after transfection.
  3. Cells ectopically expressed Flag-LDH-A were lysated by 0.3% NP-40 buffer (lysis buffer) with protein degradation inhibitors by shaking gentally at 4 °C for half an hour.
  4. Cell lysate was centrifuged 4 °C for 15 min (16,000 x g) and the supernatant was incubated with 10 μl (per 90 mm plate of 293T cells) Flag-beads for 3 hours at 4 °C by rotation slowly.
  5. And then flag-beads were washed and centrifuged at 4 °C for 1 min (400 x g) by 1 ml 0.3% NP-40 buffer for 3 times, and incubated with 250 μg/ml of flag peptide (200 μl per 90 mm plate of 293T cells) shaking for one hour, followed by centrifuge at 16,000 x g for 5 min. The supernatant was used for enzyme activity detection.
  6. Prepare the reaction buffer containing 0.2 M Tris-HCl (pH 7.3), 30 mM pyruvate and 6.6 mM NADH.
  7. For every reaction, 10 μl LDH-A enzyme solution and 290 μl reaction buffer are added into the measuring cup of F-4600 Fluorescence Spectrophotometer, and detect the fluorescence change in absorbance (340 nm) resulting from NADH oxidation at room temperature.
    Note: The reaction system could be adjusted according to your LDH-A solution concentration. And the reaction is very quick, please detect the change as soon as possible.
  8. After a reaction, the software will show the slope of fluorescence change, and this value is the speed of this reaction.

Recipes

  1. Reaction buffer
    0.2 M Tris-HCl (pH 7.3)
    30 mM pyruvate
    6.6 mM NADH
  2. 0.3% NP-40 buffer (Lysis buffer)
    50 mM Tris-HCl (pH 7.5)
    150 mM NaCl
    0.3% Nonidet P-40
    1 μg/ml aprotinin
    1 μg/ml leupeptin
    1 μg/ml pepstatin
    1 mM PMSF

Acknowledgments

This protocol has been adapted from the previously published paper Zhao et al. (2013) and is described in further detail. We thank the members of the Fudan MCB laboratory for discussions throughout this study. We also thank Dr. Liming Wei for the IEF assay. This work was supported by the Chinese Ministry of Sciences and Technology 973 (Grant No. 2009CB918401, 2011CB910600), (Grant No. NCET-09-0315), NSFC (Grant No.31271454, 81225016) and NSFC-NIH (Grant No. 81110313). This work was also supported by Chinese Ministry of Education 985 Program, 100 Talents Program of Shanghai Health and Scholar of “Dawn” Program of Shanghai Education Commission and Shanghai Key basic research program(12JC1401100) to Q.Y.L. and NIH grants (Y.X. and K.L.G.); and the Fudan University Medical School Graduate Student Ming Dao Project Funds (D.Z.). This work is dedicated to the memory of Zhen Yu, who prepared the K5 acetylation antibody.

References

  1. Koukourakis, M. I., Giatromanolaki, A., Sivridis, E., Bougioukas, G., Didilis, V., Gatter, K. C., Harris, A. L., Tumour and Angiogenesis Research, G. (2003). Lactate dehydrogenase-5 (LDH-5) overexpression in non-small-cell lung cancer tissues is linked to tumour hypoxia, angiogenic factor production and poor prognosis. Br J Cancer 89(5): 877-885.
  2. Koukourakis, M. I., Giatromanolaki, A., Sivridis, E., Gatter, K. C., Harris, A. L. and Tumour Angiogenesis Research, G. (2006). Lactate dehydrogenase 5 expression in operable colorectal cancer: strong association with survival and activated vascular endothelial growth factor pathway--a report of the Tumour Angiogenesis Research Group. J Clin Oncol 24(26): 4301-4308.
  3. Koukourakis, M. I., Kontomanolis, E., Giatromanolaki, A., Sivridis, E. and Liberis, V. (2009). Serum and tissue LDH levels in patients with breast/gynaecological cancer and benign diseases. Gynecol Obstet Invest 67(3): 162-168.
  4. Zhao, D., Zou, S. W., Liu, Y., Zhou, X., Mo, Y., Wang, P., Xu, Y. H., Dong, B., Xiong, Y., Lei, Q. Y. and Guan, K. L. (2013). Lysine-5 acetylation negatively regulates lactate dehydrogenase A and is decreased in pancreatic cancer. Cancer Cell 23(4): 464-476.

简介

LDH(乳酸脱氢酶)酶使用NAD +作为辅因子催化丙酮酸向乳酸的可逆转化。 尽管乳酸蓄积在作为细胞代谢中的死端产物的肿瘤细胞中的生理学意义目前是一个争论的话题,但长期以来已知许多肿瘤细胞表达高水平的LDH-A(Koukourakis等 et al。,2003; Koukourakis et al。,2006; Koukourakis et al。,2009)。 因此,其体外酶活性的检测对于LDH-A的研究是重要的。 最近,已报道Lys-5乙酰化可降低LDH-A酶活性(Zhao等人,2013)。

关键字:LDH, 酶的活性, 乳酸, NAD +

材料和试剂

  1. 293T细胞
  2. DMEM + 10%NCS
  3. 抑肽酶(BBI Solutions,目录号:AD0153-50mg)
  4. 亮肽素(AMRESCO,目录号:J580-25MG)
  5. 胃蛋白酶抑制剂(AMRESCO,目录号:J583)
  6. PMSF(Sangon Biotech,目录号:P0754-5g)
  7. Tris-HCl(pH7.3)(Sangon Biotech)
  8. 250μg/ml Flag肽(在PBS缓冲液中)(GL Biochem,序列:DYKDDDDK)
  9. 丙酮酸(Sigma-Aldrich,目录号:80443)
  10. NADH(Sigma-Aldrich,目录号:N8129)
  11. Flag-beads(Sigma-Aldrich,目录号:M8823)
  12. Lipofectamine 2000(Invitrogen)
  13. 反应缓冲液(参见配方)
  14. 0.3%NP-40缓冲液(裂解缓冲液)(见配方)

设备

  1. F-4600荧光分光光度计
  2. 37℃,5%CO 2培养箱
  3. 90 mm细胞培养板

程序

  1. 制备LDH-A蛋白。 你可以从ectopically过度表达和净化它从。 或在293T细胞中异位表达Flag-LDH-A质粒,随后通过Flag-珠粒进行免疫沉淀并使用Flag肽洗脱。
  2. 293T细胞在37℃下在含5%CO 2的培养箱中的DMEM + 10%NCS中培养。 使用Lipofectamine 2000或磷酸钙方法进行细胞转染。 将2μg质粒转染到90mm平板的293T细胞中。 转染后培养细胞30小时。
  3. 通过在4℃下轻轻摇动半小时,用蛋白质降解抑制剂通过0.3%NP-40缓冲液(裂解缓冲液)裂解细胞异位表达的Flag-LDH-A。
  4. 将细胞裂解物在4℃下离心15分钟(16,000×g),并将上清液与10μl(每90mm板的293T细胞)Flag-珠在4℃下通过旋转温育3小时慢慢
  5. 然后清洗旗形珠,并通过1ml 0.3%NP-40缓冲液在4℃下离心3分钟(400×g)3次,并与250μg/ml标志肽(200μl每90mm平板的293T细胞)摇动1小时,随后在16,000×g离心5分钟。将上清液用于酶活性检测
  6. 准备含有0.2M Tris-HCl(pH 7.3),30mM丙酮酸和6.6mM NADH的反应缓冲液。
  7. 对于每个反应,将10μlLDH-A酶溶液和290μl反应缓冲液加入到F-4600荧光分光光度计的量杯中,并检测在室温下由NADH氧化产生的吸光度(340nm)的荧光变化。
    注意:反应系统可以根据您的LDH-A溶液浓度进行调整。反应非常快,请尽快检测变化。
  8. 反应后,软件将显示荧光变化的斜率,该值是该反应的速度

食谱

  1. 反应缓冲液
    0.2 M Tris-HCl(pH 7.3)
    30mM丙酮酸 6.6 mM NADH
  2. 0.3%NP-40缓冲液(裂解缓冲液) 50mM Tris-HCl(pH7.5) 150mM NaCl 0.3%Nonidet P-40
    1μg/ml抑肽酶
    1μg/ml亮肽素 1μg/ml胃酶抑素
    1mM PMSF

致谢

该协议已经从先前发表的论文(等人(2013)中进行修改并且进一步详细描述。我们感谢复旦MCB实验室的成员在本研究中的讨论。我们还感谢李明伟博士的IEF测定。这项工作得到中国科学技术部973(批准号2009CB918401,2011CB910600),(批准号NCET-09-0315),NSFC(批准号No.31271454,81225016)和NSFC-NIH(批准号: 81110313)。这项工作还得到了中国教育部985计划,上海市教育委员会"黎明"上海市健康与学者100人才计划和上海市重点基础研究计划(12JC1401100)至Q.Y.L的支持。和NIH授权(Y.X.和K.L.G.);和复旦大学医学院研究生明道项目基金(D.Z.)。这项工作致力于编写K5乙酰化抗体的振宇的记忆。

参考文献

  1. Koukourakis,M.I.,Giatromanolaki,A.,Sivridis,E.,Bougioukas,G.,Didilis,V.,Gatter,K.C.,Harris,A.L.,Tumor and Angiogenesis Research,G.(2003)。 在非小细胞肺癌组织中的乳酸脱氢酶-5(LDH-5)过表达到肿瘤缺氧,血管生成因子产生和差的预后 89(5):877-885。
  2. Koukourakis,M.I.,Giatromanolaki,A.,Sivridis,E.,Gatter,K.C.,Harris,A.L。和Tumor Angiogenesis Research,G.(2006)。 乳酸脱氢酶5在可操作的结肠直肠癌中的表达:与存活和活化的血管内皮生长因子通路 - - 肿瘤血管生成研究小组的报告。 24(26):4301-4308。
  3. Koukourakis,M.I.,Kontomanolis,E.,Giatromanolaki,A.,Sivridis,E。和Liberis,V。(2009)。 患有乳腺/妇科癌症和良性疾病的患者的血清和组织LDH水平。 em> Gynecol Obstet Invest 67(3):162-168
  4. Zhao,D.,Zou,SW,Liu,Y.,Zhou,X.,Mo,Y.,Wang,P.,Xu,YH,Dong,B.,Xiong,Y.,Lei,QY and Guan,KL (2013年)。 赖氨酸-5乙酰化负调节乳酸脱氢酶A,在胰腺癌中降低。 em> Cancer Cell 23(4):464-476。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhao, D., Xiong, Y., Lei, Q. and Guan, K. (2013). LDH-A Enzyme Assay. Bio-protocol 3(19): e919. DOI: 10.21769/BioProtoc.919.
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