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物理化学学报  2018, Vol. 34 Issue (1): 73-80    DOI: 10.3866/PKU.WHXB201707043
论文     
不同条件下离体大鼠肝脏线粒体能量代谢微量热分析
袁莲1,刘玉娇1,何欢1,蒋风雷1,李会荣2,刘义1,*()
1 武汉大学化学与分子科学学院,武汉430072
2 武汉东湖学院生命科学与化学学院,武汉430212
Microcalorimetric Analysis of Isolated Rat Liver Mitochondrial Metabolism under Different Conditions
Lian YUAN1,Yu-Jiao LIU1,Huan HE1,Feng-Lei JIANG1,Hui-Rong LI2,Yi LIU1,*()
1 College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P. R. China
2 College of Life Science and Chemistry, Wuhan Donghu University, Wuhan 430212, P. R. China
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摘要:

为了探究线粒体的能量代谢过程,本文以离体大鼠肝脏线粒体为模型,利用多通道、高灵敏度的热活性检测仪TAM Ⅲ,实时监测了不同线粒体浓度、不同底物、不同缓冲液、几种呼吸抑制剂以及Ca2+和线粒体渗透转换孔抑制剂CsA存在时线粒体的能量代谢,获得了完整的热功率―时间曲线,并通过计算得到了线粒体能量代谢的热动力学参数。通过分析发现:(1)线粒体浓度越大,代谢越快;(2)直接底物琥珀酸钠使线粒体代谢更快;(3)高浓度Ca2+能够刺激线粒体快速产热,且在长期代谢进程中,线粒体渗透转换孔抑制剂CsA并不能改变Ca2+造成的影响;(4)不同缓冲液对线粒体代谢的影响基于其组分的不同,缓冲液中含有呼吸底物;(5)呼吸抑制剂都能抑制线粒体的能量代谢,尤其是复合物IV的抑制剂NaN3,高浓度下使代谢停止。

关键词: 线粒体微量热代谢速率呼吸链抑制剂线粒体渗透转换    
Abstract:

Isolated rat liver mitochondria were proposed as a model to monitor real-time heat metabolism.A high-throughput and sensitive thermal activity monitor Ⅲ (TAM Ⅲ) was used to detect the P-t curves of mitochondria under different conditions, including different mitochondrial concentrations, different substrates, different buffers, respiratory inhibitors, Ca2+, and CsA.We determined the thermokinetic parameters through calculation.The results showed that:(1) higher concentration of mitochondria led to faster energetic metabolism; (2) when succinate was the direct respiratory substrate, it promoted mitochondrial metabolism, in contrast to the condition when an indirect substrate, pyruvate, was used; (3) high concentration of Ca2+(2.5 mmol·L-1) stimulated mitochondrial metabolism, however CsA, an inhibitor of mitochondrial permeability transition pores, could not reverse the Ca2+-induced mitochondrial alteration; (4) mitochondria in various buffers displayed different rates of heat metabolism, because of the different composition of the buffers; (5) mitochondrial metabolism was inhibited by respiratory inhibitors, especially NaN3, which is an inhibitor of Complex Ⅳ and which completely stopped the mitochondrial heat release.

Key words: Mitochondria    Microcalorimetry    Metabolic rate    Respiratory inhibitor    Mitochondrial permeability transition
收稿日期: 2017-06-13 出版日期: 2017-07-04
中图分类号:  O642.3  
基金资助: 国家自然科学基金(21673166);湖北省自然科学基金项目(2015CFC892)
通讯作者: 刘义     E-mail: yiliuchem@whu.edu.cn
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引用本文:

袁莲,刘玉娇,何欢,蒋风雷,李会荣,刘义. 不同条件下离体大鼠肝脏线粒体能量代谢微量热分析[J]. 物理化学学报, 2018, 34(1): 73-80.

Lian YUAN,Yu-Jiao LIU,Huan HE,Feng-Lei JIANG,Hui-Rong LI,Yi LIU. Microcalorimetric Analysis of Isolated Rat Liver Mitochondrial Metabolism under Different Conditions. Acta Physico-Chimica Sinca, 2018, 34(1): 73-80.

链接本文:

http://www.whxb.pku.edu.cn/CN/10.3866/PKU.WHXB201707043        http://www.whxb.pku.edu.cn/CN/Y2018/V34/I1/73

BufferComponents
Buffer A220 mmol·L-1 mannitol, 70 mmol·L-1 sucrose, 20 mmol·L-1 HEPES, 2 mmol·L-1 Tris-HCl, 1 mmol·L-1 EDTA, pH 7.4
Buffer B220 mmol·L-1 mannitol, 70 mmol·L-1 sucrose, 5 mmol·L-1 Tris-HCl, 1 mmol·L-1 EDTA, pH 7.4
Media C220 mmol·L-1 mannitol, 70 mmol·L-1 sucrose, 1 mmol·L-1 EDTA, pH 7.4
MRB100 mmol·L-1 sucrose, 10 mmol·L-1 Tris, 10 mmol·L-1 MOPS, 2 mmol·L-1 MgCl2,
10 mmol·L-1 KH2PO4, 50 mmol·L-1 KCl, 10 mmol·L-1 EDTA, 2 mmol·L-1 rotenone, pH 7.4
MAB200 mmol·L-1 sucrose, 10 mmol·L-1 Tris, 10 mmol·L-1 MOPS, 1 mmol·L-1 Na3PO4,
5 mmol·L-1 succinate, 10 mmol·L-1 EGTA, 3 mg·mL-1 oligomycin, 2 mmol·L-1 rotenone, pH 7.4
Buffer H135 mmol·L-1 KAc, 5 mmol·L-1 HEPES, 10 mmol·L-1 EGTA, 10 mmol·L-1 EDTA,
2 mmol·L-1 rotenone, 1 mg·mL-1 valinomycin, pH 7.1
Buffer K135 mmol·L-1 KNO3, 5 mmol·L-1 HEPES, 10 mmol·L-1 EGTA, 10 mmol·L-1 EDTA, 2 mmol·L-1 rotenone, pH 7.1
表1  7种缓冲液的组分
图1  15 mmol?L?1琥珀酸钠作为底物时不同浓度线粒体的热量曲线
SamplesQ/Jt1/hP1/μWt2/hP2/μWk1/h-1R12k2/h-1R22k3/h-1R32
a200 mito + 15 mmol?L-1 succinate11.4328.83126.8837.37231.790.2560.99880.0990.9982-0.8440.9874
100 mito + 15 mmol?L-1 succinate11.0729.2165.1748.28144.270.2380.99890.0510.9800-0.7060.9796
b200 mito12.1121.9966.6359.2584.520.3150.98880.0120.9958-0.6720.9936
200 mito + 15 mmol?L-1 succinate11.4328.83126.8837.37231.790.2560.99880.0990.9982-0.8440.9874
200 mito + 15 mmol?L-1pyruvate11.0221.0053.9144.25158.140.3490.99890.0700.9988-0.6700.9897
ccontrol (succinate)10.4321.22127.3631.13201.950.5100.99610.0500.9952-1.3410.9635
0.1 mmol?L-1 Ca2+9.8320.83110.1430.21208.210.1960.99890.0740.9932-1.9570.9501
2.5 mmol?L-1 Ca2+16.1519.39417.280.7420.9984-0.3210.9991
10 μmol?L-1 CsA8.8620.45115.2329.56163.410.87240.99020.0540.9928-1.4670.9428
CsA + 0.1 mmol?L-1 Ca2+9.8919.79134.2728.78189.860.4520.99360.0520.9956-1.9020.9506
CsA + 2.5 mmol?L-1 Ca2+16.5019.01423.740.8030.9988-0.2930.9994
dMRB12.4722.3163.6748.28173.680.3330.99450.0470.9950-0.9810.9801
MAB13.5418.3769.1847.19218.420.2440.97900.0720.9854-0.4780.9866
H0.00--
K10.8627.3341.6476.6777.780.3250.99300.0440.9956-0.6070.9733
C11.9924.0569.1857.45100.570.3530.99730.0260.9954-0.6760.9884
PBS11.5226.2457.4167.06111.580.3010.99570.0350.9939-0.9250.9142
econtrol10.8333.34235.000.4360.9953-0.8800.9675
0.03 mmol?L-1 NaN310.9735.26244.110.2490.9943-0.7560.9945
0.3 mmol?L-1 NaN30.00--
3 mmol?L-1 NaN30.00--
40 μmol·L?1 rotenone9.8734.53264.640.2100.9896?0.9070.9964
8 mmol·L?1 malonate10.4737.27198.6622.6370.350.5160.96760.0870.9987?1.0720.9917
表2  不同条件下线粒体能量代谢的热动力学参数
图2  不同底物存在时线粒体的热量曲线
图3  Ca2+和CsA存在时线粒体的热量曲线
图4  不同缓冲液中线粒体的热量曲线
图5  呼吸链抑制剂(鱼藤酮、丙二酸钠和NaN3)存在时线粒体的热量曲线
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