Rapid fluorometric assay for mitochondrial proton adenosine triphosphatase activity for assessment of viability of liver graft tissue
Rapid fluorometric assay for mitochondrial proton adenosine triphosphatase activity for assessment of viability of liver graft tissue
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快速荧光测定线粒体质子三磷酸腺苷酶活性以评估肝移植组织的活力
DOI：
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
S. Mori
中科院分区：
文献类型：
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作者：
K. Seya;N. Ohkohchi;N. Watanabe;H. Shibuya;Y. Taguchi;S. Mori
我们建立了一种改进的测定肝脏线粒体质子三磷酸腺苷酶(ATPase)活性的方法。用3，3‘-二丙基硫代二碳菁碘(dis-C3(5))荧光法测定其活性，其激发波长为625 nm，发射波长为670 nm。这种染料将电势传递到线粒体膜内。以dis-C3(5)与线粒体(10 0μL，4~16 mg/ml蛋白质)在2ml钾缓冲液(pH 7.4)中的荧光强度为标准电位。在用琥珀酸(9μ摩尔)确认线粒体电子传递链的活性后，我们用抗霉素A(1.25μg)抑制线粒体电子传递链。5‘-三磷酸腺苷(2μ摩尔)和寡霉素(25μg)可抑制这种抑制作用。线粒体质子ATPase活性以每标准电势下ATP的荧光强度变化的百分比计算。正常新鲜大鼠肝脏线粒体质子ATPase活性为50.3±2.2%。在不同条件下的大鼠肝脏中，线粒体质子ATPase活性的两种新方法与传统比色法的相关性良好(R2=0.807)。这种方法的优点是可以在完整的线粒体中测量质子ATPase的活性，并且所有步骤都可以在40min内完成。该方法适用于肝切除和肝移植中移植肝线粒体活性的测定。©1994 Wiley-Liss，Inc.
We developed an improved determination method of mitochondrial proton adenosine triphosphatase (ATPase) activity in the liver. The activity was measured fluorometrically with a 3,3′‐dipropylthiodicarbocyanine iodide (diS‐C3(5)), which is excited at 625 nm and emits fluorescence at 670 nm. This dye transmits the electric potential across the inner mitochondrial membrane. The fluorescence intensity of diS‐C3(5) with mitochondria (100 μl, 4–16 mg/ml protein) in a 2 ml potassium buffer (pH 7.4) was regarded as a standard electric potential. After confirming the activity of the mitochondrial electron transport chain by succinic acid (9 μmol), we inhibited the chain by antimycin A (1.25 μg). Fluorescence intensity decreased by adenosine 5′‐triphosphate (ATP) (2 μmol) and oligomycin (25 μg) inhibited this depression. The value of mitochondrial proton ATPase activity was calculated as a percentage of the fluorescence intensity change by ATP per the standard electric potential. The activity of mitochondrial proton ATPase in the normal fresh rat livers was 50.3 ± 2.2%. Good correlation (r2 = 0.807) between two methods for mitochondrial proton ATPase activity, our newly developed method and a conventional colorimetric method, was obtained in the rat livers with various conditions. This method has advantages that the proton ATPase activity can be measured in intact mitochondria, and all procedures can be completed within 40 min. It is suitable for the determination of mitochondrial viability of liver graft in the hepatic resections and transplantations. © 1994 Wiley‐Liss, Inc.
甲状腺切除术对肝线粒体 ADP-ATP 易位动力学的影响。
影响因子：
3.9
作者：
Mak,IT;Shrago,E;Elson,CE
通讯作者：
Elson,CE