ACTIVITY OF PURIFIED NAD-SPECIFIC ISOCITRATE DEHYDROGENASE AT MODULATOR AND SUBSTRATE CONCENTRATIONS APPROXIMATING CONDITIONS IN MITOCHONDRIA
ACTIVITY OF PURIFIED NAD-SPECIFIC ISOCITRATE DEHYDROGENASE AT MODULATOR AND SUBSTRATE CONCENTRATIONS APPROXIMATING CONDITIONS IN MITOCHONDRIA
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DOI：
10.1016/0026-0495(86)90175-7
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发表时间：
1986-07-01
影响因子：
9.8
通讯作者：
PLAUT, GWE
中科院分区：
文献类型：
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作者：
GABRIEL, JL;ZERVOS, PR;PLAUT, GWE
NAD-特异性异柠檬酸脱氢酶从牛心脏的动力学参数进行了检查，在基板和效应器的水平接近分离的完整的心脏线粒体在不同的呼吸状态报告的浓度。在pH 7.4下，在底物D-异柠檬酸镁（0.10 mmol/L）和NAD+（3.0 mmol/L）、阳性效应物柠檬酸镁（1.0 mmol/L）和阴性效应物NADPH（1.5 mmol/L）的恒定浓度下，测量改变ADP/ATP比率（总腺嘌呤核苷酸恒定为8 mmol/L）对酶活性的影响。随着ADP/ATP比值的增加，酶活性增加，这是由于ADP浓度升高引起的激活，而不是由于ATP水平下降引起的抑制作用降低。增加ADP可降低NADPH的抑制作用，柠檬酸镁和游离Ca ~（2+）可增强这种作用。在含有所有上述效应物的温育培养基中，在总ADP/总ATP的比率为2.0至0.1时，游离Ca 2+对活性增强的S0.5为10至20 μ mol/L。该值在线粒体内游离Ca 2+浓度的范围内，但对于ADP活化的增强，其明显大于Ca 2+的S0.5（0.6至1 μ mol/L），这是在不存在其它效应物的情况下测定的。当NAD+/NADH和ADP/ATP比值均降低时，发现活性进一步下降，NAD+/NADH比值降低的作用是由于NADH的抑制（表观I 0.5 = 0.23 ± 0.01）。0.03 mmol/L），因为NAD+在检测范围内饱和。
The kinetic parameters of NAD-specific isocitrate dehydrogenase from bovine heart were examined at levels of substrates and effectors approximating the concentrations reported for isolated intact heart mitochondria in different respiratory states. The effect of changing ADP/ATP ratios (with total adenine nucleotides constant at 8 mmol/L) on enzyme activity was measured at constant concentrations of the substrates magnesium D-isocitrate (0.10 mmol/L) and NAD+ (3.0 mmol/L), the positive effector magnesium citrate (1.0 mmol/L) and the negative effector NADPH (1.5 mmol/L) at pH 7.4. Enzyme activity increased with increasing ADP/ATP ratios as a result of activation by rising ADP concentrations and not due to decreasing inhibition by falling levels of ATP. Increasing ADP decreased the inhibition by NADPH, and this effect was enhanced by magnesium citrate and by free Ca2+. In incubation media containing all of the above effectors, the S0.5 for enhancement of activity by free Ca2+ was 10 to 20 .mu.mol/L at ratios of total ADP/total ATP between 2.0 and 0.1. This value is in the range of intramitochondrial concentrations of free Ca2+, but it is appreciably larger than S0.5 of Ca2+ (0.6 to 1 .mu.mol/L) for the enhancement of ADP activation, which ws determined in the absence of other effectors. When both the NAD+/NADH and the ADP/ATP ratios were decreased, a further decline in activity was found. The effect of the decreasing NAD+/NADH ratio was due to inhibition by NADH (apparent I0.5 = 0.23 .+-. 0.03 mmol/L) since NAD+ was saturating over the range examined.