Interaction of Creatine Kinase from Monkey Brain with Substrate: Analysis of Kinetics and Fluorescence Polarization
Interaction of Creatine Kinase from Monkey Brain with Substrate: Analysis of Kinetics and Fluorescence Polarization
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猴脑肌酸激酶与底物的相互作用：动力学和荧光偏振分析
DOI：
10.1111/j.1471-4159.1983.tb04801.x
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发表时间：
1983
影响因子：
4.7
通讯作者：
S. Grossman
中科院分区：
文献类型：
--
作者：
S. Grossman
摘要：用滴定法测定了底物与猴脑肌酸激酶的解离常数，结果表明，底物与猴脑肌酸激酶的正向和反向协同作用分别为13倍和4倍。这种协同作用表现为与二元配合物相比，三元配合物中给定底物的KD降低，可能反映了底物诱导的构象变化。用两分子5 ' -碘乙酰氨基荧光素标记的肌酸激酶在与MgADP结合后显示蓝移和荧光强度降低，表明染料进入更疏水的环境并熄灭了外部荧光。MgADP结合后，丹基化脑肌酸激酶的旋转弛缓时间从212±7 ns减少到189±6 ns。丹参化肌酸激酶在0.5%十二烷基硫酸钠中的旋转松弛时间为135±6 ns。丹化肌型同工酶的旋转松弛时间不受MgADP的影响，与脑同工酶- MgADP复合物具有相同的值。用3‐（4‐马来酰苯基）‐7‐二乙胺‐4‐甲基香豆素标记的肌肉酶和脑酶在25°C下的极化值与限制极化和自由染料的极化值进行比较，表明染料的旋转在肌肉形式中受到严重限制，但在脑形式中具有自由旋转。这些结果支持了与肌肉同工酶相比，脑同工酶在活性部位更开放，整体更灵活的结论。脑肌酸激酶结合MgADP会产生一种在其一个或两个旋转轴上更紧密的蛋白质，因此类似于肌肉同工酶的构象。脑中的肌酸激酶与肌肉中的肌酸激酶不同，可能受到伴随构象改变的动力学调节。这表明脑同工酶的神经生化作用不同于肌肉同工酶的代谢功能。
Abstract: Titrimetric determination of the dissociation constants for the binding of substrates to creatine kinase from monkey brain reveals 13‐fold and 4‐fold synergism in the forward and reverse directions, respectively. This synergism is expressed as a decrease in the KD for a given substrate in the ternary complex compared with the binary complex and may be a reflection of substrate‐induced conformational change. Creatine kinase labeled with two molecules of 5′‐iodoacetamidofluorescein displays a blue shift and a decrease in fluorescence intensity upon binding of MgADP, indicative of movement of the dye into a more hydrophobic environment and quenching of the extrinsic fluorescense. Rotational relaxation times determined from analysis of fluorescence polarization of dansylated brain creatine kinase decrease from 212 ± 7 ns to 189 ± 6 ns upon MgADP binding. Dansylated creatine kinase in 0.5% sodium dodecyl sulfate has a rotational relaxation time of 135 ± 6 ns. The rotational relaxation time of dansylated muscle‐type isoenzyme is unaffected by MgADP and has the same value as the brain isoenzyme‐MgADP complex. Polarization values at 25°C for muscle and brain enzyme labeled with 3 ‐ (4 ‐ maleimidylphenyl) ‐ 7 ‐ diethylamino ‐ 4 ‐ methylcoumarin compared with limiting polarization and polarization of the free dye suggest that the dye rotation is severely restricted in the muscle form, but possesses freedom of rotation in the brain form. These results support the conclusion that compared with the muscle isoenzyme, the brain isoenzyme is more open at the active site and more flexible overall. Binding of MgADP by brain creatine kinase produces a protein more compact across one or both of its rotational axes, thus resembling the conformation of the muscle isoenzyme. It is probable that creatine kinase in the brain, unlike that from muscle, is subject to kinetic regulation accompanied by conformational modification. This suggests that the neurobiochemical role of the brain isoenzyme is distinct from the metabolic function of the muscle isoenzyme.