Hausladen A, Alscher R G
Department of Plant Pathology, Physiology and Weed Science, Virginia Polytechnic Institute, Blacksburg, USA.
Plant Physiol. 1994 May;105(1):215-23. doi: 10.1104/pp.105.1.215.
The thermal dependence of kinetic parameters has been determined in purified or partially purified preparations of cold-hardiness-specific glutathione reductase isozymes from red spruce (Picea rubens Sarg.) needles to investigate a possible functional adaptation of these isozymes to environmental temperature. We have previously purified glutathione reductase isozymes specific for nonhardened (GR-1NH) or hardened (GR-1H) needles. Isozymes that were distinct from GR-1NH and GR-1H, but appeared to be very similar to each other, were also purified from nonhardened (GR-2NH) or hardened (GR-2H) needles (A. Hausladen, R.G. Alscher [1994] Plant Physiol 105: 205-213). GR-1NH had 2-fold higher Km values for NADPH and 2- to 4-fold lower Km values for oxidized glutathione (GSSG) than GR-2NH, and a similar difference was found between GR-1H and GR-2H. However, no differences in Km values were found between the hardiness-specific isozymes GR-1NH and GR-1H. There was only a small effect of temperature on the Km(GSSG) of GR-1H and GR-2H, and no significant temperature effect on Km(NADPH) or Km(GSSG) could be found for the other isozymes. These results are discussed with respect to "thermal kinetic windows," and it is proposed that the relative independence of Km values to temperature ensures adequate enzyme function in a species that is exposed to extreme temperature differences in its natural habitat. A variety of substrates has been tested to characterize any further differences among the isozymes, but all isozymes are highly specific for their substrates, NADPH and GSSG. The reversible reductive inactivation by NADPH (redox interconversion) is more pronounced in GR-1H than in GR-2H. Reduced, partially inactive GR-1H is further deactivated by H2O2, whereas GR-2H is fully reactivated by the same treatment. Both isozymes are reactivated by GSSG or reduced glutathione. It is proposed that this property of GR-2H ensures enzyme function under oxidative conditions, and that in vivo the enzyme may exist in its partially inactive form and be activated in the presence of increased levels of GSSG or oxidants.
为了研究这些同工酶对环境温度可能的功能适应性，已在从红云杉（Picea rubens Sarg.）针叶中纯化或部分纯化的抗寒特异性谷胱甘肽还原酶同工酶制剂中测定了动力学参数的热依赖性。我们之前已纯化了非硬化针叶（GR - 1NH）或硬化针叶（GR - 1H）特异性的谷胱甘肽还原酶同工酶。还从非硬化针叶（GR - 2NH）或硬化针叶（GR - 2H）中纯化了与GR - 1NH和GR - 1H不同但彼此非常相似的同工酶（A. Hausladen，R.G. Alscher [1994] Plant Physiol 105: 205 - 213）。与GR - 2NH相比，GR - 1NH对NADPH的Km值高2倍，对氧化型谷胱甘肽（GSSG）的Km值低2至4倍，并且在GR - 1H和GR - 2H之间也发现了类似差异。然而，抗寒特异性同工酶GR - 1NH和GR - 1H之间的Km值没有差异。温度对GR - 1H和GR - 2H的Km（GSSG）只有很小的影响，而对于其他同工酶，未发现温度对Km（NADPH）或Km（GSSG）有显著影响。针对“热动力学窗口”对这些结果进行了讨论，并提出Km值对温度的相对独立性确保了在其自然栖息地中暴露于极端温度差异的物种中酶的充分功能。已测试了多种底物以表征同工酶之间的任何进一步差异，但所有同工酶对其底物NADPH和GSSG都具有高度特异性。NADPH（氧化还原相互转化）引起的可逆性还原失活在GR - 1H中比在GR - 2H中更明显。还原的、部分无活性的GR - 1H会被H2O2进一步失活，而GR - 2H通过相同处理可完全重新激活。两种同工酶都可被GSSG或还原型谷胱甘肽重新激活。有人提出，GR - 2H的这种特性确保了酶在氧化条件下的功能，并且在体内该酶可能以其部分无活性形式存在，并在GSSG或氧化剂水平升高时被激活。
