代数余子式和色氨酸大脑谷氨酸脱羧酶的可访问性和展开。
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陈锈E,马丁DL, CH
代数余子式和色氨酸大脑谷氨酸脱羧酶的可访问性和展开。
拱生物化学Biophys。2001年8月15日,392 (2):333 - 40。
- PubMed ID
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11488610 (在PubMed]
- 文摘
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代数余子式和色氨酸65 kda的可访问性形式的老鼠大脑谷氨酸脱羧酶(GAD)研究了荧光猝灭测量使用丙烯酰胺,我——,Cs +饮料。Trp残留部分暴露于溶剂。我——是无法和Cs +更能够猝灭荧光的迦得全酶的残留(holoGAD)比酶蛋白(apoGAD)。的一部分暴露Trp残留在30 - 49%的范围。相比之下,pyridoxal-P绑定到迦得的活性部位暴露于溶剂。我——是更有能力和Cs +无法熄灭的荧光pyridoxal-P holoGAD。代数余子式存在于一个带正电的微环境,使其与离子的相互作用。不同暴露的残留物和pyridoxal-P这些指控饮料建议暴露Trp残留在本质上是位于外的活性部位。可访问性的变化后的残留物pyridoxal-P绑定迦得强烈支持一个重要的构象变化。荧光强度测量也进行调查的迦得使用盐酸胍变性剂(GdnHCl)。 At 0.8-1.5 M GdnHCl, an intermediate step was observed during the unfolding of GAD from the native to the denatured state, and was not found during the refolding of GAD from the denatured to native state, indicating that this intermediate step was not a reversible process. However, at >1.5 M GdnHCl for holoGAD and >2.0 M GdnHCl for apoGAD, the transition leading to the denatured state was reversible. It was suggested that the intermediate step involved the dissociation of native dimer of GAD into monomers and the change in the secondary structure of the protein. Circular dichroism revealed a decrease in the alpha-helix content of GAD from 36 to 28%. The unfolding pattern suggested that GAD may consist of at least two unfolding domains. Unfolding of the lower GdnHCl-resisting domain occurred at a similar concentration of denaturant for apoGAD and holoGAD, while unfolding of the higher GdnHCl-resisting domain occurred at a higher concentration of GdnHCl for apoGAD than holoGAD.