盐酸胍对猪心脂酰胺脱氢酶全酶和脱辅基酶的影响。

Effect of guanidine hydrochloride on the holo- and apo-enzymes of pig heart lipoamide dehydrogenase.

作者信息

Ogasahara K, Koike K, Hamada M, Hiraoka T

出版信息

J Biochem. 1976 Apr;79(4):819-28. doi: 10.1093/oxfordjournals.jbchem.a131135.

DOI:10.1093/oxfordjournals.jbchem.a131135

PMID:931980

Abstract

The effect of guanidine hydrochloride (GuHCl) on pig heart lipoamide dehydrogenase [NADH: lipoamide oxidoreductase, EC 1.6.4.3.] was investigated by means of enzymatic activity and optical measurements (CD, absorption, and fluorescence spectra). The activity of the enzyme decreased on increasing the concentration of GuHCl and the enzyme was completely inactivated in 2.0 M GuHCl. 2. The contents of alpha-helix, beta, and unordered forms in lipoamide dehydrogenase were estimated to be 34, 14, and 52%, respectively. On increasing the concentration of GuHCl, the content of alpha-helix in lipoamide dehydrogenase decreased, whereas the content of the beta form hardly changed. 3. The native lipoamide dehydrogenase showed absorption, CD, and fluorescence spectra characteristic of bound FAD in the visible region, suggesting hydrophobic interaction between the protein moiety and FAD chromophore. The absorption, CD, and fluorescence spectra of the enzyme in 2.0 M GuHCl were similar to those of free FAD in the buffer, suggesting the release of FAD from the protein moiety. 4. The protein fluorescence spectrum of lipoamide dehydrogenase had a maximum at 350 nm blue-shifted by 8 nm from that of tryptophan in aqueous solution. The maximum of the enzyme in 2.0 M GuHCl was red-shifted to 357 nm. This suggests exposure of tryptophan residues to a polar environment. The maximum, 352nm, of the apoenzyme shifted to 350 nm on addition of FAD. These results show that the conformation in the microenvironment of some tryptophan residues in lipoamide dehydrogenase is affected by the dissociation-association of FAD. 5. The contents of alpha-helix, beta, and unordered forms in the apoenzyme were estimated to be 35, 8, and 57%, respectively. These values are similar to those of the native holoenzyme. The alpha-helical structure in the apoenzyme molecule was more sensitive to GuHCl than that in the holoenzyme. FAD and two hydrophobic probes, 8-anilinonaphthalene-1-sulfonate (ANS) and 4 benzolamido-4'-aminostilbene-2,2'-disulfonate (MBAS), which can bind to the apoenzyme, stabilized the alpha-helical structure in the apoenzyme molecule.

摘要

通过酶活性和光学测量（圆二色光谱、吸收光谱和荧光光谱）研究了盐酸胍（GuHCl）对猪心脂酰胺脱氢酶[NADH：脂酰胺氧化还原酶，EC 1.6.4.3]的影响。随着GuHCl浓度的增加，该酶的活性降低，在2.0 M GuHCl中酶完全失活。2. 脂酰胺脱氢酶中α-螺旋、β-折叠和无规形式的含量分别估计为34%、14%和52%。随着GuHCl浓度的增加，脂酰胺脱氢酶中α-螺旋的含量降低，而β-折叠形式的含量几乎不变。3. 天然脂酰胺脱氢酶在可见光区域显示出结合FAD的吸收光谱、圆二色光谱和荧光光谱特征，表明蛋白质部分与FAD发色团之间存在疏水相互作用。该酶在2.0 M GuHCl中的吸收光谱、圆二色光谱和荧光光谱与缓冲液中游离FAD的光谱相似，表明FAD从蛋白质部分释放。4. 脂酰胺脱氢酶的蛋白质荧光光谱在350 nm处有最大值，比水溶液中色氨酸的荧光光谱蓝移了8 nm。该酶在2.0 M GuHCl中的最大值红移至357 nm。这表明色氨酸残基暴露于极性环境中。脱辅基酶的最大值352 nm在加入FAD后移至350 nm。这些结果表明，脂酰胺脱氢酶中某些色氨酸残基微环境中的构象受FAD解离-缔合作用的影响。5. 脱辅基酶中α-螺旋、β-折叠和无规形式的含量分别估计为35%、8%和57%。这些值与天然全酶的值相似。脱辅基酶分子中的α-螺旋结构比全酶中的α-螺旋结构对GuHCl更敏感。FAD以及两种可与脱辅基酶结合的疏水探针8-苯胺基萘-1-磺酸盐（ANS）和4-苯甲酰胺基-4'-氨基芪-2,2'-二磺酸盐（MBAS）稳定了脱辅基酶分子中的α-螺旋结构。