氨基酰化酶在LDS溶液中的失活与去折叠的比较研究

氨基酰化酶在阴离子去污剂十二烷基硫酸溶液中的失活与去折叠的研究结果表明,在低浓度的ＬＤＳ溶液中变性时,以荧光和紫外差吸收方法监测的酶分子构象尚未发生明显变化,而酶的活力已经大部分或几乎全部丧失。当ＬＤＳ浓度达１．６ｍｍｏｌ／Ｌ时,此时酶分子的构象变化才达到最大程度。在实验使用的ＬＤＳ的浓度范围内,用远紫外ＣＤ光谱监测的二级结构没有发生明显的变化。     

超氧化物歧化酶全酶和脱辅基酶胍变性时失活与去折叠的比较研究

利用紫外差吸收光谱和荧光发射光谱等监测手段研究天然铜锌ＳＯＤ（ｈｏｌｏ－ＳＯＤ）和脱铜锌ＳＯＤ（ａｐｏ－ＳＯＤ）在不同浓度胍溶液中的去折叠及活力变化．结果表明ｈｏｌｏ－ＳＯＤ和ａｐｏ－ＳＯＤ分别在４．０和２．０ｍｏｌ／Ｌ胍溶液中去折叠,而分别在２．０和０．５ｍｏｌ／Ｌ胍溶液中其构象尚未发生明显改变时活性几乎完全丧失．提示金属离子对维持酶的整体及活性部位构象具有重要作用,脱去金属离子的酶分子的构象特别是活性部位的构象更易受到变性剂的破坏．     

超氧化物歧化酶全酶和脱辅基酶胍变性时失活与去折叠的比较研究

利用紫外差吸收光谱和荧光发射光谱等监测手段研究天然铜锌ＳＯＤ和脱铜锌ＳＯＤ在不同浓度胍溶液中的去折叠及活力变化。结果表明ｈｏｌｏ－ＳＯＤ和ａｐｏ－ＳＯＤ分别在４．０和２．０ｍｏｌ／Ｌ胍溶液中去折叠,而分别在２．０和０．５ｍｏｌ／Ｌ胍溶液中其构象尚未发生明显改变时活性几乎完全丧失。提示金属离子对维持酶的整体及活性部位构象具有重要作用,脱去金属离子的酶分子的构象特别是活性部位的构象更易受到变性剂的破坏     

水杨酸对黄瓜叶片抗氧化剂酶系的调节作用

分析了水杨酸（ＳＡ）对黄瓜（ＣｕｃｕｍｉｓｓａｔｉｖｕｓＬ．）叶片抗氧化剂酶系活性及活性氧水平的调节作用。不同浓度的ＳＡ（０．５ｍｍｏｌ／Ｌ、１ｍｍｏｌ／Ｌ、２．５ｍｍｏｌ／Ｌ、５ｍｍｏｌ／Ｌ）均能显著地提高被处理叶片超氧化物歧化酶（ＳＯＤ）和过氧化物酶（ＰＯＤ）活性,而且还能诱导同株的非处理叶片中ＳＯＤ和ＰＯＤ活性增加。用１ｍｍｏｌ／ＬＳＡ处理第一片真叶,在处理后６～７２ｈ,ＰＯＤ活性增加了２２％～６７％,同株非处理的第二片真叶ＰＯＤ活性增加了１４％～８６％,但是,在ＳＡ处理后３ｈ之前以及处理９６ｈ之后,ＰＯＤ活性没有变化。ＳＡ能够显著降低超氧物阴离子含量和提高过氧化氢水平,但它对过氧化氢酶（ＣＡＴ）活性的抑制作用很弱,表明ＳＡ提高体内过氧化氢含量的原因主要是通过提高ＳＯＤ活性而不是抑制ＣＡＴ活性。同工酶分析表明,ＳＡ不能诱导新的ＳＯＤ同工酶,但可以诱导新的ＰＯＤ同工酶。     

人肌肌酸激酶在SDS溶液中失活与构象变化的比较研究

应用紫外差吸收光谱、荧光发射光谱、ＣＤ先谱等监测手段,研究了ＳＤＳ溶液滴定人肌肌酸激酶时的构象与活力变化的关系。结果表明酶的活力丧失先于以紫外差吸收先谱、荧先发射谱和巯基暴露数目所监测到的构象变化。ＳＤＳ滴定时引起的酶的荧光发射光谱的变化在低滴定度阶段随着ＳＤＳ滴定量的增加,荧光强度下降,发射峰位红移,当ＳＤＳ浓度达到２．１ｍｍｏｌ／Ｌ时,荧光强度增大,继续增加ＳＤＳ滴定量,荧光强度又降低,发射峰位红移直至终态。紫外差吸收光谱随着ＳＤＳ溶液的加入,２８１ｎｍ．２８７ｎｍ和２９２ｎｍ的负差吸收峰增大。ＣＤ光谱结果表明在本实验所用的ＳＤＳ浓度范围内,ＳＤＳ对人肌肌酸激酶的二级结构几乎没有影响。上述结果支持了酶的活性部位构象柔性的观点。     

Cr^6＋对莼菜冬芽叶片急性毒害与保护酶系活性变化关系的研究

研究了Ｃｒ＾６＋的急性毒害对莼菜冬芽叶片的伤害程度和可溶性蛋白质、ＳＯＤ、ＣＡＴ、ＰＯＤ活性和ＭＤＡ含量变化之间的关系。在以０．５ｍｍｏｌ／Ｌ￣２．０ｍｍｏｌ／Ｌ浓度的Ｃｒ＾６＋处理时,冬芽叶片受害的程序明显地与处理浓度和处理时间呈正相关;可溶性蛋白质只在处理４ｄ时出现较大变化,其含量随处理浓度的提高而急剧增加;随着处理浓度的增加,ＳＯＤ、ＣＡＴ和ＰＯＤ的活性峰出现的时间不断后推,并且ＰＯＤ的活性     

锌离子对氨基酰化酶构象及其稳定性的影响

天然氨基酰化酶和脱谷氨基酰化酶无论在二级结构（用ＣＤ和ＦＴＩＲ监测）还是三级结构上（以荧光发射光谱监测）都有明显的差异,表明了脱锌后酶的有序度降低;当比较天然和脱锌氨基酸化酶对去圬剂的稳定性时,结果表明脱锌后酶的构象的稳定性明显降低．因此可以认为锌离子对维持酶分子活性部位的特定构象以及构象的稳定性具有重要的作用．     

脱氧雪腐镰刀菌烯醇对K~＋刺激的小麦根质膜ATP酶活性、K~＋吸收、外渗及再分配的影响

１０－８ｍｏｌ／Ｌ的ＤＯＮ毒素加入小麦根质膜制剂中可促进Ｋ＋刺激的ＡＴＰ酶活力,１０－６ｍｏｌ／Ｌ开始呈抑制效应,抑制程度随ＤＯＮ浓度加大而提高。根尖（５ｃｍ）离体根段于０．５ｍｍｏｌ／Ｌ的ＫＣｌ中,１０－８ｍｏｌ／Ｌ的ＤＯＮ能促进根段Ｋ＋吸收,１０－６ｍｏｌ／Ｌ以上浓度则Ｋ＋吸收呈抑制,１０－２ｍｏｌ／Ｌ浓度下根段的净吸收为负值,表明组织中Ｋ＋大量外渗。根段置蒸馏水中６ｈ,４ｍｍｏｌ／Ｌ的ＤＯＮ即导致振段Ｋ＋渗漏。用ＤＯＮ处理整株小麦根,浓度在０．２５ｍｍｏｌ／Ｌ以上可促进Ｋ＋从植株其它部位向根运输,而浓度在８ｍｍｏｌ／Ｌ时即抑制Ｋ＋向根富集,且根内Ｋ＋明显渗漏。     

多胺对裸大麦离体叶片活性氧代谢的影响

裸大麦离体叶片分别在光照和暗诱导下,以腐胺、亚精胺和精胺等３种多胺,分别用２ｍｍｏｌ／Ｌ,０．５ｍｍｏｌ／Ｌ,和０．２ｍｍｏｌ／Ｌ３种浓度处理,均使丙二醛累积减少,延缓过氧化氢酶和ＳＯＤ活性的下降。以ＣａＣｌ２（５ｍｍｏｌ／Ｌ）＋Ｓｐｄ（０．５ｍｍｏｌ／Ｌ）处理,可降低Ｓｐｄ（０．５ｍｍｏｌ／Ｌ）的效应,因此多胺延缓离体叶片衰老与活性氧代谢有关,并且进入细胞时,与Ｃａ发生竞争。     

无花果蛋白酶在胍溶液中的分子折叠与活力变化研究

本文研究无花果蛋白酶（ＥＣ．３．４．４．１２）在不同浓度盐酸胍溶液中分子构象与活力变化关系。酶的内源荧光光谱,圆二色光谱与酶活力的变化表明：荧光光谱呈现二个明显的变化区域,低浓度胍（低于２ｍｏｌ／Ｌ）中,荧光发射峰基本不变,但荧光强度随胍浓度上升,随胍浓度断续增大（高于２ｍｏｌ／Ｌ）,酶的最大发射波长明显红移。当胍浓度低于１ｍｏｌ／Ｌ时,不仅不会使酶失活,反而使酶激活,当胍浓度高于１ｍｏｌ／Ｌ以上时,酶逐渐失活,使酶完全失活的胍浓度为６ｍｏｌ／Ｌ酶的圆二色光谱也随着胍浓度的改变而发生复杂的变化。将荧光变化,ＣＤ谱变化及活力改变结合起来,表明活力的激活与构象的明显变化似是同步发生的,从另一角度进一步说明酶活性部位柔性是充分表现酶活力所必需。     

Inactivation and unfolding of aminoacylase during denaturation in sodium dodecyl sulfate solutions

During denaturation by sodium dodecyl sulfate (SDS), aminoacylase shows a rapid decrease in activity with increasing concentration of the detergent to reach complete inactivation at 1.0 mM SDS. The denatured minus native-enzyme difference spectrum showed two negative peaks at 287 and 295 nm. With the increase of concentration of SDS, both negative peaks increased in magnitude to reach maximal values at 5.0 mM SDS. The fluorescence emission intensity of the enzyme decreased, whereas there was no red shift of emission maximum in SDS solutions of increasing concentration. In the SDS concentration regions employed in the present study, no marked changes of secondary structure of the enzyme have been observed by following the changes in far-ultraviolet CD spectra. The inactivation of this enzyme has been followed and compared with the unfolding observed during denaturation in SDS solutions. A marked inactivation is already evident at low SDS concentration before significant conformational changes can be detected by ultraviolet absorbance and fluorescence changes. The inactivation rate constants of free enzyme and substrate-enzyme complex were determined by the kinetics method of the substrate reaction in the presence of inactivator previously described by Tsou [Tsou (1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436]. It was found that substrate protects against inactivation and at the same SDS concentrations, the inactivation rate of the free enzyme is much higher than the unfolding rate. The above results show that the active sites of metal enzyme containing Zn²⁺ are also situated in a limited and flexible region of the enzyme molecule that is more fragile to denaturants than the protein as a whole.     

Evidence for the existence of an unfolding intermediate state for aminoacylase during denaturation in guanidine solutions

The equilibrium unfolding of pig kidney aminoacylase in guanidinium chloride (GdmCl) solutions was studied by following the fluorescence and circular dichroism (CD). At low concentrations of GdmCl, less than 1.0 M, the fluorescence intensity decreased with a slight red shift of the emission maximum (from 335 to 340 nm). An unfolding intermediate was observed in low concentrations of denaturant (between 1.2 and 1.6 M GdmCl). This intermediate was characterized by a decreased fluorescence emission intensity, a red-shifted emission maximum, and increased binding of the fluorescence probe 1-anilino-8-naphthalenesulfonate. No significant changes of the secondary structure were indicated by CD measurement. This conformation state is similar to a molten globule state which may exist in the pathway of protein folding. Further changes in the fluorescence properties occurred at higher concentrations of GdmCl, more than 1.6 M, with a decrease in emission intensity and a significant red shift of the emission maximum from 340 to 354 nm. In this stage, the secondary structure was completely broken. A study of apo-enzyme (Zn2+-free enzyme) produced similar results. However, comparison of the changes of the fluorescence emission spectra of native (Holo-) enzyme with Zn2+-free (Apo-) enzyme at low GdmCl concentrations showed that the structure of the Holo-enzyme was more stable than that of the Apo-enzyme.     

Comparison of the rates of inactivation and conformational changes of creatine kinase during urea denaturation

The denaturation of creatine kinase in urea solutions of different concentrations has been studied by following the changes in the ultraviolet absorbance and intrinsic fluorescence as well as by the exposure of hidden SH groups. In concentrated urea solutions, the denaturation of the enzyme results in negative peaks at 285 nm with shoulders at 280 and 290 nm and positive peaks at 244 and 302 nm in the denatured minus native enzyme difference spectrum. The fluorescence emission maximum of the enzyme red shifts with increasing intensity in urea solutions of increasing concentrations. At least part of these changes can be attributed to direct effects of urea on the exposed Tyr and Trp residues as shown by experiments with model compounds. The inactivation of this enzyme has been followed and compared with the conformational changes observed during urea denaturation. A marked decrease in enzyme activity is already evident at low urea concentrations before significant conformational changes can be detected by the exposure of hidden SH groups or by ultraviolet absorbance and fluorescence changes. At higher urea concentrations, the enzyme is inactivated at rates 3 orders of magnitude faster than the rates of conformational changes. The above results are in accord with those reported previously for guanidine denaturation of this enzyme [Yao, Q., Hou, L., Zhou, H., & Tsou, C.-L. (1982) Sci. Sin. (Engl. Ed.) 25, 1186-1193] and can best be explained by assuming that the active site of this enzyme is situated near the surface of the enzyme molecule and is sensitive to very slight conformational changes.     

The guanidine like effects of arginine on aminoacylase and salt-induced molten globule state

Aminoacylase is a dimeric enzyme containing one Zn(2+) ion per subunit. The arginine (Arg)-induced unfolding of Holo-aminoacylase and Apo-aminoacylase has been studied by measurement of enzyme activity, fluorescence emission spectra and 1-anilino-8-naphthalenesulfonate (ANS) fluorescence spectra. Besides being the most alkaline amino acid, the arginine molecule contains a positively charged guanidine group, similar to guanidine hydrochloride, and has been used in many refolding systems to suppress protein aggregation. Our results showed that arginine caused the inactivation and unfolding of aminoacylase, with no aggregation during denaturation. A comparison between the unfolding of aminoacylase in aqueous and HCl (pH 7.5) arginine solutions indicated that the guanidine group of arginine had protein-denaturing effects similar to those of guanidine hydrochloride, which might help us understand the mechanism by which arginine suppresses incorrect refolding. The results showed that arginine-denatured aminoacylase could be reactivated and refolded correctly, indicating that arginine is as good a denaturant as the guanidine or urea for study of protein unfolding and refolding. Both the intrinsic fluorescence and the ANS fluorescence spectra showed that the arginine-unfolded aminoacylase formed a molten globule state in the presence of KCl, suggesting that intermediates exist during aminoacylase refolding. The results for the Apo-aminoacylase followed were similar to those for the Holo-enzyme, suggesting that Holo- and Apo-aminoacylase might have a similar unfolding and refolding pathway.     

Trichloroacetic acid-induced molten globule state of aminoacylase from pig kidney

The trichloroacetic acid (TCA)-induced unfolding of aminoacylase was investigated by measurement of aggregation, enzyme activity, intrinsic fluorescence, 8-anilino-1-naphthalene sulfonate (ANS) binding, circular dichroism, and native polyacrylamide gel electrophoresis. The results showed that TCA caused inactivation and unfolding of aminoacylase. Intrinsic fluorescence results demonstrated that the TCA-induced transition of aminoacylase was characterized by two distinct stages during which the fluorescence emission maxima first redshifted to 338 nm and then blueshifted to 332 nm, close to that of native protein. ANS binding measurements revealed that TCA-denatured aminoacylase had a large hydrophobic area for TCA concentration near 2 mM. Comparison of the relative changes in wavelength shift and in the ANS intensity suggested the formation of a stable molten globule state of aminoacylase with a slightly disrupted tertiary structure and more hydrophobic surface than the native protein. Far-UV circular dichroism results provided further support that TCA induced the formation of two partially folded intermediates each with an enhanced native-like secondary structure. The results collectively suggest that a TCA-induced molten globule state is formed and stabilized during unfolding of aminoacylase and that association of the molten globule state may account for precipitation of the protein when denatured by TCA.     

Refolding intermediate of guanidine hydrochloride denatured aminoacylase

The refolding of aminoacylase denatured in 6M guanidine hydrochloride (GdnHCl) has been studied by measuring enzyme activity, fluorescence emission spectra, ANS fluorescence spectra and far-UV circular dichroism spectra. The results showed that GdnHCl-denatured aminoacylase could be refolded and reactivated by dilution. A refolding intermediate was observed for low concentrations of GdnHCl (between 0.5 and 1.2M). This refolding intermediate was characterized by an increased fluorescence emission intensity, a blue-shifted emission maximum, and by increased binding of the fluorescence probe 8-anilino-1-naphthalenesulfonate (ANS). The secondary structure of the intermediate was similar to that of the native enzyme, and was therefore quite similar to the molten globule state often found in the protein folding pathway. Combined with the previous evidence of existence of an intermediate during unfolding process, we therefore proposed that the unfolding and refolding of aminoacylase might share the same pathway. A comparison of the Apo-enzyme and Holo-enzyme showed that there was little effect of the zinc ion on the refolding of the aminoacylase. Our study, the first successful report of the refolding of this metalloenzyme, also showed that lowering the concentration and the temperature of the enzyme improved the refolding rate of aminoacylase. The system therefore provides a useful model to study the refolding of proteins with prosthetic groups.     

The unfolding intermediate state of calf intestinal alkaline phosphatase during denaturation in guanidine solutions

The equilibrium unfolding of calf intestinal alkaline phosphatase in guanidinium chloride (GdmCl) solutions was studied by following the fluorescence and ultraviolet difference spectra. At low concentrations of GdmCl (< 1.6 M), the fluorescence intensity decreased with a slight red shift of the emission maximum from 332 nm to 344 nm. An unfolding intermediate state was observed at a broad concentration range of GdmCl as a denaturant (between 1.6 and 2.6 M). This intermediate was characterized by increased fluorescence emission intensity, ultraviolet difference absorption at 236 nm and 260 nm, as well as increased binding to the protein and red shift of the fluorescence probe 1-anilinonaphthalene-8-sulfonic acid.     

Aspartate-induced aminoacylase folding and forming of molten globule

Aspartate is an osmolyte found in some marine invertebrates and cyclostome fish. The aspartate-induced unfolding of N-acylamino acid amido hydrolase (aminoacylase) has been studied by measuring enzyme activity, fluorescence emission spectra, 8-anilino-1-naphthalenesulfonate (ANS) fluorescence spectra and far-UV circular dichroism (CD) spectra. The results showed that aspartate caused the inactivation and unfolding of aminoacylase. Surprisingly, increasing concentration of aspartate showed the "acid-induced folding", which used to be seen only in strong acids or salts at much lower pH. Although aspartate has the pI of 2.77 that is the lowest among all the free amino acids, it is actually a weak acid. It is thus of great interest why it causes this phenomenon to happen. The relative change of intrinsic fluorescence and ANS binding spectra have shown that there existed a stable molten globule state of aminoacylase with slightly disrupted tertiary structure and more hydrophobic surface. The molten globule state indicates that intermediates existed during aminoacylase refolding process. Unlike the other acids, such as trichloroacetic acid, there is no precipitation observed as the aspartate concentrations increased. It suggests the aspartate anions have an osmotic effect for the molten globule formed during unfolding process. Binding of aspartate anion to the protonated protein, which minimizes the intramolecular repulsion, might explain the osmotic effect of this amino acid in the nature. The results also showed the Apo-aminoacylase followed similar rules as Holo-enzyme, which suggested the zinc ion may play more important roles on activity other than structure.     

Activity change during unfolding of bovine pancreatic ribonuclease A in guanidine

Bovine pancreatic ribonuclease A loses almost completely its activity in 2-3 M guanidine, whereas only very slight conformational changes can be detected when following its unfolding by changes in its intrinsic fluorescence at 305 nm and ultraviolet absorbance at 287 nm. Reactivation on diluting out the denaturant is a time-dependent process, indicating that the inactivation is not due to inhibition by a reversible association of the enzyme with guanidine. The kinetic method of following the substrate reaction, in the presence of the denaturant previously proposed for use in the study of rapid inactivation reactions (Tian, W.X. and Tsou, C.-L. (1982) Biochemistry 21, 1028-1032), is applied to examine the inactivation rates of this enzyme during guanidine denaturation, and these have been compared with the unfolding rates as followed by fluorescence and absorbance changes. It is shown that during the unfolding of this enzyme in guanidine, the inactivation of the enzyme occurs within the dead time of mixing in a stopped-flow apparatus and is at least several orders of magnitude faster than the unfolding reaction as detected by the optical parameters. It appears that, as in the case of creatine kinase reported previously, the active site of a small enzyme stabilized by multiple disulfide linkages, such as ribonuclease A, is also situated in a region which is much more liable to being perturbed by denaturants than is the molecule as a whole.     

Effect of methanol on the activity and conformation of acid phosphatase from the prawn Penaeus penicillatus

Prawn (Penaeus penicillatus) acid phosphatase (EC 3.1.3.2) catalyzes the nonspecific hydrolysis of phosphate monoesters. The effects of some pollutants in sea water on the enzyme activity results in the loss of the biological function of the enzyme, which leads to disruption of phosphate metabolism in cells. This paper analyzes the effects of methanol on the activity and conformation of prawn acid phosphatase. The results show that low concentrations of methanol can lead to reversible inactivation. Inhibition of the enzyme by methanol is classified as non-competitive inhibition, and the inhibition constant (Ki) is 8.5%. Conformational changes of the enzyme molecule in methanol solutions of different concentrations were measured using fluorescence emission, differential UV-absorption, and circular dichroism spectra. Increased methanol concentrations caused the fluorescence emission intensity of the enzyme to increase. The ultraviolet difference spectra of the enzyme denatured with methanol had two negative peaks, at 222 and 270 nm, and a positive peak at 236 nm. The changes in the fluorescence and ultraviolet difference spectra reflected the changes of the microenvironments of tryptophan and tyrosine residues of the enzyme. The CD spectrum changes of the enzyme show that the secondary structure of the enzyme also changed some. These results suggest that methanol is a non-competitive inhibitor and the conformational integrity of the enzyme is essential for its activity.