Evidence for the Existence of Cross-Linked Intermediates during Unfolding and Refolding of CK in UGGE

IntroductionCreatine kinase ( CK) plays an important role incell energy metabolism[1] .It catalyses thereversible transfer of a phosphoryl group fromATP to creatine generating phosphocreatine andADP.Owing to its importance in bioenergetics,CK has been extensively studied.There are fourisoforms of the enzyme:muscle type( MM) ,braintype ( BB) ,hybrid type ( MB) ,and mitochondriatype( Mi Mi) .Muscle- CK is a dimer composed of twoidentical subunits,both with relative molecularmass of4.3×…     

大熊猫肌酸激酶热变性中活性与构象变化的比较

比较了大熊猫肌酸激酶在各种不同温度下热变性时，活性变化与构象变化的速度常数。结果表明在较低温度下，酶的失活速度与构象变化速度相当．而在较高温度下变性时酶的失活速度快于构象变化速度。这表明酶分子的活性部位的微区构象比整个分子的构象具有更大的易变性或柔性。     

大熊猫肌酸激酶热变性时活性与构象变化的比较

比较了热变性时大熊猫肌酸激酶的构象与活性的变化，结果表明：酶在４０℃ 及 ４０℃以下温度保温 １０ ｍｉｎ，酶的活性及构象均无明显改变； ４０℃以上酶的活性及构 象发生改变，在４５～５５℃之间，酶的活性与构象发生急剧变化，５５℃时酶活性已完全 丧失，用ＣＤ、荧光及表面流基暴露等手段监测的酶分子的构象也发生很大变化，但尚未 完全，随温度的上升继续发生变化。上述结果表明：酶活性部位的微区构象比较脆弱或 比较柔性，并且酶分子的空间结构是酶具有的活性的基础。     

乳酸对肌酸激酶活性抑制作用的研究

应用酶活测定的方法分析了肌酸激酶在不同浓度乳酸中的活性变化．实验结果说明在失活过程中没有蛋白质的聚沉；肌酸激酶的失活遵循一级反应的一相过程；失活与乳酸浓度和作用时间呈正相关．通过对比实验结果显示，乳酸是通过其解离状态的H^ 改变了机体PH值而影响肌酸激酶活性的，并且就乳酸对肌酸激酶影响及其意义进行探讨．     

磁场对肌酸激酶的影响

建立了流动注射和光纤光度法测定肌酸激酶（Ｃｋ）活性的方法。求测了Ｃｋ催化反应的米氏常数ｋｍ和反应活化能Ｅ,研究了磁场对Ｃｋ催化活性的影响。较长时间磁化Ｃｋ溶液和测活溶液,分别可使Ｃｋ加速失活和活性升高,１００ｍＴ磁场磁化处理测活液１５ｈ可使Ｃｋ催化反应速度增加４３．７％。     

Characterization of the Partially Folded Monomeric Intermediate of Creatine Kinase

IntroductionRecent studies of the protein folding pathway andintermediate states in vitro and in vivo haveinduced much interest in the importance ofunderstanding the propertiesof partially structuredintermediates[1 7] . Studies have suggested thatintermed…     

大熊猫肌肉肌酸激酶的纯化与性质

用改进的Ｋｕｂｙ等人方法从大熊猫骨骼肌中纯化了肌酸激酶，并对此酶的某些性质进行了研究。纯化倍数为 ３５．７；收率１７．３％；比活力为 ５５．３Ｕ／ｍｇ；等电点为 ６．６０。聚丙烯酰胺凝胶电泳鉴定为一条带。ＳＤＳ聚丙烯酰胺凝胶电泳测得其亚基分子量为４２ ０００，总分子量为 ８４ ０００，该酶是由两个相同亚基组成的二聚体。酶对肌酸的Ｋｍ 为 ５．２６ｍｍｏｌ／Ｌ；对ＡＴＰ的 Ｋｍ为 ０．７４ｍｍｏｌ／Ｌ，酶的最适温度是 ３０～３６℃；酶的最适ｐＨ大于８．０。     

Debaryomyces sp.甘油激酶基本动力学性质研究

通过平板初筛和摇瓶复筛,从105株菌中筛得一株甘油激酶的高产菌株,并研究了其所产甘油激酶的动力学性质.研究结果表明:在MES系统中,甘油激酶有较好的酸碱稳定性,pH稳定范围为5.5～10.5;最适pH和最适温度分别为pH 8.0和50℃,该酶催化甘油的米氏常数(Km)为3.5×10-5mol/L,催化ATP的Km为4.46×10-4mol/L.同时对一些金属离子和有机化合物对酶活性的影响也进行了研究.     

添加剂促进变性还原溶菌酶复性的作用

研究了多种添加剂促进变性还原溶菌酶复性的作用，考察了添加剂浓度及变性剂盐酸胍浓度对复性收率的影响．结果表明，精氨酸、乙酰胺、丙酮、硫脲及甘油均能有效促进变性溶菌酶复性，并且存在最佳的添加剂浓度使变性溶菌酶的复性收率最大．在促进复性中，乙酰胺等结构类似物与盐酸胍具有相同作用，因此，在降低复性液中盐酸胍浓度的同时，适当提高乙酰胺浓度即可获得较高的复性收率．当盐酸胍浓度为0．2mol／L时，复性收率达到90％时的乙酰胺浓度为2mol／L，但降低盐酸胍浓度至0．06mol／L时，达到相同变性收率的乙酰胺浓度需要4mol／L．甘油与盐酸胍存在着协同作用，在一定浓度的盐酸胍存在下，添加适量的甘油能获得较高的复性收率．     

血清磷酸肌酸激酶BB的薄层等电聚焦分析法

通过对载体两性电解度,电极液,电泳功率和时间及加样方法,灵敏度等影响因素比较分析,对薄层等电聚焦血清磷酸肌酸激酶－ＢＢ的实验方法进行了探讨,并在此基础上对脑缺血病变时ＣＫ－ＢＢ亚型变化进行了初探。     

大鼠离体心脏缺灌和再灌早期乳酸脱氢酶和肌酸激酶的释放规律

用Ｌａｎｇｅｎｄｏｅｆｆ法灌流雄性Ｗｉｓｔａｒ大鼠心脏，平衡１０ｍｉｎ，缺灌１０ｍｉｎ，于再灌３ｍｉｎ内每隔１５ｓｅｃ收集一次冠脉流份，连续检测冠脉流份中ＣＫ、ＬＤＨ以及ＧＯＴ的活性（Ｕ／Ｌ），比较它们释放的动态特征。结果表明，再灌３ｍｉｎ内，３种酶都呈双相释放。第１峰出现在再灌１５ｓｅｃ，酶活性为对照值的５一９倍，它主要反映缺灌损伤。再灌９０一１２０ｓｅｃ又出现第Ⅱ峰，主要代表再灌损伤。无论是再灌３ｍｉｎ内酶释放总量，还是第Ⅰ，第Ⅱ峰的峰值，依酶活性高低排列的次序都是ＣＫ＞ＬＤＨ＞ＧＯＴ。此外，在部分动物身上进行了反复两次缺灌再灌实验，结果发现，第二次缺灌后ＣＫ释放量明显高于第一次缺灌值。ＬＤＨ的Ⅰ峰和Ⅱ峰出现时间与第一次无差别，只是３分钟内酶释放总量高于第一次。说明在本实验条件下，未见预缺灌处理对随后再次缺灌有保护作用。缺灌期心率逐渐减慢，收缩力逐渐减弱，但多数标本仍有微弱的收缩。这使缺灌期冠脉流份的酶活测定成为可能。实验发现，早在缺灌１ｍｉｎ时心肌细胞释放的酶，其活性可达对照值的２倍（ｐ＜０．０５），至缺灌１０ｍｉｎ时ＣＫ和ＬＤＨ的值可达对照值的１０倍左右。这一现象进一步证实，再灌１５ｓｅｃ内大量酶的     

Unfolding and Inactivation of Green Crab ( Scylla Serrata) Alkaline Phosphatase during Thermal Denaturation *

ＩｎｔｒｏｄｕｃｔｉｏｎＴｈａｔｉｎａｃｔｉｖａｔｉｏｎｏｃｃｕｒｓｂｅｆｏｒｅｎｏｔｉｃｅａｂｌｅｃｏｎｆｏｒｍａｔｉｏｎａｌｃｈａｎｇｅｓｃａｎｂｅｄｅｔｅｃｔｅｄｄｕｒｉｎｇｄｅｎａｔｕｒａｔｉｏｎｏｆｃｒｅａｔｉｎｅｋｉｎａｓｅａｎｄｏｔｈｅ...     

Unfolding of C2A Domain of Synaptotagmin I in the Presence of Guanidine Hydrochloride

The C2 domain originally referred to the second of four constant structural motifs in protein kinase C (PKC). Now this domain represents a large structural family sharing a homologous dimensional structure in many proteins that play important roles in many organisms. The C2A domain is one of the two C2 domains of synaptotagmin I involved in the Ca^2 regulation of exocytosis. This domain is mostly composed of β-sheet except for a small fraction of α-helix, and therefore provides an ideal model for a protein folding study. In this report, the unfolding equilibrium of the C2A domain in guanidine hydrochloride (GdnHCI) containing solutions has been studied using ultraviolet (UV) difference spectrum, fluorescence spectrum, size exclusion chromatography (SEC), and circular dichroism (CD) spectrum. The results suggest that unfolding of the C2A domain occurs as a two-state process during GdnHCI titration. By examining the changes of both tertiary structure and secondary structure, no intermediates could be detected during this unfolding study. However, it has been found that the native state of the C2A domain has a large hydrophobic surface. This result suggests that as a fragment of a protein, the C2A domain itself may exist in a state with large hydrophobic surface. This hydrophobic surface may be the molecular basis for interaction between domains in the whole protein.Furthermore, the hydrophobic behavior may play a role during the oligomerization of svnaptotagmin.     

Activity and Structure Changes of Arginine Kinase from Shrimp Feneropenaeus chinensis Muscle in Trifluoroethanol Solutions

Trifluoroethanol has often been used in protein folding studies. The changes in activity and unfolding of arginine kinase from shrimp Feneropenaeus Ghinensis muscle during denaturation in different concentrations of trifuoroethanol were investigated using far-ultraviolet circular dichroism and fluorescence emission spectra. Arginine kinase was inactivated in trifluoroethanol solutions. The tertiary and secondary structures of arginine kinase were also destroyed in the trifluoroethanol solutions. The unfolding and inactivation courses were measured and compared. Inactivation occurred prior to unfolding, which suggests that the arginine kinase active site is more easily damaged by the denaturant than the enzyme as a whole. The result also indicates that the arginine kinase active site is situated in a limited and flexible region of the enzymemolecule.     

利多卡因和碘甘油治疗RAU的临床疗效观察

目的:评价利多卡因,碘甘油联合治疗复发性阿弗他溃疡(RAU)的疗效。方法:选择到我科就诊的83例RAU患者,随机分成两组。治疗组43例用利多卡因和碘甘油联合治疗,对照组40例用复方氯己定含漱液治疗,分别在治疗后3、5、7 d复诊观察临床症状和体征,并进行评定。结果:利多卡因,碘甘油联合治疗RAU的有效率为97.7%,高于复方氯己定含漱液治疗组的有效率(57.5%),经秩和检验,差异有统计学意义(P<0.05)。结论:利多卡因,碘甘油联合治疗RAU效果良好。     

丙酮缩甘油的合成研究

以甘油和丙酮为原料,经缩酮反应制备丙酮缩甘油.探讨了原料比、反应时间、催化剂用量、带水剂的选择及用量对产率的影响.合适的反应条件为:原料甘油:丙酮=1:3(物质的量比),反应时间5 h,催化剂用量为甘油质量的6%,丙酮作为带水剂,用量为V(甘油):V(丙酮)=1:4.5,在最佳条件下,该步反应取得了85%左右的较高收率...