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.     

Extraction and DNA Digestion of 5′-Phosphodiesterase from Malt Root

This study investigated the extraction of 5＇-phosphodiesterase from malt root and the degradation of nucleic acids by this enzyme. The extraction used grade precipitation with ammonium sulfate and enzymatic hydrolysis. Samples were assayed using the modified Bradford method and high performance liquid chromatography. The results show that 5＇-phosphodiesterase is isolated by grade precipitation with 30% and 80% saturation of ammonium sulfate and can be utilized to degrade deoxyribonucleic acid. The hydrolysate has four kinds of deoxynucleotides： 5＇-dCMP, 5＇-dTMP, 5＇-dAMP, and 5＇-dGMP. The optimum reaction temperature is 70℃, and the optimum pH is 5.5-6.0 for the reaction. The percentage of deoxynucleotides indicated by the China Pharmacopoeia （2000 edition） in the product is over 70%. The extraction of 5＇-phosphodiesterase from malt root is shown to be possible and economical. Products from the enzYmatic hydrolysate of DNA meet the pharmacopoeia.     

Low Temperature Induced Conformation Changes of Aminoacylase

Introduction Aminoacylase (N-acylamino acid amido hydrolase. EC 3.5.1.14), which exists in mammalian kidneys and microorganisms, catalyzes the reversible hydrolysis of L-acylamino acids[1]. The enzyme is dimeric with a relative molecular mass of 8.6?04, containing one Zn2 ion per subunit, which is essential for enzyme activity[2,3]. The two subunits of the enzyme are identical in sequence. Although the primary structure of aminoacylase has been determined by cDNA sequencing[4,5], no X-ray…     

鲨鱼皮胶原蛋白肽的成分分析及对血管紧张素转化酶活力的影响

以鲨鱼皮为原料,采用现代生物化学技术制备胶原蛋白肽水解液,经过喷雾干燥,获得鲨鱼皮胶原蛋白肽干粉.对获得的胶原蛋白肽样品进行成分分析,测定了粗蛋白、粗脂肪、水分、灰分以及K、Na、Ca、Fe、Cu、Zn、Pb等金属元素的含量,应用氨基酸自动分析仪测定样品的氨基酸组成,并用化学分析法测定羟脯氨酸含量为7.08%,应用MALDI-TOF分析胶原蛋白肽分子量的分布,表明我们得到的鲨鱼皮胶原蛋白的水解产物胶原蛋白肽的分子量主要分布在2～3 ku,为水溶性多肽混合物.研究鲨鱼皮胶原蛋白肽对血管紧张素转化酶(ACE)的抑制效应,表明该样品对ACE有较强的抑制作用,其IC50为4.8 mg/mL.本文还探讨了鲨鱼皮胶原蛋白的吸湿性和保湿性.     

Effects of Glycerol in the Refolding and Unfolding of Creatine Kinase

IntroductionProtein folding is the process by which the aminoacid sequence of a protein determines the three-dimensional conformation of the functionalprotein[1 ] . The elucidation of the molecularmechanism of protein folding from a disorderedpolypeptide chain to the specific native stateremains one of the major challenges inbiochemistry[2 ,3] ,namely,deciphering the secondhalf of the genetic code[4 ] .Molecular chaperonesplay an importantrole in protein folding in vivo aswell asin vitro.A cha…     

Low Temperature Induced Conformation Changes of Amlnoacylase

Control of aggregation, by lowering temperature and protein concentrations, can enhance the extent of successful refolding. The low temperature has been used in protein folding studies, as undesired aggregations often occur at higher temperatures. Therefore, it is very important to study the effects of low temperature on the native enzyme to help understand the factors that affect the structure of the proteins. In this paper, aminoacylase was studied at different temperatures by measuring enzyme activity, fluorescence emission spectra, and ultraviolet difference spectra. The results show that aminoacylase conformation changes as the temperature changes, becoming more compact at low temperatures, and having more secondary structural content. However, the activity is very low at low temperature, and totally diminishes at 4℃. Aminoacylase tends therefore to be more condense, with less residues exposed and low enzyme activities at low temperature. This observation might explain the self-protection of organisms under conditions of extreme temperature.     

从猪胸腺提取胸腺素α1的工艺

为了提高猪胸腺的利用价值,探索猪胸腺素活性物质的提取工艺.选取猪胸腺为原料,采用匀浆、凝胶过滤、离子交换层析和丙酮分级沉淀的方法纯化胸腺素α1, 并用Bradford法检测蛋白含量,用SDS-PAGE电泳测定蛋白纯度.将猪胸腺组织匀浆、三次冻融和80 ℃热变性纯化后,上清采用Sephadex G-50柱填料凝胶过滤,然后进行阴离子交换树脂DEAE-C32离子交换层析和丙酮分级沉淀,通过真空冷冻干燥,最后得到胸腺素α1, 纯度为95%以上, 得率为3.84×10 -5.最终得到提取胸腺素α1的有效工艺.