测定麦芽糖转糖苷反应体系组成的高效液相色谱法

建立了分析低聚异麦芽糖组成的高效液相色谱法，采用Spherisorb-NH2色谱柱，示差折光检测器，乙腈－水（体积比70：30）为流动相，外标法定量测定；结果显示各糖质量浓度在0．1－10g/L范围内与峰面积呈良好线性关系，相关系数为0．999 0－0．999 7；应用法跟踪了pH5．0的柠檬酸－柠檬酸钠缓冲溶液中以α－葡萄糖转苷酶为催化剂在58℃温度下的麦芽糖转糖苷反应，分析了反应体系组成随时间的变化，得到了上述反应条件下麦芽糖最大限度地转化为低聚异麦芽糖的最佳反应时间为24h；该法快捷、简单、准确，可用于低聚异麦芽糖生产的质量控制。     

超两亲分子在药物转运体系构筑中的应用

由超分子两亲化合物自组装形成的具有刺激响应性的纳米药物转运体系由于其能够有效提高药物的生物利用率、延长药物在血液中的循环和滞留时间、增加体系的稳定性等优点,近年来在以恶性肿瘤为代表的疾病的治疗和研究领域倍受关注.针对几类大环化合物的结构特征,主要概述了近年来基于其构筑的超分子两亲体在智能药物转运体系中的应用概况,分析了现阶段超分子纳米药物转运体系的优缺点,并指出其未来发展面临的机遇与挑战.     

铜/镍金属伴侣蛋白的研究进展

含铜/镍金属酶的成熟需要一系列的铜/镍金属伴侣蛋白,这些铜/镍金属伴侣蛋白分别参与铜或者镍的转运,对维持细胞体内铜/镍金属平衡至关重要,同时金属酶完成金属催化活性中心的组装也依赖于这类伴侣蛋白。近年来关于铜/镍金属蛋白的研究取得可喜的进展,这些研究为进一步认识体内铜/镍平衡体系提供了重要依据。本文首先简要地介绍铜的摄取和细胞内平衡体系,接着着重介绍三个重要的铜转运蛋白Atox1、Cox17和CCS关于结构和功能的进展,以及这些铜转运蛋白和药物相互作用的机理。然后详细介绍在氢化酶和脲酶成熟路径中参与了镍的摄取、调节、转运和存储,维持细胞内镍金属平衡的镍伴侣蛋白,并介绍了脲酶、氢化酶这两条成熟路径之间的联系。     

关于饴糖中还原糖的分析方法问题

目前,生产饴糖的工厂对产品中还原糖的分析方法不一致。在使用斐林氏法的过程中,好多单位在计算结果时采用的是葡萄糖的系数(当量),致使产品对比和技术交流没有统一的标准。本人意见认为,饴糖中含的还原糖实质上是麦芽糖,所以采用麦芽糖的系数(当量)来计算比较切合实际。然而,直接用分析试剂麦芽糖来标定斐林氏溶液还需先行水解,这样既麻烦又会造成较大误差,这是因为麦芽糖由二个分子的葡萄糖组成,其中只有一个分子的葡萄搪具有自由的羰基能与斐林氏     

麦芽糖作选择剂西酞普兰手性毛细管电泳分析及拆分机理研究

研究了以麦芽糖为选择剂的毛细管电泳手性拆分方法。以抗抑郁药物西酞普兰对映体的分离和定量测定为实例,考察了分离条件,在40%(m/m)麦芽糖、8.0×10-2mol/L磷酸盐运行缓冲液(pH5.0)中,分离电压20kV时,西酞普兰对映体分离度达2.3。测定S-( )-西酞普兰中R-(-)异构体的含量,在0.05~4.00g/L浓度范围内线性关系良好。R-(-)-西酞普兰与S-( )-西酞普兰的检出限分别为0.0453g/L和0·0473g/L,线性相关系数均>0.9978。以荧光光谱法对西酞普兰与麦芽糖的相互作用进行了考察,并较系统地对拆分机理进行了研究。证明麦芽糖的手性识别能力与其浓度有关,当麦芽糖达到一定浓度后将形成聚集体,而麦芽糖的拆分作用就主要体现在其聚集体疏水空腔的立体作用上。     

meso-四(4-羧基苯基)卟啉及金属配合物的合成和对糖类的选择识别

用微波辐射和溶剂热方法合成了meso-四(4-羧基苯基)卟啉(TCPP)及其锌配合物,分别采用紫外-可见光谱滴定法和荧光光谱法测定了目标化合物对5种糖类化合物(D-葡萄糖、D-果糖、蔗糖、麦芽糖和乳糖)的分子识别作用.结果表明,两种卟啉化合物对麦芽糖有较好的识别作用,用紫外-可见光谱方法计算出TCPP和ZnTCPP与麦芽糖分别形成1∶2和1∶1的络合物,结合常数分别为8.1×107和8.1×103L·mol-1.     

meso-四(对-磺酸基苯基)卟啉对麦芽糖的分子识别

ｍｅｓｏ－四（对－磺酸基苯基）卟啉对麦芽糖的分子识别高爽,王杏乔,于连香,曹锡章（吉林大学化学系,长春,１３００２３）关键词卟啉,麦芽糖,分子识别分子识别在生物和生命活动中起着重要作用。近年来,以卟啉及其配合物为接受体对生物分子的人工分子识别研究日趋...     

以十二烷基麦芽糖为模板合成导电聚苯胺纳米线

以绿色表面活性剂十二烷基麦芽糖为软模板,在不同pH值条件下合成了导电聚苯胺纳米线,研究了不同酸度对所得材料电导率的影响,发现电导率随酸度的增加成上升趋势.这是具有良好生物兼容性十二烷基麦芽糖作为表面活性剂首次用于导电聚合物纳米材料的合成.     

金属巯蛋白与人体内铜锌元素的代谢

金属巯蛋白是一类富含金属的化学结构极为特殊的内源性蛋白。它构成机体内金属元素的新陈代谢中快速应答的缓冲体系。尤其在铜、锌两种元素的吸收、转运、排泄方面发挥着重要作用。     

红参加工过程中梅拉德初级反应产物的研究

利用电喷雾质谱技术分析红参水层提取物,发现有不同于鲜参的成分,经进一步分析证明在红参加工过程中鲜参中的麦芽糖和氨基酸发生了梅拉德反应,生成了新的氨基酸衍生物.通过产物的模拟合成,电喷雾串联质谱以及傅立叶变换离子回旋共振质谱分析,对这几种成分进行了深入研究,鉴定出它们是麦芽糖分别与精氨酸,谷氨酸和天冬氨酸反应生成的双糖苷,是红参在加工过程中产生的特有成分.     

Visualization of interactions between siderophore transporters and the energizing protein TonB by native PAGE

Horizontal nondenaturing electrophoresis of proteins in polyacrylamide gels was used to observe specific interactions between membrane proteins. The method was particularly well suited for solubilized transporters of the outer membrane of Gram-negative bacteria, and allowed specific complexes of transporter and the inner-membrane protein TonB to be isolated. We have used this method to investigate the interactions between four different outer-membrane transporters, and the TonB proteins from two different organisms. The results show that a stable complex can be isolated on gels for all the proteins studied, but can depend in some cases of the detergent used for solubilization. Furthermore, we observe cross-species interaction as TonB from a given organism can interact with transporters from another organism.     

基于时间分辨方法的LicT蛋白荧光动力学特性

使用时间分辨荧光方法,结合紫外吸收光谱和稳态荧光光谱技术,测量了LicT蛋白中色氨酸残基的荧光动力学特性,进而对LicT蛋白质激活前后的局部微环境和结构变化进行了研究。LicT蛋白质的激活态使得有关糖类利用的基因转录过程继续进行,促进机体新陈代谢。通过色氨酸残基的荧光发射和寿命的差异判断出激活型蛋白AC 141和野生型蛋白Q 22不同的结构性质和微环境差异。在此基础上,通过衰减相关光谱(DAS)和时间分辨发射光谱(TRES)阐释了两种蛋白色氨酸残基和溶剂的相互作用,说明了激活型AC 141的比野生型Q 22的结构更加紧密。此外,TRES还说明了蛋白中的色氨酸残基存在连续光谱弛豫过程。各向异性结果则对残基和整个蛋白的构象运动进行了阐述,说明了色氨酸残基在蛋白质体系内有独立的局部运动,且在激活型蛋白中该运动更加强烈。     

Design, synthesis, and delivery properties of novel guanidine-containing molecular transporters built on dimeric inositol scaffolds

We have developed a novel class of synthetic molecular transporters that contain eight residues of guanidine with an inositol dimer as the scaffold. The dimers were prepared by connecting two units of myo- or scyllo-inositol via a carbonate or amide linkage, and the multiple units of the guanidine functionality were constructed on the inositol scaffold by means of peracylation with omega-aminocarboxylate derivatives of varying length. Bioassays based on confocal laser scanning microscopy and fluorescence-activated cell sorter analyses indicated that these transporters display a varying degree of membrane translocating ability, and the intracellular localization and mouse-tissue distribution studies strongly suggested that these transporters undergo substantially different mechanistic processes from those of peptide transporters reported to date. It was also shown that doxorubicin, an anticancer antibiotic, can be efficiently delivered into mouse brain by aid of this type of transporter.     

Development of an ATP force field for coarse-grained simulation of ATPases and its application to the maltose transporter

The biological functions of ATPases, such as myosin, kinesin, and ABC transporter, are due to large conformational motions driven by energy obtained from ATP. Elucidation of the mechanisms underlying these ATP-driven movements is one of the greatest challenges in computational chemistry. It has been shown that the MARTINI coarse-grained method is a promising tool for the investigation of large conformational motions in various proteins. However, this method has not yet been applied to ATPases because of the lack of a force field for the ATP molecule. Here, we developed force field parameters for the ATP molecule and conducted simulations using these parameters for the subunits (MalK₂) and the full-length structure (MalFGK₂-E) of a maltose transporter. It was found for both targets that the dimerization of the nucleotide binding domains (NBDs) is induced upon ATP binding. Moreover, for the full-length transporter, the conformational transition from the pre-translocation state to the outward-facing state was observed and was accompanied by an initial transport motion of the substrate. It is expected that coarse-grained simulations utilizing the parameters for the ATP molecule developed here will serve as a powerful tool for investigating other ATPases as well. © 2019 Wiley Periodicals, Inc.     

Dynamics of large proteins through hierarchical levels of coarse-grained structures

Elastic network models have been successful in elucidating the largest scale collective motions of proteins. These models are based on a set of highly coupled springs, where only the close neighboring amino acids interact, without any residue specificity. Our objective here is to determine whether the equivalent cooperative motions can be obtained upon further coarse-graining of the protein structure along the backbone. The influenza virus hemagglutinin A (HA), composed of N = 1509 residues, is utilized for this analysis. Elastic network model calculations are performed for coarse-grained HA structures containing only N/2, N/10, N/20, and N/40 residues along the backbone. High correlations (>0.95) between residue fluctuations are obtained for the first dominant (slowest) mode of motion between the original model and the coarse-grained models. In the case of coarse-graining by a factor of 1/40, the slowest mode shape for HA is reconstructed for all residues by successively selecting different subsets of residues, shifting one residue at a time. The correlation for this reconstructed first mode shape with the original all-residue case is 0.73, while the computational time is reduced by about three orders of magnitude. The reduction in computational time will be much more significant for larger targeted structures. Thus, the dominant motions of protein structures are robust enough to be captured at extremely high levels of coarse-graining. And more importantly, the dynamics of extremely large complexes are now accessible with this new methodology.     

Single-molecule motions of oligoarginine transporter conjugates on the plasma membrane of Chinese hamster ovary cells

To explore the real-time dynamic behavior of molecular transporters of the cell-penetrating-peptide (CPP) type on a biological membrane, single fluorescently labeled oligoarginine conjugates were imaged interacting with the plasma membrane of Chinese hamster ovary (CHO) cells. The diffusional motion on the membrane, characterized by single-molecule diffusion coefficient and residence time (tau R), defined as the time from the initial appearance of a single-molecule spot on the membrane (from the solution) to the time the single molecule disappears from the imaging focal plane, was observed for a fluorophore-labeled octaarginine (a model guanidinium-rich CPP) and compared with the corresponding values observed for a tetraarginine conjugate (negative control), a lipid analogue, and a fluorescently labeled protein conjugate (transferrin-Alexa594) known to enter the cell through endocytosis. Imaging of the oligoarginine conjugates was enabled by the use of a new high-contrast fluorophore in the dicyanomethylenedihydrofuran family, which brightens upon interaction with the membrane at normal oxygen concentrations. Taken as a whole, the motions of the octaarginine conjugate single molecules are highly heterogeneous and cannot be described as Brownian motion with a single diffusion coefficient. The observed behavior is also different from that of lipids, known to penetrate cellular membranes through passive diffusion, conventionally involving lateral diffusion followed by membrane bilayer flip-flop. Furthermore, while the octaarginine conjugate behavior shares some common features with transferrin uptake (endocytotic) processes, the two systems also exhibit dissimilar traits when diffusional motions and residence times of single constructs are compared. Additionally, pretreatment of cells with cytochalasin D, a known actin filament disruptor, produces no significant effect, which further rules out unimodal endocytosis as the mechanism of uptake. Also, the involvement of membrane potential in octaarginine-membrane interaction is supported by significant changes in the motion with high [K(+)] treatment. In sum, this first study of single transporter motion on the membrane of a living cell indicates that the mode by which the octaarginine transporter penetrates the cell membrane appears to either be a multimechanism uptake process or a mechanism different from unimodal passive diffusion or endocytosis.     

Coordination of Na(+) by monoamine ligands in dopamine, norepinephrine, and serotonin transporters

The reuptake of neurotransmitters by dopamine, norepinephrine, and serotonin transporters during neuronal transmission requires a sodium gradient. An "ionic mode" of binding proposes that aspartate anchors the ligand's positive charge but ignores the direct role of sodium in ligand binding seen in the only representative structure, the prokaryotic leucine transporter LeuT. Here, we built structural models of human transporters of dopamine, norepinephrine, and serotonin using the LeuT structure. The ligand and sodium-binding sites are highly conserved. We examined the possibilities for ligand binding given the available experimental evidence, including examples of catechol-cation chelates in X-ray structures of protein and other complexes. We conclude that a "chelation mode" of binding with direct interaction between the catechol hydroxyls and sodium is a valid alternative, with consequences for pharmaceutical design. In the modeled serotonin transporter complexes, Y95 is placed where it could select for serotonin through hydrogen bonding to the indole nitrogen.