Solution structure of the B-chain of insulin as determined by 1H NMR spectroscopy Comparison with the crystal structure of the insulin hexamer and with the solution structure of the insulin monomer

The solution structure of the isolated B-chain of bovine insulin has been determined by ¹H NMR spectroscopy combined with simulated annealing calculations. Complete sequence-specific assignments for the proton resonances are reported together with a set of 309 NOES used in the structure calculations. Chemical-shift variations from random coil values provide support for the existence of helical regions in the polypeptide chain, as do a characteristic series of d_αp(i,i+ 3) NOES from residues B8 to B17. While there is some evidence for a limited degree of conformational averaging over the helical region, in general the helix is relatively well defined and corresponds closely to the helical region seen in the X-ray crystal structure of the insulin hexamer. Other similarities with the crystal structure include turn-like conformations at the carboxy terminal end of the helix and extended strands at both the amino and carboxy termini of the peptide. These similarities between the crystal structure and the isolated B-chain suggest that this peptide has intrinsic folding properties, which allow it to adopt its characteristic structure in intact insulin without the need for extensive cooperative interactions with the A-chain. Despite these general similarities, an important difference between the isolated B-chain and the intact protein occurs in the carboxy terminal region. This region appears significantly more mobile in the isolated B-chain. As a conformational change involving the carboxy terminus has been implicated in receptor binding, the current study of the isolated B-chain provides valuable information on the extent of this region's intrinsic mobility. © Munksgaard 1995.     

Molecular basis of insulin action

Insulin is the main anabolic and anticatabolic hormone in mammals. The stimulatory effect of insulin on glucose uptake in muscle and adipose tissue is a consequence of the rapid translocation of GLUT4 glucose transporters from an intracellular site to the cell surface. The actions of insulin are initiated by hormone binding to its cell surface receptors. Insulin receptors are ligand-stimulated protein tyrosine kinases and phosphorylate a number of proteins, known as insulin receptor substrate proteins. Insulin resistance has been recognized as a main pathogenic factor in the development of type 2 diabetes, and has been associated with dyslipidemia, hypertension, endothelial dysfunction, inflammation and coagulative state. The current challenge is the study of impaired insulin signaling pathways leading to beta-cell dysfunction and its progression to type 2 diabetes, as well as control of chronic inflammation processes that may improve insulin action.     

Molecular perspective on tight-junction assembly and epithelial polarity

Apical-basal polarity and a highly organized actin cytoskeleton are main characteristics of epithelial cells that support exchange of ions and nutrients from one body compartment to another. The junctional complexes, localized to the apical end of the basolateral domain of the plasma membrane, are not simply epithelial barriers in paracellular transport or fences preventing diffusion of integral proteins in the plasma membrane, but also contain proteins involved in the maintenance of the physiologic epithelial cell state and signal transduction. Claudin-based tight junctions and E-cadherin-based adherens junctions have been extensively studied. Nectins, along with a unique scaffolding protein, afadin, form homophilic and heterophilic trans-dimers and play a key role in identifying cell partners in the primordial cell-cell adhesion. Nectin-based cell-cell adhesion participates in the epithelial morphogenesis, both independently and cooperatively with claudin-based tight junctions and cadherin-based adherens junctions. This review discusses how these adhesion systems interact with each other to form apical junctional complexes, and how they reorganize the actin cytoskeleton in a multistage process of cell adhesion, migration, and polarization.     

高效液相色谱法测定胰岛素及其制剂中胰岛素的效价

目的建立用高效液相色谱法测定胰岛素及其制剂的效价,并将之与已有的小鼠血糖法比较。方法色谱柱:C18柱(岛津VP-ODS,4.6 mm×150 mm,5μm);流动相:0.2 mol/L硫酸盐缓冲液(用乙醇胺调pH至2.3)-乙腈(73∶27);检测波长:214 nm;流速:1 mL/min。结果胰岛素在10~50 u/mL范围内线性关系良好,回归方程为:Y=0.001 4X+0.222 7,r=0.999 8;精密度、重现性良好。结论此法灵敏度高、专属性强、准确、可靠,较生物测定法方便,适于胰岛素及其制剂中胰岛素的效价测定。     

Molecular basis of insulin receptor function

Insulin regulates a wide spectrum of metabolic processes in a variety of tissues. In addition, insulin is a potent growth factor, promoting normal cell growth and division in cultured cells, though the physiological importance of such actions in man is less certain. Some of the effects of insulin are very rapid and apparent within seconds, for example those on transport processes and the activity of regulatory enzymes. Other effects, such as those on enzyme concentrations and cell growth, may be manifest only over a period of many minutes or even hours. Such differences in time dependence may be more a reflection of the nature of the processes themselves than an indication of different signalling mechanisms. However, it is still unclear whether the regulation of diverse metabolic processes by insulin involves just one or multiple distinct primary intracellular signals. We will review here what is at present known about the structure and activity of the receptor, and current ideas on signalling mechanisms.     

Purification and primary structure of ostrich insulin

Insulin has been isolated from ostrich pancreas by a procedure of acid ethanol extraction, adsorption onto SP-Sephadex, gel permeation chromatography and HPLC. The primary structure of the ostrich insulin is identical to that reported for the chicken hormone. The isoelectric point as determined by polyacrylamide gel isoelectric focusing was significantly higher than that of the bovine hormone.     

A new B‐chain mutant of insulin: comparison with the insulin crystal structure and role of sulfonate groups in the B‐chain structure

Abstract: The solution structure of a new B‐chain mutant of bovine insulin, in which the cysteines B7 and B19 are replaced by two serines, has been determined by circular dichroism, 2D‐NMR and molecular modeling. This structure is compared with that of the oxidized B‐chain of bovine insulin [Hawkins et al. (1995) Int. J. Peptide Protein Res. 46 , 424–433]. Circular dichroism spectroscopy showed in particular that a higher percentage of helical secondary structure for the B‐chain mutant is estimated in trifluoroethanol solution in comparison with the oxidized B‐chain. 2D‐NMR experiments confirmed, among multiple conformations, that the B‐chain mutant presents defined secondary structures such as a α‐helix between residues B9 and B19, and a β‐turn between amino acids B20 and B23 in aqueous trifluoroethanol. The 3D structures, which are consistent with NMR data and were obtained using a simulated annealing protocol, showed that the tertiary structure of the B‐chain mutant is better resolved and is more in agreement with the insulin crystal structure than the oxidized B‐chain structure described by Hawkins et al. An explanation could be the presence of two sulfonate groups in the oxidized insulin B‐chain. Either by their charges and/or their size, such chemical groups could play a destructuring effect and thus could favor peptide flexibility and conformational averaging. Thus, this study provides new insights on the folding of isolated B‐chains.     

Human insulin genome sequence map,biochemical structure of insulin for recombinant DNA insulin

Insulin is a essential molecule for type I diabetes that is marketed by very few companies. It is the first molecule, which was made by recombinant technology; but the commercialization process is very difficult. Knowledge about biochemical structure of insulin and human insulin genome sequence map is pivotal to large scale manufacturing of recombinant DNA Insulin. This paper reviews human insulin genome sequence map, the amino acid sequence of porcine insulin, crystal structure of porcine insulin, insulin monomer, aggregation surfaces of insulin, conformational variation in the insulin monomer, insulin X-ray structures for recombinant DNA technology in the synthesis of human insulin in Escherichia coli.     

Molecular connectivity: treatment of the electronic structure of amino acids.

A molecular connectivity model of atomic charges on the second-row atoms in alpha-amino acids is discussed. A correlation was found between the valence delta (delta v) index from molecular connectivity theory and the charges on atoms that belong to the same class, whereas different classes of carbon atoms had the same slopes on the Q versus sigma v matrix, where Q is the calculated charge distribution. A similar correlation was found for nucleotide bases.     

胰岛素及其类似物的研究进展

自胰岛素首次用于治疗糖尿病以来,随着对糖尿病病理生理和发病机制认识的深入,胰岛素的应用越来越广泛。综述近年来相关文献中有关胰岛素及其类似物制剂的分类、给药途径、体内吸收代谢、临床应用和不良反应等方面内容,为进一步研究、开发和合理利用胰岛素及其类似物提供参考。     

胰岛素硬脂酸纳米粒的制备与结构分析

目的：研究胰岛素硬脂酸纳米粒的结构及抗消化酶特点。方法：采用透射电镜和激光散射法测定其形态和粒径大小：荧光扫描、聚丙烯酰胺凝胶电泳和抗消化酶降解等方法研究胰岛素在硬脂酸纳米粒中的包封情况。结果：胰岛素硬脂酸纳米粒为球形，平均粒径为55．3nm，多分散性指数为0．643。荧光扫描显示胰岛素硬脂酸纳米粒中无胰岛素荧光发射峰；聚丙烯酰胺凝胶电泳图谱中未见胰岛素条带；胰岛素硬脂酸纳米粒中的胰岛素可抵抗消化酶降解。结论：胰岛素被包裹在硬脂酸纳米粒中。     

包裹在脂质体内部胰岛素的二级结构

目的以单纯胰岛素为对照，采用傅立叶红外光谱（Fourier transform infrared， FTIR）研究包裹在脂质体内部胰岛素二级结构的变化。方法分别对单纯胰岛素、胰岛素与空白脂质体混合物（样品I）及包裹胰岛素的脂质体（II）样品进行测定。结果样品I和样品II中胰岛素的FTIR谱图形状与单纯胰岛素相比基本一致，仅α-螺旋百分比略有下降（由36.09%分别下降到31.68%和31.45%），β-折叠百分比略有增加（由47.83%分别增加到53.29%和51.36%）。样品I和样品II中胰岛素的二级结构无明显差别（α-螺旋百分比为31.68%和31.45%，β-折叠百分比为53.29%和51.36%）。结论包裹在脂质体内部胰岛素的二级结构与单纯胰岛素相比无明显变化。     

Molecular engineering of conotoxins: the importance of loop size to alpha-conotoxin structure and function

Alpha-conotoxins are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). The majority of currently characterized alpha-conotoxins have a 4/7 loop size, and the major features of neuronal alpha-conotoxins include a globular disulfide connectivity and a helical structure centered around the third of their four cysteine residues. In this study, a novel "molecular pruning" approach was undertaken to define the relationship between loop size, structure, and function of alpha-conotoxins. This involved the systematic truncation of the second loop in the alpha-conotoxin [A10L]PnIA [4/7], a potent antagonist of the alpha7 nAChR. The penalty for truncation was found to be decreased conformational stability and increased susceptibility to disulfide bond scrambling. Truncation down to 4/4[A10L]PnIA maintained helicity and did not significantly reduce electrophysiological activity at alpha7 nAChRs, whereas 4/3[A10L]PnIA lost both alpha7 nAChR activity and helicity. In contrast, all truncated analogues lost approximately 100-fold affinity at the AChBP, a model protein for the extracellular domain of the nAChR. Docking simulations identified several hydrogen bonds lost upon truncation that provide an explanation for the reduced affinities observed at the alpha7 nAChR and AChBP.