还原型胰岛素A、B链相互作用的初步研究

本文报道在chance所用“一步法”重组胰岛素的类似条件下,还原型A、B链相互作用研究的初步结果。在pH10.6对S—磺酸型A及B链加入1.2倍SH/SSO_3—的二巯基苏糖醇,在一小时内A、B链与DTT的混合物较之单独的A、B链与DTT的混合物,紫外差光谱显示295、245nm负差峰,峰值随时间增大;园二色性测定观察到混合链的α螺旋含量比单独的A、B链有所增加。这表明在A、B链的巯基氧化生成二硫链前,还原型A、B链间的相互作用导致Tyr残基逐渐内埋,肽链构象的调整使有序二级结构增加。根据此时还原程度的测定,讨论了DTT与S—磺酸型A、B链作巯基交换的可能机制,过多的还原试剂易破坏已正确配对的二硫键,也妨碍肽链构象的调整。     

人胰岛素A、B链基因的合成、克隆及其在大肠杆菌中的表达

根据人胰岛素A、B链的多肽顺序,设计并用一种简便快速的多肽基因合成了A、B链基因。合成的A 链和B 链基因分别克隆到pWR590质粒上,构建了表达型质粒pWR590-HIA和pWR 590-HIB,它们能够表达由β-半乳糖苷酶N-端约590个氨基酸残基与A 链或B 链组成的融合蛋白(两者之间由Met 连接)。A 链或B 链融合蛋白经BrCN 降解,磺化及分离纯化等步骤,得到了磺化A、B 链。磺化A、B链体外重组得到人胰岛素。     

S-硫甲基型胰岛素A及B链相互作用的200MHz~1H NMR研究

前已报道,S-硫甲基型胰岛素A及B链相混后,α螺旋等有序二级结构含量增加,部分酪氨酸残基由相对亲水的环境逐渐转移到相对疏水的环境,整个过程在1.5～2小时达到稳定,说明在还原型A、B链能重组成有活力的胰岛素的相同条件下,两肽链间确实存在着次级键相互作用。本文报道以200兆赫NMR~1H谱所测S-硫甲基型A及B链的图谱,对其主要谱峰作了初步识别。在pH10.8时,A及B链以克分子比1.5:1相混后,Tyr的(2,6)位及(3,5)位质子峰较之单独链有低场位移,提示Tyr的酚羟基解离减少,Tyr转移至相对疏水的环境;也可能是肽链构象变化,使Tyr受去屏蔽效应所致。在A、B链相混后,每半小时累加的结果表明,不仅Tyr的两个低场峰随时间有低场位移,甲基峰随时间也有连续的低场位移,在1.5小时后达到稳定。此时间过程与前者报道的紫外差光谱、园二色性的观察结果一致,说明构象的调整涉及整个肽链。不同pH下的~1H谱测定显示,在一段pH区间都存在着A、B链间的次级键相互作用。上述结果表明,或许正是这种链间的次级键相互作用,才使A、B链之巯基有可能正确配对,重组成有生物活力的胰岛素分子。     

霍乱毒素B亚单位与胰岛素B链融合基因的克隆及原核表达分析

构建了霍乱毒素B亚单位(choleratoxinBsubunit,CTB)与胰岛素(insulin)B链的融合基因CTB-INSB,将该融合基因克隆到大肠杆菌表达载体pET-30a(+)中,获得重组质粒pETCIB;并将该质粒转入大肠杆菌菌株BL21(DE3)中;重组菌株经IPTG诱导后的表达产物经15％SDS-PAGE分析表明可以表达融合蛋白,其分子量约为15.4kDa,且主要以包涵体形式存在,约占全菌蛋白的30％。含CTB-INSB重组蛋白的包涵体经变性和复性后,可在体外自组装成五聚体结构。Westernblotting分析结果显示CTB-INSB可分别被霍乱毒素的抗体和胰岛素的抗体识别,表明该蛋白具有霍乱毒素B亚单位与胰岛素的双重抗原性。同时GM1-ELISA分析结果表明CTB-INSB在体外可与神经节苷脂GM1(monosialoganglioside)特异结合,进一步证实了它能够形成类似CTB五聚体的高级结构,具有生物活性。     

B27K—去B链C端三肽胰岛素的制备、生物活力与自聚合性质

液相合成五肽Gly Phe Phe Tyr Lys(Boc)Obut,与B链C端去八肽胰岛素酶促缩合得到B2 7K 去B链C端三肽胰岛素(B2 7K DTrI,B2 7K destripeptideinsulin)。用小鼠惊厥法和小鼠降血糖法测得B2 7K DTrI的整体生物活力为标准胰岛素的 80 % ,B2 7K DTrI与人胎盘细胞膜胰岛素受体结合能力为标准胰岛素的 ( 12 5± 13 ) %。用凝胶过滤法证明B2 7K DTrI自聚合性质降低 ,具有与去B链C端五肽胰岛素相同的单体性质。在B2 7K DtrI结构中 ,B2 7T被K取代 ,其优点是在酵母中表达其前体后 ,可以很方便地通过胰蛋白酶水解获得     

B链氨端去－肽（B1）羧端去五肽（B26—30）猪胰岛素的初步晶体学研究

应用悬滴气相扩散法在含６％氯化钠和１２．５％丙酮的柠檬酸缓冲体系中,获得可供Ｘ射线结构分析用的Ｂ链氨端去－肽（Ｂ１）羧端去五肽（Ｂ２６－３０）猪胰岛素单晶体。晶体属四方晶系,空间群为Ｐ４１２２或Ｐ４３２２,晶胞参数为：ａ＝ｂ＝３６．０Ａ,ｃ＝１２０．０Ａ,α＝β＝γ＝９０度。单位晶胞中每个结晶学不对称单位含有２个ＤｅｓＢ１－ＤＰＩ分子。     

蛋白质酪氨酸磷酸酶1B（PTP1B）与2型糖尿病及肥胖的关系

蛋白质酪氨酸磷酸酶1B（PTP1B）是一种在体内广泛表达的胞内蛋白质酪氨酸磷酸酶,在调节胰岛素敏感性和能量代谢的过程中起着重要作用。通过抑制PTP1B可增加胰岛素和瘦蛋白（leptin）的活性, 为寻找2型糖尿病、肥胖的治疗提供了光明前景。     

去B链羧端七肽人胰岛素的分离纯化及性质研究

在大肠杆菌温度诱导体系中以非融合方式进行去Ｂ链羧端七肽人胰岛素原基因的表达,获得去Ｂ链羧端七肽人胰岛素原,表达产物占细胞总蛋白量的１３％,表达产物经ＳｅｐｈａｄｅｘＧ－５０柱层析分离及胰蛋白酶和羧肽酶Ｂ的酶促转化等步骤,可得到纯度达９４％以上的去Ｂ链羧端七肽人胰岛素,其氨基酸组成与预期值相符,受体活性是标准猪胰岛素的１％．     

The ribonuclease inhibitors from porcine thyroid and liver are slow, tight-binding inhibitors of bovine pancreatic ribonuclease A

Ribonuclease inhibitors were purified from the latent ribonuclease fractions of porcine thyroid and liver and used to test the hypothesis that their inhibition of bovine pancreatic ribonuclease A is correctly described by tight-binding rather than Michaelis-Menton kinetics. Both proteins were found to act as slow, tight-binding inhibitors of the enzyme. These steady-state velocities also showed that both the thyroid and liver inhibitors were competitive inhibitors of bovine pancreatic ribonuclease A with Ki's of 0.1 and 0.4 nM, respectively. In contrast to interpretations based on Michaelis-Menton assumptions that show non-competitive inhibition, these results suggest that an enzyme:inhibitor:substrate complex does not exist.     

Evidence for multiple molecular weight forms of the chick intestinal 1,25-dihydroxyvitamin D3 receptor

Studies from many laboratories have reported apparent molecular weights for the chick intestinal 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] receptor varying from 47,000 to 67,000 daltons. We report here that in the presence of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF, 0.3 mM) and in the presence or absence of ligand, the apparent molecular weight of the receptor is 99,700 ± 9,400 (SD) daltons (as determined by gel filtration). In the absence of PMSF, however, the unoccupied receptor migrates with an apparent molecular weight of 51,400 ± 5,700 (SD) daltons. This smaller form of the 1,25(OH)₂D₃ receptor, upon incubation with [³H]-1,25(OH)₂D₃ in the presence of PMSF, then migrates with an apparent molecular weight of 95,900 ± 7,300 (SD) daltons. These results suggest the presence of heretofore unappreciated multiple molecular forms of the chick intestinal 1,25(OH)₂D₃ receptor.     

The Calcium-Dependent and Calcium-Independent Membrane Binding of Synaptotagmin 1: Two Modes of C2B Binding

The Ca²⁺-independent membrane interactions of the soluble C2 domains from synaptotagmin 1 (syt1) were characterized using a combination of site-directed spin labeling and vesicle sedimentation. The second C2 domain of syt1, C2B, binds to membranes containing phosphatidylserine and phosphatidylcholine in a Ca²⁺-independent manner with a lipid partition coefficient of approximately 3.0 × 10² M^− 1. A soluble fragment containing the first and second C2 domains of syt1, C2A and C2B, has a similar affinity, but C2A alone has no detectable affinity to phosphatidylcholine/phosphatidylserine bilayers in the absence of Ca²⁺. Although the Ca²⁺-independent membrane affinity of C2B is modest, it indicates that this domain will never be free in solution within the cell. Site-directed spin labeling was used to obtain bilayer depth restraints, and a simulated annealing routine was used to generate a model for the membrane docking of C2B in the absence of Ca²⁺. In this model, the polybasic strand of C2B forms the membrane binding surface for the domain; however, this face of C2B does not penetrate the bilayer but is localized within the aqueous double layer when C2B is bound. This double-layer location indicates that C2B interacts in a purely electrostatic manner with the bilayer interface. In the presence of Ca²⁺, the membrane affinity of C2B is increased approximately 20-fold, and the domain rotates so that the Ca²⁺-binding loops of C2B insert into the bilayer. This Ca²⁺-triggered conformational change may act as a switch to modulate the accessibility of the polybasic face of C2B and control interactions of syt1 with other components of the fusion machinery.