海洋微生物来源吲哚酮纤溶化合物影响纤溶因子构象特性的研究
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摘要
目的研究FGFC1对各纤溶因子构象的影响及FGFC1促进纤溶反应的机制。方法采用圆二色谱法研究FGFC1对各纤溶因子结构的影响,采用发色底物法研究FGFC1对各纤溶因子活性的影响,采用SDSPAGE电泳法研究FGFC1在纤溶酶原和单链尿激酶型纤溶酶原激活剂构成的相互活化反应体系中的作用。结果远紫外区的CD结果表明FGFC1在23~115μmol·L-1的浓度范围内均使各纤溶因子的二级结构发生变化,这变化都导致酶分子柔性增加、更易发生反应;近紫外区的CD结果表明,FGFC1使纤溶酶原在285nm处的摩尔旋光度增大。CD研究结果表明FGFC1对单链尿激酶型纤溶酶原激活剂和尿激酶型纤溶酶原激活剂结构的影响较为微弱,对纤溶酶及纤溶酶原结构的影响复杂而深刻。运用发色底物法检测加入不同浓度FGFC1后各纤溶因子的酶活力,排除了FGFC1对纤溶因子的结构影响而导致的纤溶因子失活的可能性。在体外构筑的纤溶反应体系中,SDS-PAGE的结果显示FGFC1的加入促进FITC-纤维蛋白原的降解,表明FGFC1加速纤溶酶原和单链尿激酶型纤溶酶原激活剂的相互活化作用。结论 FGFC1作用于纤溶酶原并辅以改变单链尿激酶型纤溶酶原激活剂的二级结构发挥纤溶促进作用。
Objective To investigate the influences of FGFC1 on spatial structure of the fibrinolytic activity factor and explore the mechanisms of FGFC1 to promote fibrinolysis.Methods Circular dichroism spectroscopy(CD)was used to investigate the influences of FGFC1 on spatial structure of the fibrinolytic activity factor,and polyacrylamide gel electrophoresis(SDS-PAGE)was used to study the role of FGFC1 in mutual activation reaction system constituted by single chain urokinase-type plasminogen activator and plasminogen,and chromogenic substrate method was used to evaluate the effects of FGFC1 on the enzymatic activity of fibrinolytic factors.Results Far-ultraviolet CD spectra showed that in the concentration of 23μmol·L-1to 115μmol·L-1,FGFC1 changed the secondary structures of these enzymes which could be responsible for the increased molecular flexibility and more susceptible to react with substrate.Near-ultraviolet CD spectra showed that there was an increase in the ellipticity of the plasminogen with an enhancement of absorption about at 285 nm.The results of far and near-ultraviolet CD spectra exposed that FGFC1 slightly influenced the conformation of single chain urokinase-type plasminogen activator and urokinase and highly influenced the conformation of plasminogen and plasmin.The results of chromogenic substrate method ruled out the possibility of deactivation due to effects of FGFC1 on the structure of the fibrinolytic factors.SDS-PAGE results showed FGFC1 promoted the degradation of FITC-fibrinogen which indicated that FGFC1 accelerated the mutual activation reaction between single chain urokinase-type plasminogen activator and plasminogen.Conclusion Based on CD spectra and SDS-PAGE,the present study explained FGFC1 mainly reacted with plasminogen/plasmin,not with the single-chain urokinase-type plasminogen activator.As a result,the FGFC1 could influence the fibrinolytic process.
Objective To investigate the influences of FGFC1 on spatial structure of the fibrinolytic activity factor and explore the mechanisms of FGFC1 to promote fibrinolysis.Methods Circular dichroism spectroscopy(CD)was used to investigate the influences of FGFC1 on spatial structure of the fibrinolytic activity factor,and polyacrylamide gel electrophoresis(SDS-PAGE)was used to study the role of FGFC1 in mutual activation reaction system constituted by single chain urokinase-type plasminogen activator and plasminogen,and chromogenic substrate method was used to evaluate the effects of FGFC1 on the enzymatic activity of fibrinolytic factors.Results Far-ultraviolet CD spectra showed that in the concentration of 23μmol·L-1to 115μmol·L-1,FGFC1 changed the secondary structures of these enzymes which could be responsible for the increased molecular flexibility and more susceptible to react with substrate.Near-ultraviolet CD spectra showed that there was an increase in the ellipticity of the plasminogen with an enhancement of absorption about at 285 nm.The results of far and near-ultraviolet CD spectra exposed that FGFC1 slightly influenced the conformation of single chain urokinase-type plasminogen activator and urokinase and highly influenced the conformation of plasminogen and plasmin.The results of chromogenic substrate method ruled out the possibility of deactivation due to effects of FGFC1 on the structure of the fibrinolytic factors.SDS-PAGE results showed FGFC1 promoted the degradation of FITC-fibrinogen which indicated that FGFC1 accelerated the mutual activation reaction between single chain urokinase-type plasminogen activator and plasminogen.Conclusion Based on CD spectra and SDS-PAGE,the present study explained FGFC1 mainly reacted with plasminogen/plasmin,not with the single-chain urokinase-type plasminogen activator.As a result,the FGFC1 could influence the fibrinolytic process.
引文
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[14]List K,Jensen O N,Bugge T H,et al.Plasminogen-independent initiation of the pro-urokinase activation cascade in vivo.Activation of pro-urokinase by glandular kallikrein(mGK-6)in plasminogen-deficient mice[J].Biochem,2000,39(3):508-515.
[15]姝亚,吴文惠,汪之和,等.纤溶化合物促进血栓溶解作用的初步研究[J].食品科学,2007,28(8):471-475.
[16]Wu W,Hasumi K,Peng H,et al.Fibrinolytic compounds isolated from a brown alga,Sargassum fulvellum[J].Mar drugs,2009,7(2):85-94.
[17]张艳,吴文惠,周培根,等.海洋微生物来源纤溶活性化合物的筛选及其分离[J].中国海洋药物,2008,27(6):39-43.
[18]瞿佳华,田宏,吴文惠,等.纤溶化合物促进大鼠肺血栓溶解作用的研究[J].食品科学,2009,23:410-414.
[2]王幸,吴文惠,孙立春,等.具有纤溶活性作用的海洋真菌代谢产物的分离与菌株鉴定[J].天然产物研究与开发,2012,24(1):57-61.
[3]Su T,Wu W,Yan T,et al.Pharmacokinetics and tissue distribution of a novel marine fibrinolytic compound in Wistar rat following intravenous administrations[J].J.Chromatogr B,2013,942:77-82.
[4]吴文惠,包斌.具有纤溶促进作用的低分子天然化合物的研究[J].食品科学,2006,26(10):34-42.
[5]林凯蕾.圆二色光谱预测蛋白质二级结构组成方法评析[D].上海:复旦大学,2012.
[6]郝斐,吴文惠,瞿佳华,等.葡萄糖甘油二脂对纤溶酶原和纤溶酶原激活剂二级结构的影响[J].光谱学与光谱分析,2010,8:39.
[7]姚占全.应用圆二色光谱研究电场对蛋白质(酶)构象的影响[D].呼和浩特:内蒙古大学,2005.
[8]Guinn L,Johnson J,Doctor V M.Ionic modulation of the effects of heparin and 6-aminohexanoic acid on plasminogen activation by streptokinase:The role of ionic strength,divalent cations and chloride[J].Eur J Drug Metab Pharmacokinet,2003,28(2):161-166.
[9]沈国华,华颖,刘大群,等.纤溶酶SPFE-Ⅱ的纤溶机制和酶反应动力学研究[J].中国食品学报,2010,1:48-54.
[10]沈琼,黄滨,邵嘉亮,等.运用圆二色谱研究酶与化合物相互作用的机理[J].中山大学学报(自然科学版),2006,45(4):62-64.
[11]Brown K L,Zou X,Chen G,et al.Solution structure,enzymatic,and non-enzymatic reactivity of 3-isoadenosylcobalamin,a structural isomer of coenzyme B12 with surprising coenzymic activity[J].J Inorg Biochem,2004,98(2):287-300.
[12]Tello-Solís S R,Jiménez-Guzmn J,Sarabia-Leos C,et al.Determination of the secondary structure of Kluyveromyces lactisβ-galactosidase by circular dichroism and its structureactivity relationship as a function of the pH[J].J Agr Food Chem,2005,53(26):10200-10204.
[13]Cang-Rong J T,Pastorin G.The influence of carbon nanotubes on enzyme activity and structure:investigation of different immobilization procedures through enzyme kinetics and circular dichroism studies[J].Nanotechnol,2009,20(25):255102.
[14]List K,Jensen O N,Bugge T H,et al.Plasminogen-independent initiation of the pro-urokinase activation cascade in vivo.Activation of pro-urokinase by glandular kallikrein(mGK-6)in plasminogen-deficient mice[J].Biochem,2000,39(3):508-515.
[15]姝亚,吴文惠,汪之和,等.纤溶化合物促进血栓溶解作用的初步研究[J].食品科学,2007,28(8):471-475.
[16]Wu W,Hasumi K,Peng H,et al.Fibrinolytic compounds isolated from a brown alga,Sargassum fulvellum[J].Mar drugs,2009,7(2):85-94.
[17]张艳,吴文惠,周培根,等.海洋微生物来源纤溶活性化合物的筛选及其分离[J].中国海洋药物,2008,27(6):39-43.
[18]瞿佳华,田宏,吴文惠,等.纤溶化合物促进大鼠肺血栓溶解作用的研究[J].食品科学,2009,23:410-414.