本文選題：胰島素 + 過氧亞硝酸根��； 參考：《華中科技大學(xué)》2006年博士論文

【摘要】： 糖尿病是一種常見的易發(fā)性代謝疾病。越來越多的證據(jù)表明,活性氧和活性氮介導(dǎo)糖尿病及其并發(fā)癥的病理過程。蛋白質(zhì)酪氨酸硝化是一種重要的蛋白質(zhì)翻譯后修飾,它與炎癥、心血管疾病和神經(jīng)退行性疾病等多種病癥相關(guān)。過氧亞硝酸根(ONOO-)路徑是促使蛋白質(zhì)硝化最主要的一種途徑,其反應(yīng)為自由基機(jī)理。與糖尿病相關(guān)的蛋白質(zhì)硝化已有較多文獻(xiàn)報(bào)道,這不僅表現(xiàn)在硝基酪氨酸(NT)水平的增高,也有特定的硝化蛋白被發(fā)現(xiàn)。胰島素是由脊椎動(dòng)物胰腺β細(xì)胞分泌的一種多功能蛋白質(zhì)激素,是治療糖尿病最有效的藥物。ONOO-可能會(huì)在β細(xì)胞內(nèi)形成,對(duì)細(xì)胞造成損傷,而胰島素也可能成為氧化應(yīng)激條件下ONOO-的一個(gè)靶點(diǎn)。目前有關(guān)ONOO-對(duì)胰島素及其信號(hào)轉(zhuǎn)導(dǎo)系統(tǒng)影響的報(bào)道還較少。 本文研究了體外ONOO-對(duì)胰島素的硝化反應(yīng),探討了硝化對(duì)胰島素結(jié)構(gòu)、功能的影響,并對(duì)抗氧化劑等因素對(duì)胰島素硝化作用的影響進(jìn)行了研究,取得了以下主要結(jié)果: ⑴采用紫外可見光譜、凝膠電泳、免疫印跡以及質(zhì)譜等對(duì)胰島素的硝化產(chǎn)物進(jìn)行了表征,并考察了pH值、CO2、鐵配合物等因素以及白蛋白對(duì)該反應(yīng)的影響。結(jié)果顯示ONOO-可以硝化胰島素酪氨酸(Tyr)殘基,且隨濃度的不斷增大,胰島素4個(gè)酪氨酸殘基都有可能被硝化。ONOO-硝化胰島素的反應(yīng)與pH值密切相關(guān),在生理pH值條件下,ONOO-的硝化作用最強(qiáng)。CO2對(duì)胰島素的硝化具有催化作用。乙二胺四乙酸鐵配合物可催化胰島素硝化,而二乙三胺五乙酸鐵配合物對(duì)胰島素硝化無影響。白蛋白對(duì)胰島素的硝化具有競(jìng)爭(zhēng)抑制作用,但在高濃度的白蛋白存在時(shí),胰島素依然可以被硝化。 ⑵采用高效液相色譜分離制備得到了胰島素的單硝化和二硝化組分。運(yùn)用凝膠電泳、高效液相色譜和質(zhì)譜等技術(shù),結(jié)合A、B鏈拆分和V8蛋白酶酶解方法,對(duì)硝化胰島素的硝化位點(diǎn)進(jìn)行了分析。還原條件下硝化組分的常規(guī)聚丙烯酰胺凝膠電泳以及色譜分離出的單硝化胰島素A鏈的質(zhì)譜結(jié)果表明胰島素的A鏈優(yōu)先B鏈被硝化。V8蛋白酶酶切單硝化胰島素后的質(zhì)譜結(jié)果表明單硝化胰島素的硝化位點(diǎn)是Tyr-A14。結(jié)合電泳的結(jié)果和晶體結(jié)構(gòu)分析,Tyr-A19較B鏈上的Tyr殘基更容易硝化,而Tyr-B26可能較Tyr-B16更容易硝化。 ⑶運(yùn)用光譜學(xué)方法研究了硝化對(duì)胰島素二級(jí)結(jié)構(gòu)的影響。綜合熒光光譜、圓二色光譜和傅立葉變換紅外光譜的測(cè)試結(jié)果,發(fā)現(xiàn)單硝化和二硝化胰島素與胰島素相比α-螺旋的含量下降,A鏈的硝化可能使A(12-17)的310螺旋肽段逐漸向不規(guī)則螺旋和無規(guī)結(jié)構(gòu)方向變化,但對(duì)胰島素與受體結(jié)合面的影響不大。同時(shí)硝基的引入使得單硝化和二硝化硝化胰島素趨向于聚集,而二硝化胰島素中Tyr-A19的硝化可能會(huì)改變胰島素與受體的結(jié)合面疏水區(qū)的疏水性質(zhì)。 采用循環(huán)伏安法研究了Tyr、NT以及Tyr、NT與白蛋白相互作用后的伏安行為,結(jié)果表明硝基的引入將會(huì)改變酚羥基氫鍵的作用方式,影響Tyr與白蛋白的相互作用,這從一個(gè)側(cè)面反映了硝化可能會(huì)影響胰島素與受體的結(jié)合。 ⑷測(cè)定了單硝化胰島素的生物活力,并對(duì)硝化胰島素刺激胰島素受體磷酸化進(jìn)行了初步的討論。單硝化胰島素的受體結(jié)合能力約為胰島素的70%,動(dòng)物實(shí)驗(yàn)與此結(jié)果相一致。 ⑸采用紫外可見光譜測(cè)定胰島素與ONOO-反應(yīng)后NT產(chǎn)量的變化,研究了谷胱甘肽(GSH)和Ebselen等抗氧化劑對(duì)ONOO-硝化胰島素的影響,并采用高效液相色譜和質(zhì)譜等手段對(duì)GSH和Ebselen之間相互作用及其對(duì)ONOO-硝化胰島素的影響機(jī)理進(jìn)行了分析。結(jié)果表明,油酸和花生四烯酸對(duì)胰島素的硝化具有很強(qiáng)的抑制作用,不飽和脂肪酸的抑制作用隨其不飽和度的增大而增強(qiáng)。在實(shí)驗(yàn)濃度范圍內(nèi),亞硒酸鈉對(duì)胰島素的硝化無明顯影響;單獨(dú)的GSH、抗壞血酸、Ebselen和維生素E等抗氧化劑對(duì)胰島素的硝化均有不同程度的抑制作用;但在一定濃度范圍內(nèi),GSH和Ebselen之間存在相互拮抗作用,原因是GSH和Ebselen可以生成加合物Ebselen-Se-SG。
[Abstract]:Diabetes mellitus is a common metabolic disease . More and more evidence suggests that reactive oxygen and active nitrogen mediate the pathological process of diabetes and its complications . The pathway of protein tyrosine nitration is one of the most important pathways for protein nitration . It is a kind of multi - functional protein hormone secreted by beta cells of vertebrate pancreas . Insulin is one of the most effective drugs for treating diabetes . Insulin is one of the most effective drugs for treating diabetes . Insulin is one of the most effective drugs in the treatment of diabetes . Insulin is one of the targets of ONOO - in the condition of oxidative stress .






In this paper , the nitrating reaction of insulin in vitro was studied . The effects of nitrification on the structure and function of insulin were studied . The effects of the factors such as antioxidants on the nitrification of insulin were studied . The following main results were obtained :






The nitrating of insulin was characterized by UV - Vis spectroscopy , gel electrophoresis , Western blot and mass spectrometry . The results showed that ONOO - nitrated insulin tyrosine ( Tyr ) residue and the effect of albumin on the reaction . The results showed that ONOO - nitrated insulin tyrosine ( Tyr ) residue could be nitrated .






The nitrating of insulin was determined by high performance liquid chromatography ( HPLC ) . The nitrating sites of nitrated insulin were analyzed by means of gel electrophoresis , high performance liquid chromatography ( HPLC ) and mass spectrometry ( MS ) .






The effects of nitrification on the secondary structure of insulin were studied by means of spectroscopy . The results showed that the content of 偽 - helix was decreased compared with that of insulin , and the nitration of A - chain could make the 310 - helix peptide segment of A ( 12 - 17 ) gradually change to irregular spiral and random structure direction , but the effect of nitration of the nitro group on the binding surface of insulin and acceptor was not large . The nitration of nitro group led to the tendency of single nitrification and dinitrifying nitrating insulin to accumulate , while the nitration of Tyr - A19 in the dinitrogen insulin could alter the hydrophobic property of the binding surface hydrophobic region of insulin and acceptor .






Cyclic voltammetry was used to study the voltammogram behavior of Tyr , NT and Tyr , NT and albumin . The results showed that the introduction of nitro group could alter the action of hydroxyl hydrogen bond of phenolic hydroxyl group , which influenced the interaction between Tyr and albumin , which reflected nitrification could affect the binding of insulin and acceptor .






( 4 ) The biological activity of mono - nitrated insulin was measured , and the phosphorylation of insulin receptor stimulated by nitrated insulin was discussed . The receptor binding capacity of mono - nitrated insulin was about 70 % of insulin , and animal experiment was consistent with this result .






( 5 ) The effects of anti - oxidants such as glutathione ( GSH ) and ebselen on ONOO - nitrating insulin were studied by UV - Vis spectroscopy . The effects of glutathione ( GSH ) and ebselen on the effects of antioxidant on insulin were studied . The results showed that oleic acid and arachidonic acid had a strong inhibitory effect on the nitrification of insulin .
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R363

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 宗萬松;環(huán)境氧化應(yīng)激誘發(fā)蛋白質(zhì)（多肽）氧化損傷評(píng)價(jià)新方法的研究[D];山東大學(xué);2011年

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本文編號(hào)：1969732