本文選題：蛋白質(zhì)巰基亞硝化修飾 + Biotin　； 參考：《浙江大學(xué)》2008年碩士論文

【摘要】： 蛋白質(zhì)巰基(S-)亞硝基化修飾是一種新近發(fā)現(xiàn)的蛋白質(zhì)翻譯后修飾方式,即一氧化氮(nitric oxide,NO)作用于蛋白質(zhì)半胱氨酸巰基(-SH)生成巰亞硝基(-SNO)的過(guò)程。研究表明NO通過(guò)蛋白質(zhì)S-亞硝基化修飾影響蛋白質(zhì)的活性與功能,屬于一種氧化還原信號(hào)的轉(zhuǎn)導(dǎo)機(jī)制。特別是在缺血性心臟病中,提高心肌組織中的蛋白S-亞硝基化修飾水平,能發(fā)揮一定的心肌保護(hù)作用。目前,對(duì)心肌組織中潛在的S-亞硝基化修飾靶點(diǎn)還缺乏系統(tǒng)全面的研究。一些在臨床上廣泛應(yīng)用,且能提高組織中NO水平的抗心肌缺血藥物,其藥理效應(yīng)是否與心肌蛋白S-亞硝基化修飾水平的改變有關(guān)也尚不清楚。為此,本文開(kāi)展了以下研究工作: 采用NO供體—巰基-亞硝基-谷胱甘肽(GSNO)對(duì)大鼠心肌蛋白進(jìn)行體外孵育,采用Biotin switch法純化S-亞硝基化蛋白,經(jīng)雙向電泳(2-DE)分離,基質(zhì)輔助電離激光解吸-飛行時(shí)間-串聯(lián)二級(jí)質(zhì)譜(MALDI—TOF-MS/MS)以及候選Western blot技術(shù)進(jìn)行蛋白鑒定。結(jié)果共確定了10種蛋白,其中GAPDH用Westernblot法進(jìn)行了驗(yàn)證。其中,腺苷酸激酶1(AK1)是一個(gè)新發(fā)現(xiàn)的S-亞硝�；揎棸悬c(diǎn),其活性可以被GSNO,而不是GSSG所抑制;且這種抑制作用可以被DTT逆轉(zhuǎn),說(shuō)明S-亞硝基化修飾可能是一種新的AK活性的調(diào)節(jié)機(jī)制。 采用10mg·kg~(-1)劑量的硝酸甘油腹腔注射,誘導(dǎo)大鼠心肌蛋白S-亞硝基化修飾。采用Biotin switch法純化S-亞硝基化修飾蛋白,2-DE分離,MALDI—TOF-MS/MS鑒定,共確定17種蛋白。其中磷酸甘油酸激酶1、醛縮酶A、抗氧化蛋白1、3和6、NDPK、transthyretin等為首次發(fā)現(xiàn)的S-亞硝基化修飾蛋白。這些所發(fā)現(xiàn)的蛋白在調(diào)節(jié)心肌收縮,清除氧自由基,改善能量代謝等方面發(fā)揮著重要作用,而且S-亞硝基化修飾往往是調(diào)控這些酶活性的關(guān)鍵。因此推測(cè)硝酸甘油在體內(nèi)發(fā)揮的藥理作用與調(diào)節(jié)蛋白S-亞硝基化修飾有關(guān)。 采用大鼠心肌缺血/再灌注損傷模型,以血清肌酸激酶(CK)、乳酸脫氫酶(LDH)為指標(biāo),考察參麥方抗心肌缺血再灌注損傷的作用,Griess法測(cè)定血清NO含量,Western blot法檢測(cè)心肌組織內(nèi)皮型一氧化氮合酶(eNOS)的表達(dá)變化;并分別采用Biotin switch法和DAN熒光法進(jìn)行半定量和定量檢測(cè)心肌蛋白S-亞硝基化水平。結(jié)果表明,與模型組相比,參麥方給藥組血清損傷指標(biāo)CK、LDH均明顯下降;與假手術(shù)組和模型組相比,參麥方給藥組血清NO含量顯著升高,心肌組織eNOS表達(dá)上調(diào),心肌蛋白S-亞硝基化水平由4.42+0.60 nmol·mg~(-1)上升至8.78±1.37nmol·mg~(-1),以及在90-117 kD分子量范圍發(fā)生S-亞硝基化的心肌蛋白明顯增多,說(shuō)明參麥方可能通過(guò)促進(jìn)蛋白S-亞硝基化而發(fā)揮心肌保護(hù)作用。 本研究首次運(yùn)用蛋白質(zhì)組學(xué)方法對(duì)大鼠心肌可能發(fā)生S-亞硝基修飾的蛋白質(zhì)進(jìn)行了較為全面的分析,共發(fā)現(xiàn)22個(gè)S-亞硝基修飾靶點(diǎn),且S-亞硝基化修飾可能是一種新的AK活性的調(diào)節(jié)機(jī)制,并發(fā)現(xiàn)參麥方抗心肌缺血/再灌注損傷的作用可能與提高心肌蛋白S-亞硝基化修飾水平有關(guān)。
[Abstract]:Nitroso modification of protein sulfhydryl (S-) is a newly discovered post-translational modification of protein, that is, nitric oxide (NO) acts on protein cysteine sulfhydryl (-SH) to produce mercapto nitroso (-SNO). The study shows that NO can affect the activity and function of protein through the modification of protein S-, which belongs to a kind of oxygen. The transduction mechanism of reduced signal, especially in ischemic heart disease, improves the level of S- nitroso modification in myocardial tissue and can play a certain role in myocardial protection. At present, a systematic and comprehensive study of potential S- nitroso modification targets in myocardial tissue is still lacking. Some are widely used in clinical and can improve tissue. It is not clear whether the NO level of anti myocardial ischemia drugs is related to changes in the level of S- nitroso modification. The following work has been carried out in this paper.
The NO donor - thiol - nitroso - glutathione (GSNO) was used to incubate the rat cardiac muscle protein in vitro. The Biotin switch method was used to purify the S- nitroso protein and be separated by two-dimensional electrophoresis (2-DE). The matrix assisted ionization laser desorption - flight time series two - mass spectrometry (MALDI - TOF-MS/MS) and the candidate Western blot technology were used to identify the protein. Results a total of 10 proteins were identified, of which GAPDH was verified by Westernblot. Adenylate kinase 1 (AK1) was a newly discovered S- subnitrosation target, and its activity could be inhibited by GSNO rather than GSSG; and this inhibition could be reversed by DTT, suggesting that S- nitroso modification may be a new AK activity. Section mechanism.
10mg / kg~ (-1) dose of nitroglycerin was intraperitoneally injected to induce S- nitroglycerin modification in rat cardiac muscle protein. The Biotin switch method was used to purify the S- nitroso modified protein, 2-DE separation, MALDI TOF-MS/MS identification, and 17 proteins were identified, including glyceric acid kinase 1, aldehyde contraction enzyme A, antioxidant protein 1,3 and 6 S- nitroso modified proteins are found. These proteins play an important role in regulating myocardial contraction, removing oxygen free radicals, and improving energy metabolism, and S- nitroso modification is often the key to regulate the activity of these enzymes. Therefore, the pharmacological effects of nitroglycerin in the body and the regulation of protein S- nitroso are suggested. It is related to chemical modification.
The rat myocardial ischemia / reperfusion injury model was used to investigate the effect of Shenmai recipe (CK) and lactate dehydrogenase (LDH) on the anti myocardial ischemia and reperfusion injury. The content of serum NO was measured by Griess method. The expression of eNOS in myocardial tissue was detected by Western blot, and Biotin switch was used respectively. The method of semi quantitative and quantitative determination of the level of S- nitroso of myocardial protein was carried out by method and DAN fluorescence method. The results showed that compared with the model group, the serum damage indexes of the Shenmai prescription group, CK, LDH were obviously decreased, and the serum NO content of the Shenmai prescription group was significantly higher than that of the sham operation group and the model group, the expression of eNOS in the myocardial tissue was up and the myocardial protein S- subnitrate was increased. The level of 4.42+0.60 nmol. Mg~ (-1) increased to 8.78 + 1.37nmol. Mg~ (-1), as well as the increase of myocardial protein of S- nitroso in the range of 90-117 kD molecular weight, indicating that Shenmai Fang may play a myocardial protective effect by promoting protein S- nitroylation.
In this study, the proteomics method was used for the first time to analyze the possible S- nitroso modified protein in rat myocardium. 22 S- nitroso modification targets were found, and S- nitroso modification may be a new regulation mechanism for the activity of AK, and the effect of Shenmai Fang on myocardial ischemia / reperfusion injury may be found. It is related to improving the level of myocardial protein S- nitroso modification.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R341

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 高華;巰基亞硝基化修飾調(diào)控NF-κB轉(zhuǎn)錄活性的研究[D];大連理工大學(xué);2011年

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本文編號(hào)：1876871