肌酸激酶
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肌酸激酶 (Creatine Kinase, CK) (ATP: Creatine N-phosphotransferase EC 2.7.3.2)通常存在于動(dòng)物的心臟、肌肉以及腦等組織的細(xì)胞漿和線粒體中，是一個(gè)與細(xì)胞內(nèi)能量運(yùn)轉(zhuǎn)、肌肉收縮、ATP再生有直接關(guān)系的重要激酶[1,2]，它可逆地催化肌酸與ATP之間的轉(zhuǎn)磷?；?/a>反應(yīng)?！ ?/p>
目錄 |
形式
肌酸激酶有四種同功酶形式：肌肉型(MM)、腦型(BB)、雜化型(MB)和線粒體型(MiMi)。MM型主要存在于各種肌肉細(xì)胞中，BB型主要存在于腦細(xì)胞中，MB型主要存在于心肌細(xì)胞中，MiMi型主要存在于心肌和骨骼肌線粒體中。肌肉型肌酸激酶分子是由兩個(gè)相同的亞基組成的二聚體。根據(jù)目前已經(jīng)測(cè)定的兔、人、雞、鼠肌酸激酶的一級(jí)結(jié)構(gòu)[3-6]，M型亞基由387個(gè)氨基酸殘基組成，分子量為43 KDa左右，分子內(nèi)有8個(gè)巰基，但無(wú)二硫鍵。大熊貓肌肉型肌酸激酶也是二聚體酶，每個(gè)亞基由376個(gè)氨基酸殘基組成，分子量為42 KDa[7]?！ ?/p>
臨床價(jià)值
肌酸激酶的同功酶在臨床診斷中有十分重要的意義[2，8-10]，在各種病變包括肌肉萎縮和心肌梗塞發(fā)生時(shí)，人的血清中肌酸激酶水平迅速提高，目前認(rèn)為在心肌梗塞的診斷中測(cè)定肌酸激酶的活性比做心電圖更為可靠。心肌梗死時(shí)，肌酸激酶在起病6小時(shí)內(nèi)升高，24小時(shí)達(dá)高峰，3-4日內(nèi)恢復(fù)正常。其中肌酸激酶的同工酶CK-MB診斷的特異性最高。肌酸激酶因其具有重要的生理功能和臨床應(yīng)用價(jià)值已引起人們廣泛的重視和深入的研究?！ ?/p>
實(shí)驗(yàn)作用及理由
肌酸激酶作為研究蛋白質(zhì)折疊的理想模型基于以下理由：i) 肌肉型肌酸激酶分子是由兩個(gè)相同的亞基組成的二聚體，目前兔肌CK的2.35 ?高分辨率晶體結(jié)構(gòu)已經(jīng)解出[11]，每個(gè)亞基具有一個(gè)小的N-末端結(jié)構(gòu)域和一個(gè)大的C-末端結(jié)構(gòu)域。人肌CK的3.5?分辨率晶體結(jié)構(gòu)也已經(jīng)得到[12]。ii)多種條件下變性或修飾后的CK在體外仍可再折疊為天然構(gòu)象[13-16]。iii). CK是一個(gè)大的二聚體蛋白質(zhì)，比小的二聚體或單體蛋白質(zhì)分子更復(fù)雜，再折疊過(guò)程中可以得到更多的中間體[16-18]，聚沉與正確折疊之間的競(jìng)爭(zhēng)也被觀察到[19,20]。
天然的肌酸激酶分子是一個(gè)緊密的球狀結(jié)構(gòu)。近來(lái)關(guān)于肌酸激酶構(gòu)象變化和活力變化關(guān)系的研究顯示了酶分子活性部位構(gòu)象的柔性[17，21，22]，即酶分子活性部位的微區(qū)構(gòu)象在變性劑作用下易發(fā)生改變而導(dǎo)致酶分子快速失活，此時(shí)酶分子整體構(gòu)象尚未發(fā)生明顯變化。周海夢(mèng)等人[23]用熒光探針標(biāo)記兔肌肌酸激酶的活性部位，監(jiān)測(cè)了熒光衍生物微區(qū)構(gòu)象變化與相應(yīng)酶活力喪失速度，發(fā)現(xiàn)二者幾乎一致，為酶活性部位柔性的假說(shuō)提供了有力的證據(jù)?！ ?/p>
參考文獻(xiàn)
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[2] Seraydrarian M W and Abbot B C. The role of the creatine phosphokinase system in muscle. J. Mol. Cell. Cardiol. 1976, 8: 741~746
[3] Shain S A. Creatine kinase and lactate dehydrogenase: stability of isoenzymes and their activity in stored human plasma and prostatic tissue extracts and effect of sample dilution. Clin. Chem., 1983, 29: 832~835
[4] Kwiatkowski R W, Schweinfest C W and Dottin R P. Molecular cloning and the complete nucleotide sequence of the creatine kinase-M cDNA from chicken. Nucleic. Acids Res. 1984, 12: 6952~6934
[5] Pickering L, Pang H, Biemann K, et al. Two tissue-specific isozymes of creatine kinase have closely matched amino acid sequences. Proc. Natl. Acad. Sci. USA, 1985, 82: 2310~2314
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[12] Shen Y Q, Tang L, Zhou H M et al. and Lin Z J. Structure of human muscle creatine kinase. ACTA CRYSTALLOGR D-BIOL CRYST, 2001, 57:1196-1200.
[13]Bickerstaff, G.F., Paterson, C., and Price, N.C. 1980. The refolding of denatured rabbit muscle creatine kinase. Biochim. Biophys. Acta 621: 305–314.
[14]Hou, L.X., Zhou, H.M., Yao, Q.Z., and Tsou, C.L. 1983. A comparative study of renaturation and reactivation kinetics of the guanidine denatured creatine kinase. Acta Biochim. Biophys. Sin. 15: 393–397.
[15]Grossman, S.H. 1984. Fluorescence analysis of denaturation and reassembly of dansylated creatine kinase. Biochim. Biophys. Acta 785: 61–67.
[16]Zhou, H.M. and Tsou, C.L. 1986. Comparison of activity and conformation changes during refolding of urea-denatured creatine kinase. Biochim. Biophys. Acta 869: 69–74.
[17]Wang, Z.F, Yang, Y., and Zhou, H.M. 1995. Conformational changes of active sites during refolding of urea-denatured creatine kinase. Biochimie 77: 953–956.
[18]Yang, Y., Park, Y.D., Yu, T.W., and Zhou, H.M. 1999. Reactivation and refolding of a partially folded creatine kinase modified by 5,5_-Dithio-bis(2-nitrobenzoic acid). Biochem. Biophys. Res. Commun. 259: 450–454.
[19]Webb, T., Jackson, P.J., and Morris, G.E. 1997. Protease digestion studies of an equilibrium intermediate in the unfolding of creatine kinase. Biochem. J. 321: 83–88.
[20]Zhou, J.M., Fan, Y.X., Kihara, H., Kimura, K., and Amemiya, Y. 1997. Unfolding of dimeric creatine kinase in urea and guanidine hydrochloride as measured using small angle X-ray scattering with synchrotron radiation. FEBS Lett. 415: 183–185.
[21] Yao Q Z, Zhou H M, Hou L X, et al. A comparison of denaturation rates of creatine kinase in guanidine solution. Sci. Sin. Ser. B. (Engl. Ed.), 1982, 25: 1296~1302
[22 Wang Z F, Huang M Q, Zou X M, et al. Unfolding , conformational change of active site and inactivation of creatine kinase in SDS solutions. Biochim. Biophys. Acta, 1995, 1251: 109~114
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