肌酸激酶
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肌酸激酶 (Creatine Kinase, CK) (ATP: Creatine N-phosphotransferase EC 2.7.3.2)通常存在于動物的心臟、肌肉以及腦等組織的細胞漿和線粒體中，是一個與細胞內能量運轉、肌肉收縮、ATP再生有直接關系的重要激酶[1,2]，它可逆地催化肌酸與ATP之間的轉磷酰基反應。
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胞中，BB型主要存在于腦細胞中，MB型主要存在于心肌細胞中，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相同的亞基組成的二聚體。根據目前已經測定的兔、人、雞、鼠肌酸激酶的一級結構[3-6]，M型亞基由387個氨基酸殘基組成，分子量為43 KDa左右，分子內有8個巰基，但無二硫鍵。大熊貓肌肉型肌酸激酶也是二聚體酶，每個亞基由376個氨基酸殘基組成，分子量為42 KDa[7]。
臨床價值
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生時，人的血清中肌酸激酶水平迅速提高，目前認為在心肌梗塞的診斷中測定肌酸激酶的活性比做心電圖更為可靠。心肌梗死時，肌酸激酶在起病6小時內升高，24小時達高峰，3-4日內恢復正常。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用價值已引起人們廣泛的重視和深入的研究。
實驗作用及理由
肌酸激酶作為研究蛋白質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 ?高分辨率晶體結構已經解出[11]，每個亞基具有一個小的N-末端結構域和一個大的C-末端結構域。人肌CK的3.5?分辨率晶體結構也已經得到[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象[13-16]。iii). CK是一個大的二聚體蛋白質，比小的二聚體或單體蛋白質分子更復雜，再折疊過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爭也被觀察到[19,20]。
天然的肌酸激酶分子是一個緊密的球狀結構。近來關于肌酸激酶構象變化和活力變化關系的研究顯示了酶分子活性部位構象的柔性[17，21，22]，即酶分子活性部位的微區構象在變性劑作用下易發生改變而導致酶分子快速失活，此時酶分子整體構象尚未發生明顯變化。周海夢等人[23]用熒光探針標記兔肌肌酸激酶的活性部位，監測了熒光衍生物微區構象變化與相應酶活力喪失速度，發現二者幾乎一致，為酶活性部位柔性的假說提供了有力的證據。
參考文獻
[1] Lehninger A L. Bioenergetics, 2nd.. Benjamin, Menlo Park. 1977. 67-77
[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
[6] Muhlebach S M, Gross M, Wirz T, et al. Sequence Homology and Structure Predictions of the Creatine-Kinase Isoenzymes. Mol.Cell. Biochem., 1994, 133: 245~262
[7] Benfield P A. Isolation and sequence analysis of cDNA clones coding for rat skeletal muscle creatine kinase. J. Biol. Chem., 1984, 259: 14979~14984
[8] Sobel B E, Markham J and Roberts R. Factors influencing enzymatic estimates of infarct size. Am. J. Cardiol., 1977, 39: 130~132
[9] Sobel B E. Applications and limitations of estimation of infarct size from serial changes in plasma creatine phosphokinase activity. Acta Med. Scand. Suppl., 1976, 587: 151~167
[10] Kouttinen A. Purification of human and canine creatine kinase isozymes. Acta Med.Scand.Suppl. 1978, 623: 115~117.
[11] Rao J K, Bujacz G, and Alexander W. 1998. Crystal structure of rabbit muscle creatine kinase. FEBS Lett. 439: 133–137.
[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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