Zhang Jian-Wei, Guo Qin, Zhao Tong-Jin, Liu Tao-Tao, Wang Xi-Cheng
Department of Biological Sciences and Biotechnology, School of Life Science and Engineering, Tsinghua University, Beijing 100084, China.
Biochemistry (Mosc). 2006 Sep;71(9):983-8. doi: 10.1134/s0006297906090069.
Two fused proteins of dimeric arginine kinase (AK) from sea cucumber and dimeric creatine kinase (CK) from rabbit muscle, named AK-CK and CK-AK, were obtained through the expression of fused AK and CK genes. Both AK-CK and CK-AK had about 50% AK activity and about 2-fold K(m) values for arginine of native AK, as well as about 50% CK activity and about 2-fold K(m) values for creatine of native CK. This indicated that both AK and CK moieties are fully active in the two fused proteins. The structures of AK, CK, AK-CK, and CK-AK were compared by collecting data of far-UV circular dichroism, intrinsic fluorescence, 1-anilinonaphthalene-8-sulfonate binding fluorescence, and size-exclusion chromatography. The results indicated that dimeric AK and CK differed in the maximum emission wavelength, the exposure extent of hydrophobic surfaces, and molecular size, though they have a close evolutionary relationship. The structure and thermodynamic stability of AK, CK, AK-CK, and CK-AK were compared by guanidine hydrochloride (GdnHCl) titration. Dimeric AK was more dependent on the cooperation of two subunits than CK according to the analysis of residual AK or CK activity with GdnHCl concentration increase. Additionally, AK and CK had different denaturation curves induced by GdnHCl, but almost the same thermodynamic stability. The two fused proteins, AK-CK and CK-AK, had similar secondary structure, tertiary structure, molecular size, structure, and thermodynamic stability, which indicated that the expression order of AK and CK genes might have little effect on the characteristics of the fused proteins and might further verify the close relationship of dimeric AK and CK.
通过表达融合的精氨酸激酶（AK）基因和肌酸激酶（CK）基因，获得了两种融合蛋白，分别是来自海参的二聚体精氨酸激酶（AK）与来自兔肌肉的二聚体肌酸激酶（CK）融合而成的AK-CK和CK-AK。AK-CK和CK-AK都具有约50%的AK活性，其对精氨酸的米氏常数（K(m)）约为天然AK的2倍，同时具有约50%的CK活性，其对肌酸的K(m)值约为天然CK的2倍。这表明AK和CK部分在这两种融合蛋白中均具有完全活性。通过收集远紫外圆二色性、内源荧光、1-苯胺基萘-8-磺酸盐结合荧光以及尺寸排阻色谱的数据，对AK、CK、AK-CK和CK-AK的结构进行了比较。结果表明，二聚体AK和CK虽然具有密切的进化关系，但在最大发射波长、疏水表面暴露程度和分子大小方面存在差异。通过盐酸胍（GdnHCl）滴定比较了AK、CK、AK-CK和CK-AK的结构和热力学稳定性。根据随着GdnHCl浓度增加时剩余AK或CK活性的分析，二聚体AK比CK更依赖于两个亚基的协同作用。此外，AK和CK由GdnHCl诱导的变性曲线不同，但热力学稳定性几乎相同。两种融合蛋白AK-CK和CK-AK具有相似的二级结构、三级结构、分子大小、结构和热力学稳定性，这表明AK和CK基因的表达顺序可能对融合蛋白的特性影响很小，并且可能进一步证实二聚体AK和CK的密切关系。
