Olsen Birgitte B, Rasmussen Tine, Niefind Karsten, Issinger Olaf-Georg
Institut for Biokemi og Molekylaer Biologi, Syddansk Universitet, Campusvej 55, 5230, Odense, Denmark.
Mol Cell Biochem. 2008 Sep;316(1-2):37-47. doi: 10.1007/s11010-008-9824-3. Epub 2008 Jun 24.
Altogether 2 holoenzymes and 4 catalytic CK2 constructs were expressed and characterized i.e. CK2alpha(2)1-335 beta2; CK2alpha'-derived holoenzyme; CK2alpha1-335; MBP-CK2alpha'; His-tagged CK2alpha and His-tagged CK2alpha'. The two His-tagged catalytic subunits were expressed in insect cells, all others in Escherichia coli. IC50 studies involving the established CK2 inhibitors DMAT, TBBt, TBBz, apigenin and emodin were carried out and the Ki values calculated. Although the differences in the Ki values found were modest, there was a general tendency showing that the CK2 holoenzymes were more sensitive towards the inhibitors than the free catalytic subunits. Thermal inactivation experiments involving the individual catalytic subunits showed an almost complete loss of activity after only 2 min at 45 degrees C. In the case of the two holoenzymes, the CK2alpha'-derived holoenzyme lost ca. 90% of its activity after 14 min, whereas CK2alpha2(1-335) beta2 only showed a loss of ca. 40% by this time of incubation. Gel filtration analyses were performed at high (500 mM) and low (150 mM) monovalent salt concentrations in the absence or presence of ATP. At 500 mM NaCl the CK2alpha'-derived holoenzyme eluted at a position corresponding to a molecular mass of 105 kDa which is significantly below the elution of the CK2alpha(2)1-335 beta2 holoenzyme (145 kDa). Calmodulin was not phosphorylated by either CK2alpha2(1-335) beta2 or the CK2alpha'-derived holoenzyme. However, in the presence of polylysine only the CK2alpha(2)1-335 beta2 holoenzyme could use calmodulin as a substrate such as the catalytic subunits, in contrast to the CK2alpha'-derived holoenzyme which only phosphorylated calmodulin weakly. This attenuation may be owing to a different structural interaction between the catalytic CK2alpha' subunit and non-catalytic CK2beta subunit.
总共表达并表征了2种全酶和4种催化性CK2构建体，即CK2α(2)1 - 335β2；源自CK2α'的全酶；CK2α1 - 335；MBP - CK2α'；His标签化的CK2α和His标签化的CK2α'。两种His标签化的催化亚基在昆虫细胞中表达，其他所有构建体在大肠杆菌中表达。进行了涉及已确立的CK2抑制剂DMAT、TBBt、TBBz、芹菜素和大黄素的IC50研究并计算了Ki值。尽管所发现的Ki值差异不大，但总体趋势表明CK2全酶比游离催化亚基对抑制剂更敏感。涉及单个催化亚基的热失活实验表明，在45℃仅2分钟后活性几乎完全丧失。对于两种全酶，源自CK2α'的全酶在14分钟后丧失约90%的活性，而CK2α2(1 - 335)β2在此时的孵育中仅显示约40%的活性丧失。在不存在或存在ATP的情况下，在高（500 mM）和低（150 mM）单价盐浓度下进行凝胶过滤分析。在500 mM NaCl时，源自CK2α'的全酶在对应于105 kDa分子量的位置洗脱，这明显低于CK2α(2)1 - 335β2全酶（145 kDa）的洗脱位置。钙调蛋白既不被CK2α2(1 - 335)β2也不被源自CK2α'的全酶磷酸化。然而，在存在聚赖氨酸的情况下，与仅微弱磷酸化钙调蛋白的源自CK2α'的全酶相反，只有CK2α(2)1 - 335β2全酶能够像催化亚基一样将钙调蛋白用作底物。这种减弱可能归因于催化性CK2α'亚基与非催化性CK2β亚基之间不同的结构相互作用。
