Overview:
CDK4 is a member of the cyclin-dependent protein kinase family and is involved in the control of cell proliferation during the G1 phase of cell cycle. CDK4 forms a complex with the D-type cyclins and is inhibited by p16 (cyclin-dependent kinase inhibitor-2). CDK4 can mediate phosphorylation of the C-terminal region of RB protein leading to an active transcriptional repression of E2F complex (1). CDC37 and HSP90 can preferentially associate with the fraction of CDK4 not bound to D-type cyclins. SMAD3 is a major physiologic substrate of the G1 cyclin-dependent kinases CDK4 and CDK2 (2).
Gene Aliases:
CDK4: CMM3; PSK-J3; MGC14458CyclinD1: BCL1, PRAD1, U21B31, D11S287E
Genbank Number:
NM_000075NM_053056
References:
1.Harbour, J W. et al: Cdk phosphorylation triggers sequential intramolecular interactions that progressively block Rb functions as cells move through G1. Cell 98: 859-869, 1999.2.Matsuura, I. et al: Cyclin-dependent kinases regulate the antiproliferative function of Smads. Nature 430: 226-231, 2004.
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1.对于抑制剂筛选工作(求ic50)是不是体系内酶与底物的量(底物应该是过量的)对实验结果影响不大。
2.如果要求算Km值,是不是需要知道反应产物的绝对量。反应时间文献上都是5分钟,反应速度就用反应产物量除以反应时间即可。
3.酶是进口分装的,规格5U,一次用不完,用PBS稀释后如何保存
谢谢
1、测定酶比活力:底物需要过量么?测定时间多长?是否可以加入过量的底物,然后测定3min吸光度的增加值,从吸光度的变化值计算比活力。
2、在酶抑制剂筛选的过程中,是否需要保证底物过量?还是要水浴一定时间让反应完全?我看到文献说用终浓度为0.1μmol/mL的底物,终浓度为45U/ml的酶,我想问,假设总体积为1ml,那么终浓度为45U/ml的酶岂不是每分钟能转化4.8μmol的底物?那么0.1μmol/mL的底物不就几秒钟就反应完了?那么怎么测定初速度?
2、可逆抑制剂:包括
a、竞争性抑制剂,抑制剂与底物竞争性结合酶反应中心,使Km增大,而Vmax不变 b、非竞争性抑制剂,酶与抑制剂结合后还能与底物结合,但活性降低,使Vmax减小,而Km不变
c、反竞争性抑制剂,酶只能与底物结合后才能与抑制剂结合,Vmax与Km都减小
可逆抑制剂可用透析等方法除去,使酶恢复作用
《血管紧张素转换酶抑制剂在心血管病中应用的中国专家共识》.PDF(242.69k)

