
Overview:
CAMKK2 (CAMKKβ) is a member of the CAMKK family. It is broadly distributed in tissues with highest levels in brain, thymus, spleen, and testis (1). CaMKK2 undergoes intramolecular autophosphorylation, is regulated by Ca2+/calmodulin and phosphorylates CaMKI and CaMKIV on Thr177 and Thr200, respectively. CaMKK2 activates both CaMKI and CaMKIV when coexpressed in Jurkat T cells. CaMKK2 also phosphorylates and regulates the activity of AMPK, which is an important regulator of cellular metabolism in response to metabolic stress (2).
Gene Aliases:
CAMKK; CAMKKB; KIAA0787; MGC15254
Genbank Number:
NM_172226
References:
1.Anderson, K.A. et al: Components of a calmodulin-dependent protein kinase cascade. Molecular cloning, functional characterization and cellular localization of Ca2+/calmodulin-dependent protein kinase kinase beta.2.Hurley, R.L. et al: The Ca2+/calmodulin-dependent protein kinase kinases are AMP-activated protein kinase kinases.J Biol Chem. 2005 Aug 12;280(32):29060-6.
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《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)
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
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
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?

