Kimura Yoshio, Kakemizu Aoi, Matsubara Yuuko, Takegawa Kaoru
Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan.
J Biosci Bioeng. 2009 Jan;107(1):10-5. doi: 10.1016/j.jbiosc.2008.08.002.
Two Ser/Thr protein kinases, SpkA and SpkB, selected from Myxococcus xanthus based on amino acid sequence similarities with the catalytic subunits of cAMP-dependent protein kinases (PKA) were synthesized using a cell-free protein synthesis system. In various protein kinase assays, purified StkA and StkB showed their highest protein kinase activities in a PKA assay using the selective PKA substrate Kemptide and in a protein kinase C (PKC) assay using the selective PKC substrate neurogranin((28-43)), respectively. SpkA had apparent K(m) values of 45 microM and 37 microM for Kemptide and ATP, respectively. Phosphorylation of Kemptide was inhibited by a specific PKA inhibitor peptide, PKI(5-24), and the IC(50) and K(i) values for inhibition of the SpkA activity were 117 nM and 36 nM, respectively.
基于与环磷酸腺苷依赖性蛋白激酶（PKA）催化亚基的氨基酸序列相似性，从黄色粘球菌中筛选出两种丝氨酸/苏氨酸蛋白激酶SpkA和SpkB，并使用无细胞蛋白质合成系统进行合成。在各种蛋白激酶测定中，纯化的StkA和StkB分别在使用选择性PKA底物肯普肽的PKA测定和使用选择性PKC底物神经颗粒蛋白（(28 - 43)）的蛋白激酶C（PKC）测定中表现出最高的蛋白激酶活性。SpkA对肯普肽和ATP的表观K(m)值分别为45 microM和37 microM。肯普肽的磷酸化受到特异性PKA抑制肽PKI(5 - 24)的抑制，抑制SpkA活性的IC(50)和K(i)值分别为117 nM和36 nM。
