Ueki H, Mitsugi S, Kawashima Y, Motoyashiki T, Morita T
Department of Biochemistry, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Hiroshima, Japan.
Endocrinology. 1997 Jul;138(7):2784-9. doi: 10.1210/endo.138.7.5224.
Involvement of protein kinases in the stimulation of cGMP-inhibited cAMP phosphodiesterase (PDE) activity by orthovanadate (vanadate) was studied. When the fat pads were incubated with 2 mM vanadate or 10 nM insulin, the stimulation of myelin basic protein kinase (MBPK) activity in the particulate by vanadate reached a maximum at 60 min. In contrast, insulin showed a transient increase at 20 min. A 60-min incubation of the fat pads with vanadate stimulated all activities of protein tyrosine kinase (PTK), MBPK, and PDE in the particulate, in a similar dose-dependent manner. Amiloride, a PTK inhibitor, inhibited the stimulations of three enzymes by vanadate in a similar concentration range. Enzyme fractions, which were separated from the solubilized particulate, were subjected to the immunoblot analysis. A fraction of MBPK was identified to contain a major protein of mol wt (44K) and a minor one (42K), both of which are immunoreactive with a mitogen-activated protein kinase (MAPK) antibody. The partially purified PDE activity was stimulated by the addition of the partially purified MBPK. The further stimulation was observed with the PTK-activated MBPK. These results suggest that vanadate stimulates in part the PDE activity through the activation of the particulate MBPK, probably MAPKs, by PTK sensitive to vanadate.
研究了蛋白激酶在原钒酸盐（钒酸盐）刺激环磷酸鸟苷抑制的环磷酸腺苷磷酸二酯酶（PDE）活性中的作用。当脂肪垫与2 mM钒酸盐或10 nM胰岛素一起孵育时，钒酸盐对微粒体中髓鞘碱性蛋白激酶（MBPK）活性的刺激在60分钟时达到最大值。相比之下，胰岛素在20分钟时出现短暂增加。脂肪垫与钒酸盐孵育60分钟，以类似的剂量依赖性方式刺激微粒体中蛋白酪氨酸激酶（PTK）、MBPK和PDE的所有活性。PTK抑制剂阿米洛利在类似浓度范围内抑制钒酸盐对三种酶的刺激。从溶解的微粒体中分离出的酶组分进行免疫印迹分析。鉴定出一部分MBPK含有一种主要蛋白质（分子量44K）和一种次要蛋白质（42K），两者均与丝裂原活化蛋白激酶（MAPK）抗体发生免疫反应。添加部分纯化的MBPK可刺激部分纯化的PDE活性。用PTK激活的MBPK可观察到进一步的刺激。这些结果表明，钒酸盐可能通过对钒酸盐敏感的PTK激活微粒体MBPK（可能是MAPK），部分刺激PDE活性。
