牛感光细胞视杆外段中的磷脂酰肌醇3激酶

Phosphatidylinositol 3-kinase in bovine photoreceptor rod outer segments.

作者信息

Guo X, Ghalayini A J, Chen H, Anderson R E

机构信息

Oklahoma Center for Neuroscience, University of Oklahoma Health Sciences Center, Oklahoma City, USA.

出版信息

Invest Ophthalmol Vis Sci. 1997 Aug;38(9):1873-82.

PMID:9286278

Abstract

PURPOSE

Phosphatidylinositol 3-kinase (PI 3-kinase) plays important roles in mitogenesis, vesicular trafficking, actin rearrangement, and prevention of apoptotic cell death in nonocular tissues. This investigation looked for whether PI 3-kinase is present in bovine rod photoreceptors and if light has any effect on its activity.

METHODS

Bleached (BROS) and dark-adapted (DROS) rod outer segments were prepared from frozen bovine retinas and immunoblotted or immunoprecipitated with antibodies against the regulatory (p85) or catalytic (p110) subunits of PI 3-kinase. Kinase activity was measured in the immunoprecipitates and the reaction products were identified by high-performance liquid chromatography (HPLC). The amount of PI 3-kinase in membrane and cytosol fractions was determined by densitometry of immunoblots.

RESULTS

Immunoblot analysis showed the presence of 85 and 110 kDa proteins in ROS. PI 3-kinase immunoprecipitated by anti-p85 antibody converted PI to PI-3-P and PI-4-P to PI-3,4-P2, as determined by HPLC analysis of the deacylated products. The PI 3-kinase activity in these ROS preparations was sensitive to wortmannin, a potent inhibitor of PI 3-kinase, at low concentrations (IC50 3 nM). Immunoprecipitates (IPs) showed activity twice as high in BROS as in DROS. The IPs of ROS membranes but not cytosol maintained the light-dark difference. However, measurement of anti-p85 and anti-p110 immunoreactivities on western blots of ROS, ROS membranes, and ROS cytosol did not show any light-dark differences.

CONCLUSIONS

PI 3-kinase is present in bovine rod outer segments and its activity appears to be greater in light-adapted retinas.

摘要

目的

磷脂酰肌醇3激酶（PI 3激酶）在有丝分裂、囊泡运输、肌动蛋白重排以及非眼组织中凋亡性细胞死亡的预防过程中发挥着重要作用。本研究旨在探究PI 3激酶是否存在于牛视杆光感受器中，以及光对其活性是否有影响。

方法

从冷冻的牛视网膜制备漂白（BROS）和暗适应（DROS）的视杆外段，并用抗PI 3激酶调节亚基（p85）或催化亚基（p110）的抗体进行免疫印迹或免疫沉淀。在免疫沉淀物中测量激酶活性，并通过高效液相色谱（HPLC）鉴定反应产物。通过免疫印迹的光密度测定法确定膜和胞质溶胶组分中PI 3激酶的含量。

结果

免疫印迹分析显示视杆外段中存在85 kDa和110 kDa的蛋白质。通过对去酰化产物的HPLC分析确定，抗p85抗体免疫沉淀的PI 3激酶将PI转化为PI-3-P，并将PI-4-P转化为PI-3,4-P2。这些视杆外段制剂中的PI 3激酶活性在低浓度（IC50 3 nM）下对PI 3激酶的强效抑制剂渥曼青霉素敏感。免疫沉淀物（IPs）显示BROS中的活性是DROS中的两倍。视杆外段膜而非胞质溶胶的IPs保持了明暗差异。然而，对视杆外段、视杆外段膜和视杆外段胞质溶胶的western印迹上的抗p85和抗p110免疫反应性测量未显示任何明暗差异。