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Proteome analysis of motor proteins in rat parotid acinar cells

大鼠腮腺腺泡细胞运动蛋白的蛋白质组分析

基本信息

批准号：
15591982
负责人：
NASHIDA Tomoko
金额：
$ 2.3万
依托单位：
The Nippon Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591982/
关键词：
parotid motor protein cytoskeleton secretory granule amylase kinesin myosin dynein

项目摘要

Motor proteins in rat parotid glands were analyzed by proteome analysis and Western blotting.The roles of the motor proteins and the cytoskeletons were investigated in the process of amylase secretion from the secretory granules and the secretory vesicles.RT-PCR indicated that KifC1, C2, C3, C4, and 5B (ubiquitous kinesin) were present in the rat parotid glands. Kif 5B was degradated to show the two protein bands about 80KDa and 50KDa in the cytosol fraction prepared from rat parotid gland homogenate.Myosins, ubiquitous kinesin (Kif 5B), dynein/dynactin p150/dynactin p50 complex were detected on the secretory granule membranes. Tubulin and actin were also detected on the secretory granule membranes. Actin was related to the secretion by β-adrenergic stimulation, but tubulin was not. Myosin was not involved to the process of the amylase secretion. These indicated that a microtubule- and motor protein-independent mechanism may be involved in the β-adrenergic process in parotid acinar cells. On the other hand, microtubules and myosin were related to the muscarinic stimulated-amylase release. These results were reported in Arch Oral Biol (49:975-82, 2004).Secretory vesicle fraction was separated by Redi Grad -gradient centrifugation of the crude granule/vesicle fraction. Tubulin and actin were detected in the secretory vesicle fraction. Immunoreactive band was observed with anti-KIFC2 antibody at 50KDa but not at the adequate position. Dynein/dynactin complex was not detected in the fraction. Amylase release under non-stimulated conditions may be attributed to the secretory vesicles. The non-stimulated amylase release was decreased by jasplakinolide but not decreased by colchicine. The myosin ATPase inhibitor 2,3-butanediene monoxime2,3-butanediene monoxime did not affect the non-stimulated amylase secretion. The cytoskeleton and the motor protein played different roles in the non-stimulated and the regulated secretion.

用蛋白质组分析和Western blotting分析了大鼠腮腺中的运动蛋白，研究了运动蛋白和细胞骨架在分泌颗粒和分泌囊泡分泌淀粉酶过程中的作用。RT-PCR结果表明，大鼠腮腺中存在KifC1、C2、C3、C4和5B(普遍存在的激动素)。KIF 5B被降解后，在大鼠腮腺匀浆的胞浆中出现约80 KDa和50 KDa的两条蛋白质带，并在分泌颗粒膜上检测到肌球蛋白、无处不在的动蛋白(KIF 5B)、动力蛋白/动力蛋白P150/动力蛋白P50复合体。在分泌颗粒膜上也检测到微管蛋白和肌动蛋白。肌动蛋白与β-肾上腺素能刺激的分泌有关，而微管蛋白与之无关。肌球蛋白不参与淀粉酶的分泌过程。这些结果表明，腮腺腺泡细胞的β-肾上腺素能过程可能涉及微管和运动蛋白非依赖性机制。另一方面，微管和肌球蛋白与M胆碱刺激的淀粉酶释放有关。这些结果发表在《Arch Oral Biol》(49：975-82,2004)上。在分泌囊泡中检测到微管蛋白和肌动蛋白。抗KIFC2抗体在50 KDa处可见免疫反应条带，但在适当位置未见阳性条带。组分中未检测到动力蛋白/动力蛋白复合体。在非刺激条件下，淀粉酶的释放可能归因于分泌囊泡。茉莉花内酯可减少非刺激性淀粉酶的释放，而秋水仙碱不能减少非刺激淀粉酶的释放。肌球蛋白ATPase抑制剂2，3-丁二烯-2，3-丁二烯-单肟不影响非刺激性淀粉酶的分泌。细胞骨架和运动蛋白在非刺激性和调节性分泌中起不同作用。