Proteome analysis of motor proteins in rat parotid acinar cells

采用蛋白质组学和免疫印迹法对大鼠腮腺运动蛋白进行分析。研究了运动蛋白和细胞骨架在淀粉酶从分泌颗粒和分泌囊泡分泌过程中的作用。RT-PCR结果显示，大鼠腮腺中存在KifC1、C2、C3、C4和5B（泛在激酶）。对Kif 5B进行降解，在大鼠腮腺匀浆制备的细胞质组分中显示约80KDa和50KDa的两条蛋白带。分泌颗粒膜上检测到肌球蛋白、泛在运动蛋白（kif5b）、动力蛋白/动力蛋白p150/动力蛋白p50复合物。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-肾上腺素能刺激分泌有关，而微管蛋白与β-肾上腺素能刺激分泌无关。肌球蛋白不参与淀粉酶分泌过程。提示腮腺腺泡细胞β-肾上腺素能过程可能有一种不依赖微管和运动蛋白的机制参与。另一方面，微管和肌球蛋白与毒蕈碱刺激淀粉酶释放有关。这些结果发表在Arch Oral Biol（49:975- 82,2004）。粗颗粒/囊泡组分采用Redi梯度离心分离法分离分泌囊泡组分。分泌囊泡部分检测到微管蛋白和肌动蛋白。抗kifc2抗体在50KDa处观察到免疫反应带，但没有在适当的位置。未检出动力蛋白/动力蛋白复合物。在非刺激条件下，淀粉酶的释放可能归因于分泌囊泡。茉莉烯内酯降低了非刺激淀粉酶的释放，秋水仙碱没有降低淀粉酶的释放。肌球蛋白atp酶抑制剂2,3-丁二烯单肟对非刺激淀粉酶分泌无影响。细胞骨架和运动蛋白在非刺激和调节分泌中发挥着不同的作用。

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.