2005 Fiscal Year Final Research Report Summary
Proteome analysis of motor proteins in rat parotid acinar cells
| Project/Area Number |
15591982
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional basic dentistry
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| Research Institution | The Nippon Dental University |
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Principal Investigator |
NASHIDA Tomoko The Nippon Dental University, School of Dentistry at Niigata, Lecturer, 新潟歯学部, 講師 (10133464)
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| Co-Investigator(Kenkyū-buntansha) |
IMAI Akane The Nippon Dental University, School of Dentistry at Niigata, Lecturer, 新潟歯学部, 講師 (60180080)
SHIMOMURA Hiromi The Nippon Dental University, School of Dentistry at Niigata, Professor, 新潟歯学部, 教授 (40139259)
YOSHIE Sumio The Nippon Dental University, School of Dentistry at Niigata, Professor, 新潟歯学部, 教授 (30095278)
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| Project Period (FY) |
2003 – 2005
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| Keywords | parotid / motor protein / cytoskeleton / secretory granule / amylase / kinesin / myosin / dynein |
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| Research Abstract |
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.
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