1991 Fiscal Year Final Research Report Summary
Immunoelectron microscopical analysis of intracellular processing of hormones by use of ultracryosection.
| Project/Area Number |
02670004
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
General anatomy (including Histology/Embryology)
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| Research Institution | Gunma University |
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Principal Investigator |
TANAKA Shigeyasu Dept. Morph., Inst. Endocrinol., Res. Assoc., 内分泌研究所, 助手 (90146233)
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| Co-Investigator(Kenkyū-buntansha) |
KUROSUMI Kazumasa Dept. Morph., Inst. Endocrinol., Prof., 内分泌研究所, 教授 (20008593)
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| Project Period (FY) |
1990 – 1991
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| Keywords | Pituitary gland / Pro-opiomelanocortin / Immunocytochemistry / Intracellular processing / Ultracryomicrotomy / Immunogold method / Corticotroph |
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| Research Abstract |
To obtain antibodies specific for precursor pro-opiomelanocortiti(POMC)and its cleaved peptides, we prepared synthetic peptides corresponding to the cleavage site(ST-1)between ACTH and beta-lipotropic hormone moieties of POMC and to amidated joining peptide(JP ; ST-3)and generated their polyclonal antibodies in rabbits. The anti-ST-1 serum immunoprecipitated only POMC in the extract of AtT-20 cells. In addition to. a similar immunoprecipitation examination, immunoabsorption test showed that anti-ST-3 serum recognizes cleaved peptides, 16K amino-terminal peptide, and JP, but not POMC precursor. In ultracryosections of pituitary corticotrophs two distinct types of secretory granules were observed : one is small dense granules, and the other is large and lucent granules. The former was found not only near the Golgi apparatus, but also among the peripheral secretory granules arranged in a single row along the cell membrane. The latter was observed mainly among the peripheral secretory granules. Double-immunolabeling with gold particles of different sizes for the precursor POMC and its cleaved peptides revealed that the former was localized in electron-dense secretory granules, whereas the latter was found in the electron-lucent secretory granules in the same ultrathin cryosections. Colocalization of POMC and its cleaved peptides in the same secretory granules was rarely observed. Only POMC immunolabeling was detected in the Golgi cisternae. These results suggest that a certain step of proteolytic processing of POMC occurs in the secretory granules during the transition between the two distinct stages of secretory granule maturation in the rat anterior pituitary corticotroph.
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