| Co-Investigator(Kenkyū-buntansha) |
IKEGAME Mamoru Institute for Comprehensive Medical Science, Division of Molecular Biology, Assi, 総合医科学研究所, 助手 (00176083)
FUJITA Ko Institute for Comprehensive Medical Science, Division of Molecular Biology, Lect, 総合医科学研究所, 講師 (90173426)
|
|---|
| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1991: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1990: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1989: ¥600,000 (Direct Cost: ¥600,000)
|
|---|
| Research Abstract |
A unclear protein designated as SMP (slow migrating protein) with a MW of about 30 kDa in PC12 cells is known to be increased in its phosphorylation by NGF. IN this study, N-terminal amino acid, production of monoclonal antibody and immunocytochemistry of the nuclear protein were investigated. METHOD Nuclear prteins were electrophoresed on acid-urea polyacrylamidegels. ICR mice were immunized against the 3OkDa protein which were separated on the gels. After the antibody generated spleen cells were fused with NS-1 myeloma cells. IgG was purified DEAE-cellulose column chromatograpgy. Amino acid analysis was directly carried out by using PVDF membrane which was electroblotted from the gels. RESULTS 1. N-terminal amino acid sequence of 28 residues has considerable homology with those of ribosomal protein L2 and KD4 of slime mold (Dictyostelium discoildeum) and yeast (Schizosaccharomyces pombe), respectively. 2. The 30 kDa protein immunoreactivity was appeared mainly on nuclear membranes, and occasionlly on nuclei and cytoplasms in PCl2 cells. 3. Rat adrenals and superior cervical ganglion were stained by indirect immunofluorescent stain using the anti-30 kDa protein, and the other organs examined such as submaxillary gland, heart, liver, spleen, muscle, and kidney were not. 4. In the rat brain, Purukinje cells were stained by horseradish peroxidase-DAB. 5. NGF-treated PC12 cells were stained much stronger as compared to untreated PC12 cells, while EGF, which is a weak mitogen for PC12 cells, and which increases in phosphorylation of the 3OkDa nuclear protein, did not affect on the immunoreactivity. CONCLUSION The antigen(s) which are recognized by the monoclonal antibody may exist specifically in neural crest-derived cells and tissues, and might be related to differentiation and proliferation of PC12 cells.
|
