PURIFICATION AND CHARACTERIZATION OF PROTEIN KINASE C FROM A HIGHER PLANT (Brassica campestris L.) AND ANALYSIS FOR ITS BIOCHEMICAL ROLE.
| Project/Area Number |
05454131
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Applied molecular and cellular biology
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| Research Institution | KOBE UNIVERSITY |
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Principal Investigator |
NANMORI Takashi KOBE UNIV.AGRICULTURE ASSISTANT PROF., 農学部, 助教授 (00180555)
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| Co-Investigator(Kenkyū-buntansha) |
FUKAMI Yasuo KOBE UNIV.LAB.OF MOLECULAR BIOLOGY ASSISTANT PROF., 遺伝子実験施設, 助教授 (00156746)
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| Project Period (FY) |
1993 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥5,000,000 (Direct Cost: ¥5,000,000)
Fiscal Year 1994: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1993: ¥2,900,000 (Direct Cost: ¥2,900,000)
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| Keywords | PROTEIN KINASE C / PLANT PROTEIN KINASE C / PLANT PROTEIN KINASE / SIGNAL TRANSDUCTION / PLANT SIGNAL TRANSDUCTION / NITRATE REDUCTASE |
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| Research Abstract |
Protein kinase C (PKC) was partially purified from Brassica campestris L., by successive chromatographies on DEAE-cellulose membrane, hydroxyapatite and pheny1-5PW columns. The purified preparation showed typical characteristics of the conventional type of mammalian PKC that responds to Ca^<2+>, phosphatidylserine, and diacylglycerol or the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. The plant PKC activity was apparently associated with a 75-kDa polypeptide that was recongnized by an antibody against the catalytic domain of rat PKC.Substrate specificity of the plant PKC was similar to that of the rat PKC.A synthetic peptide corresponding to residues 4-14 of myelin basic protein, which is a selective substrate for the mammalian PKC,was phosphorylated efficiently by the plant PKC.These results indicate the existence of a PKC equivalent in higher plant cells.
The plant PKC activity was mainly detected in the leaf and the root of this plant. A PKC was also partially puri
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fied from the plant leaf according to the procedures described above.
It was well-known that protein phosphortlayion and dephosphorylation are important events during signal transductions in mammalian cells. On the other hand, in plant cells, metabolic regulation by phosphorylation/dephosphorylation have not been elucidated before recent years. Nitrate reductase (NR), which is the key enzyme of nitrate assimilation in higher plant, seems to be one of the enzymes regulated by phosphorylation and dephosphorylation. Our co-workers showed that the NR in Brassica campestris leaf is phosphorylated in vivo in response to environmental light conditions.
The PKC fractions partially purified from the plant leaf were allowed to react to the NR purified from the leaf with the presence of gamma-^<32>PATP as phosphorus donor. SDS-PAGE and autoradiography analysis revealed that the NR was phosphorylated only when the Ca2+, phosphatidylserine, and diacylglycerol were present in the reaction mixtures. The result support the idea that the plant PKC involved in phosphorylation of the NR protein. Further studies would be need to clearify the PKC involevement in the regulation of NR activity in response to environmental conditions. In summary of this Grant-in Aid for Scientific Research, we point out not only that PKC homologues exist in the higher plant but also that they have the important role during the plant signal transduction process. Less
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Report
(3 results)
Research Products
(4 results)
