1987 Fiscal Year Final Research Report Summary
Function of lymphocyte mitogen receptor and cellular components involved in its regulation
| Project/Area Number |
61571045
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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 |
Biological pharmacy
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| Research Institution | University of Tokyo |
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Principal Investigator |
TOSYOSHIMA Satoshi Faculty of Pharmaceutical Sciences, University of Tokyo, 薬学部, 助教授 (40092283)
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| Project Period (FY) |
1986 – 1987
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| Keywords | lymphocyte / mitogen receptor / inositolphospholipid / phospholipase C. / phosphatidylinositol 4 / 5-bisphosphate |
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| Research Abstract |
To investigate the function of lymphocyte mitogen-receptor, inositolphospholipid-specific phospholipase C (PI-PLase C), a key enzyme of inositolphospholipid turnover which is a receptor coupled phenomenon, was purified and the regulation mechanism of its activity was studied. Frist, membrane-bound and soluble PI-PLase C from murine thymocytes were partially purified and characterized. At neutral pH and low Ca^<2+> concentration (1 <micrn>M), PI-PLase C hydrolyzed phosphatidylinositol 4, 5-bisphosphate (PIP_2) in preference to phosphatidylinositol. The result suggests that in mitogen-stimulated lymphocytes, PI-PLase C essentially hydrolyzes PIP_2.
However, the intracellular Ca^<2+> concentration of mitogen-stimulated lymphocytes was 0.2-0.4 <micrn>M which was still low to account for the increase of PI-PLase C activity. Then, to study the regulatory mechanism of PI-PLase C further, soluble PI-PLase C of calf thymocytes was purified and characterized. The purified enzyme was homogenous on SDS-PAGE and its molecular weight was estimated to be 68 KDa. GTP_rS-binding activity was co-purified with PI-PLase C activity and these two activities were finally separated by the column chromatography on Sephadex G-100 in the presence of 1 % sodium cholate. Furthermore, GTP and GTP_rS could enhance PIP_2 hydrolysis of the partially purified enzyme but not that of the purified enzyme without GTP-binding activity. Properties of the GTP-binding protein were studied and shown to be a novel one composed of 54, 41 and 27 KDa polypeptides. The 27 KDa polypeptide showed GTP-binding activity and enhanced PIP_2 hydrolysis by the purified enzyme. Elucidation of the physiological role of this GTP-binding protein in the signal transduction is the next improtant target of further study.
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