1990 Fiscal Year Final Research Report Summary
Purification of Growth Inhibitory Material from Bovine Brain
| Project/Area Number |
01571210
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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 | Hiroshima University |
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Principal Investigator |
IDE Toshinori Hiroshima Univ. Sch. Med Prof., 医学部, 教授 (60012746)
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| Project Period (FY) |
1989 – 1990
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| Keywords | Cell growth inhibition / Cell surface material / Bovine brain |
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| Research Abstract |
The purpose of this project is to purify a growth inhibitory material from the cell surface of growth-arrested cells.
Bovine brain was minced and suspended to get brain cell suspension. Growth-inhibiting activity was released from the cell surface by treatment with proteolytic enzymes or with phospholipase. Recovery of the activity was found to be less effective but more reproducible when brain cell suspension was treated with phospholipase C than with proteases. However, since phospholipase C was expensive we chose proteases for the further experiments. Recovery of the activity varied among repeated experiment when brain suspension was treated with protease, and this problem could not be solved even after an extensive trial for improvement. When we got good recovery, the further purification was carried out. The cell line used for the assay of the activity was also examined. Finally we chose rat normal fibroblast line and human normal fibroblast line. These cells were first growth arre
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sted and then growth-stimulated with growth factors followed by the addition of the test materials. Growth inhibition was monitored by the incorporation of radioactive thymidine into DNA. Growth inhibitory activity recovered from the brain cell surface was further extracted with organic solvents and the resulted precipitate was lyophilyzed. The material was dissolved in the buffer and applied on gel filtration column. The activity was detected both in the void volume (high molecular weight fraction) and in fractins retained in the column. The void volume was recovered and concentrated. Finally, the activity that adsorbed on the affinity column for a sugar was recovered. This final material had an activity to reduce an induction of DNA synthesis in growth-arrested human and rat normal fibroblasts. However, this material still contained several kinds of proteins as revealed by polyacrylamidgel electrophoresis and silver staining. Since obtained materials were too small amount to purify further, the final purification should be awaited. Less
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