| Project/Area Number |
60571054
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
Biological pharmacy
|
|---|
| Research Institution | Kitasato University |
|---|
Principal Investigator |
KOBAYASHI Bonro Dept. Pharmaceutical Sciences , Professor, 薬学部・生理化学, 教授 (90050319)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
ISHIHARA Noriko Dept. Pharmaceutical Sciences , Instructor, 薬学部・生理化学, 助手 (10050585)
WATANABE Yasuko Dept. Pharmaceutical Sciences , Assistant Professor, 薬学部・生理化学, 助教授 (80050409)
小林 凡郎 , 薬学部生理化学, 教授 (90050319)
|
|---|
| Project Period (FY) |
1986 – 1987
|
| Project Status |
Completed (Fiscal Year 1987)
|
| Budget Amount *help |
¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1987: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1986: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1985: ¥600,000 (Direct Cost: ¥600,000)
|
|---|
| Keywords | Platelet "40 k" protein / Tyrosine-specific protein kinase / PMA / Monoclonal antibody / チロシン残基(特異的)リン酸化酵素 |
|---|
| Research Abstract |
We have shown that relatively high tyroshine kinase activity is present in normal rabbit platelets 3nd that 48 k protein phosphorylation is stimulated by PMA. The difference in timesequences of total and alkali-stable phosphorylations after stimulation by PMA suggested that tyrosine kinase activation might be an earlier event than that of C-kinase after the PMA-stimulation. Four kinds of monoclonal anti 48 k-platelet proteins were prepared and their specificity was tested by immunostaining of antigens, after blotting, of two-dimensionally electrophoresed proteins from PMA-treated platelets. The antibodies recognized actin in common, and differentially the 48k-sub group proteins. Ten 30 ug of antibody " J " was added to 32Pi-labeled platelet suspension an dincubated for 5 minutes and then stimulated by PMA. The inhibition of 48 k protein phosphorylatin was shown only after alkali-treatment, while any inhibitory effect of total phosphorylation was not observed. Trifuluoperazine(TFP), known as a Ca^<++>-calmodulin kinase inhibitor, reduced 48 k protein phosphorylation of platelets stimulated by PMA but no difference from the control in alkali-stable 48k protein phosphorylation was observed. These 48 k proteins separated by 20EP were named 48k-3 and 48k-5 phosphoproteins and were shown to have pIvalues of 7.2 and 7.4. After 5 sec-stimulation by PMA, 48k-3 phosphoprotein clearly showed an increase in alkali-stability. Other^*48 k proteins were later phosphorylated, and 48k-1,5,6,7 phosphoproteins were detected when the stimulation was continued for 30 sec. or more. These results suggested that the 48k-3 phosphoprotein is the product of eariest stimulation of tyrosine kinase, and that other 48 5-phosphoprotein were those of the followed stimulation of serine, threonine kinases. ^*less alkali-stable
|
