DEVELOPMENT OF CATALYTIC ANTIBODIES AGAINST PHOSPJOLIPASE A_2 AND ELUCIDATION OF ITS MECHANISM OF ACTION
| Project/Area Number |
06807165
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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 |
Chemical pharmacy
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| Research Institution | MEIJO UNIVERSITY |
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Principal Investigator |
HARUNA Mitsumasa MEIJO UNIVERSITY,FACULTY OF PHARMACY,ASSOCIATE PROFESSOR, 薬学部, 助教授 (10076755)
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| Co-Investigator(Kenkyū-buntansha) |
ITO Kazuo MEIJO UNIVERSITY,FACULTY OF PHARMACY,PROFESSOR, 薬学部, 教授 (00076697)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1995: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1994: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | Catalytic antibody / Phospholipase A_2 / Artificial enzyme / Hydrolysis / Phospholipid / Recognition / Vesicles / Monoclonal antibody |
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| Research Abstract |
Preparation of catalytic antibodies against Phospholipase A_2 (PLA_2) by designed racemic transition analog 1 containing C10-carbon chains at C_1 and C_2 in phospholipid as hapten, and its structural recognition of several substrates were investigated.
Hapten 1, synthesized in twelve steps from solketal, was conjugated directly to the carrier protein KLH.Fifty monoclonal antibodies specific for hapten 1 were generated using standard protocols and purified form ascites by protein G affinity chromatography. twenty-two antibodies were found to accelerate the hydrolytic degradation of didecanoylphosphatidylcholine 16, and the most effective antibody 7B3 was characterized in further detail. The kinetic parameters of 7B3 were afforded values of Km=347muM,V_<max>=2.52muM/min, kcat=0.126min^<-1>, and Ki=449muM,respectively.
Moreover hydrolytic activity of 7B3 against several substrates was studied for the determination of structural recognition. Substrate 15 (C_1=C_2=C16), 17 (C_1=C10, C_2=C16), and 18 (C_1=C16, C_2=C10) were not hydrolyzed by 7B3. Further hydrolytic activity of 7B3 against substrate 19, replaced choline ammonium group of 16 to bromine, was reduced to about half times. Therefore antibody 7B3 recognized not only the chain length of carbon at C_1 and C_2, but also the phospholic and ammonium ions of the phosphorylcholine at C_3 in substrate, respectively. Further optically active L-27a was hydrolyzed by 7B3, whereas the D-27b was not affected to that. The kinetic parameters for the hydrolysis of L-27a by 7B3 were afforded values of Km=68.7muM,V_<max>=2.52 muM/min, and k_<cat>=0.126min^<-1>, respectively.
From the hydrolytic activities of 7B3 to lipozomes as small unilamellar vesicles (SUV) and multilamellar vesicles (MLV) in the presence or absence of sodium deoxycholate, the action of antibody was proved that effected a single substrate rather than substrate in state of aggregation.
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Report
(3 results)
Research Products
(2 results)
