| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1987: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1986: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
(1) Five heterobifunctional reagents, N-(bromoacetamido-n-alkanoyloxy)-succinimides were synthesized. N-(<beta>-bromoacetamido-n-propionoyloxy)-succinimides, one of reagents, was examined as a reagent for the preparation of horseradish peroxidese-insulin conjugates. A simple <beta>-bromo-acetamido-n-propionoyl (BAP) group was first introduced into GlyAl,-PheBl-dicitraconylinsulin through the <epsilon>-amino group of the LysB29 residue and the product was subjected to decitraconylation to obtain LysB29-BAPinsulin. The insulin was reacted with thiolated horseradish peroxidase (HRP)to give HRP-insulin (1:1) conjugate. (2) Five [3-(2-pyridyldithio)propionoy] insulins (PDP-insulins) were separated from reaction mixture of insulin with N-succinimidyl-3-(2-pyridyldithio)-propionate by means of DEAE anion-exchange HPLC. GlyAl-HRP-insulin, LysB29-HRP-insulin and GlyAl,LysB29-HRP-insulin were prepared by the reaction with thiolated HRP and the corresponding PDP-insuling and, purified by gel-permeation HPLC. (3) The method for the estimation of insulin-binding capacity of insulin receptor was developed by using LysB29-HRP-insulin and rat liver membrane fraction. Furthermore, New competitive "enzyme receptor assay" using phospholipase C (PL-C)-treated rat liver was developed. The out line of the procedure is as follows: an 80-<micrn>l of the membrane solution (29 <micrn>g protein) was added to 20-<micrn>l of 300 ng/ml of PL-C solution. The solution was incubated at 23゜C for 90 min, and 50-<micrn>l of LysB29-HRP-insulin solution was added and incubated at 10゜C for 15 h. The suspension was filtered and the membranes trapped on the filter was washed, and then the HRP activity on the filter was measured fluorimetrically. A 2-600 ng of insulin can be measured by the new method.
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