|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1991 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1990 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1989 : ¥800,000 (Direct Cost : ¥800,000)
Purification and biochemical characteristics of intracellular pratease ; insulin degrading enzyme(IDE)in erythrocyte were investigated.
1. Purification of IDE
IDE in erythrocyte was labile and easily lost activity the same as other intracellular proteases. To purify IDE effectively and in a -short time, 3 step purification method was carried out using ion-change chromatography(DEAE Sephrose), gel filtration(Superdex 200 pg)and chromatofocusing(Mono P). IDE was purified to a single band on polyacrylamide gel electrophoresis(PAGE). However, it was noted that IDE was decomposed under acidic condition of chromatofocusing and the last step of purification was changed to hydroxyapatite to moderate the reactive condition. By this procedure, IDE was purified with keeping activity.
2. Biochemical characteristics
Purified IDE showed molecular weight 107K and isoelectric point 5.0 on SDS-PAGE. Optimal pH of IDE activity was pH 7.2 in HEPES and phosphate buffer. IDE activity was inhibited by EDTA, but activated by 1mM of Ca- and Mg-salt. From above results, optimal reactive condition of IDE activity was set up in 2OmM HEPES, pH 7.2'and 0.5mM CaCl_2- Under this condition, measurement of IDE activity using human monocomponent insulin as substrate showed good reproducibility with 5% C. V.