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目的:从猪肾脏中寻找并纯化furin样酶的天然抑制剂。方法:通过酵母分泌表达系统获得furin样酶的重组Kexin。从猪肾丙酮粉中抽提并纯化抑制剂组分。抑制剂活力在荧光分光光度计上,用荧光底物Boc-Arg-Val-Arg-Arg-MCA测定。结果:纯化到的抑制剂组分是一等电点超过9.5的碱性蛋白。测定了其N末端22个残基的序列。该序列与非组蛋白HMG-17高度同源,后者含有4个易被furin样酶裂解的双碱性氨基酸位点。因此,该非组蛋白可与荧光底物强烈竞争。若将酶与非组蛋白长时间温育,其抑制剂活力将最终丧失。结论:猪肾中纯化的非组蛋白是furin样酶的自杀性底物抑制剂。
OBJECTIVE: To find and purify the natural inhibitor of furin-like enzyme from porcine kidney. Methods: Furin - like recombinant Kexin was obtained through yeast secretory expression system. Extract and purify inhibitor components from porcine kidney acetone powder. Inhibitor activity was measured on a fluorescence spectrophotometer using the fluorescent substrate Boc-Arg-Val-Arg-Arg-MCA. Results: The purified inhibitor component is a basic protein with an EP of more than 9.5. The sequence of its N-terminal 22 residues was determined. This sequence is highly homologous to the non-histone HMG-17, which contains four di-basic amino acid sites that are easily cleaved by furin-like enzymes. Therefore, this non-histone protein can compete strongly with fluorescent substrates. If the enzyme and non-histone incubation for a long time, its inhibitor activity will eventually lose. Conclusion: The purified non-histone protein from porcine kidney is a suicide substrate inhibitor of furin-like enzyme.