Rapid and Quantitative Detection of Aspartic Proteinase in Animal Tissues by Radio-labeled Pepstatin A

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A new radio-derivative of pepstatin A was developed and was shown to be used as a probe for rapidly and quantitatively detecting aspartic proteinases in animal tissues. The carboxyl group of pepstatin A was activated by the water soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and was then coupled with N-hydroxysulfosuccinimide (Sulfo NHS). [^<35>S]-Methionyl-pepstatin with a relatively high specific activity was obtained by coupling the Sulfo NHS-pepstatin with L-[^<35>S]-methionine. Binding specificity of the pepstatin A derivative was characterized using pepsin as a test enzyme. Binding experiments showed that the radio-labeled pepstatin can be used as a probe which binds specifically to aspartic proteinases and detects them. The probe could detect as low as 0.1 nmoles of pepsin. To know whether the radio-labeled probe can be actually used to detect aspartic proteinases in animal tissues, it was applied to the tail tissue of metamorphosing amphibian tadpole which had been known to show high activity of cathepsin D. The assay demonstrated marked increase in aspartic proteinases at the climax stage. It was concluded that the radio-labeled pepstatin derivative developed by the present study is useful for quick and quantitative determination of pepstatin-reactive enzymes in animal tissues.
社団法人日本動物学会の論文
1994-04-15