Purification and biochemical function of Astragalus membtanaceus pathogenesis-related protein 10 / 中国中药杂志

ABSTRACT

Astragalus membranaceus pathogenesis-related protein 10 (AmPR-10) is largely expressed in case of environmental pressure and pathogen invasion. This study aims to explore the biochemical functions of AmPR-10. The dried root of Astragalus membranaceus was mechanically homogenized and extracted by Tris-HCl buffer to obtain its crude extract, which was then purified by anion exchange chromatography and gel filtration chromatography to obtain electrophoretically pure AmPR-10. The nuclease activity of AmPR-10 was tested with different RNAs by detecting the absorption value at 260 nm. The results demonstrated potent nuclease activity toward yeast tRNA, yeast RNA, Poly (A) and Poly (C). The optimum reaction temperature was 50 °C and pH was 7-8. EDTA showed no effect on its activity, while Mg²⁺ exhibited potent activation effect on the activity, and Co²⁺, Ca²⁺ and Zn²⁺ manifested moderately inhibition of the activity. Since AmPR-10 had no sequence homology with other known nucleases, AmPR-10 was probably a novel nuclease. The inhibition kinetic data against papain was analyzed by Lineweaver-Burk plots, and the results showed that the inhibition of papain followed noncompetitive-type kinetics. AmPR-10 played an important role in Astragalus membranaceus defense mechanism against environmental pressure and pathogen invasion, which may be achieved by inhibiting cycteine enzymes activity.