商陆根中抗真菌蛋白的分离和特性研究

从美洲商陆(Phytolacca americana L.)夏天采集的2—4年生宿根中分离了二种抗真菌蛋白,称为PAFP-R_1和PAFP-R_2。分离程序包括用盐溶液提取,经CM-Sephadex离子交换层析、凝胶过滤层析和羟基磷灰石柱层析纯化。在PGA培养基上,0.1mg/ml蛋白明显抑制木霉菌丝的生长;但对细菌的增殖,即使1mg/ml也无抑制作用。用SDS-PAGE测得二者的相对分子量各为13kD和15kD,单多肽链,等电点约为5.8。用酚-硫酸法未测出含糖。二种蛋白均高含半胱氨酸(19mol/mol)。用Edman降解法测得二者的N末端均为Ala。秋天采集的根中,这二种蛋白含量均很低,但富含由二条PAFP-R_1肽链以双硫键联结的二聚体,它无抗真菌活性。冬天宿根中抗真菌蛋白主要成分是Mr为17kD的单肽链蛋白。上述蛋白对于人红血球均无凝集活性,因此不是PWM的成分。以上结果说明,商陆根中有多种具抗真菌活性的蛋白,成分随季节发生变化,它们都是不含糖,高含半胱氨酸,分子量小于20kD的单肽链蛋白。

ISOLATION AND CHARACTERIZATION OF ANTIFUNGAL PROTEINS FROM THE ROOTS OF PHYTOLACCA AMERICANA (POKEWEED)

From summer harvested roots of 2-4 years old plants of Pokeweed, two antifungal proteins named PAFP-R,and PAFP-R2were isolated by using a procedure including extraction with saline solution, purification with ion exchange chromatography on CM-sephadex, gel filtration on sephadex G50 and chromatography on hydroxyapatite. Both proteins at 0.1 mg/ml inhibited obviously the growth of Trichoderma viride mycelia on a PGA medium plates, but even at 1 mg / ml did not inhibit the multiplication of bacteria E. cola and B. subtilis. They are both a single polypeptide with Mr of 13 kD and 15 kD respectively, with a pI of about 5.8 and with Ala as the N-terminus. Both proteins are rich in Cys, i.e, 19 mol / mol, and in them no carbohydrate was detected by phenol-sulfate reaction. Differing from summer harvested roots, late autumn harvested roots contained much less PAFP-R1and R2, but more dimer of PAFP-R1 linked by disulfide bonds which is of no antifungal activity. In winter harvested roots, the main antifungal component was found to be a protein of 17 kD. These proteins are all of no hemagglutination activity to human erythrocytes even at 2 mg / ml, they are not the components of pokeweed mitogen (PWM).