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目的:分离纯化苦瓜核糖核酸酶,并进行酶学性质及生物学性质研究,为其生物活性蛋白研究提供依据。方法:采用乙醇沉降、C_(18)反相高效液相色谱、Tricine-SDS-PAGE和等电聚焦等方法分离纯化与鉴定,以酵母tRNA为底物测定核糖核酸酶酶活性。结果 :从苦瓜中分离纯化获得一种核糖核酸酶,命名为MC-RNase。Tricine-SDS-PAGE和C18反相液相色谱鉴定表明该酶具有较高纯度。其N-端氨基酸序列为VNEFDYYQVVLQWQPAT,相对分子质量为14 ku,等电点PI值为8.1;酶学性质表明,该酶的最适反应温度为50℃,最适反应pH值为5,金属离子Zn~(2+)、Fe~(2+)、Fe~(3+)、Mg~(2+)对该酶均有抑制作用,而Pb~(2+)可以增强该酶活性;细胞试验结果表明,该酶对人正常细胞L-02及肝癌细胞Hep-G2并没有明显的抑制作用。结论:从新鲜苦瓜组织中分离并鉴定出一种新的核糖核酸酶。
OBJECTIVE: To isolate and purify the ribonucleoprotein from Momordica charantia and to study its enzymological and biological properties and provide the basis for the study of its bioactive protein. Methods: Purification and identification were carried out by ethanol precipitation, C 18 reversed-phase high performance liquid chromatography, Tricine-SDS-PAGE and isoelectric focusing. The ribonuclease activity was determined using yeast tRNA as a substrate. Results: A kind of ribonuclease was isolated and purified from balsam pear and named MC-RNase. Tricine-SDS-PAGE and C18 reverse phase liquid chromatography identification showed that the enzyme has higher purity. The N-terminal amino acid sequence was VNEFDYYQVVLQWQPAT, the relative molecular mass was 14 ku, and the isoelectric point PI value was 8.1. The enzymatic properties showed that the optimal reaction temperature was 50 ℃, the optimum reaction pH was 5, Zn 2+, Fe 2+, Fe 3+ and Mg 2+ could inhibit the activity of the enzyme, while Pb 2+ could enhance the activity of the enzyme. Cell test The results showed that the enzyme had no obvious inhibitory effect on human normal L-02 and Hep-G2 cells. Conclusion: A new ribonuclease was isolated and identified from fresh bitter melon tissue.