南京林业大学学报(自然科学版) ›› 1991, Vol. 15 ›› Issue (01): 15-22.doi: 10.3969/j.jssn.1000-2006.1991.01.003

• 研究论文 • 上一篇    下一篇

毛竹笋中脱氧核糖核酸酶的提纯和特性

陆宪辉   

  1. 南京林业大学林学系
  • 出版日期:1991-03-18 发布日期:1991-02-18

PURIFICATION AND PROPERTIES OF DEOXYRIBONUCLEASE FROM PHYLLOSTACHYS PUBESCENS SHOOTS

Lu Xianhui   

  1. Department of Forestry
  • Online:1991-03-18 Published:1991-02-18

摘要: <正>毛竹笋中的脱氧核糖核酸酶(DNasa)经热处理、硫酸铵沉淀、SephadexG-100和DEAE-纤维素柱层析被提纯27.2倍,SDS-聚丙烯酰胺凝胶电泳显示单一的带。这种酶的分子量为5.4×10~4,最适pH为5.0~5.2,最适温度为65℃,它对DNA、RNA和5~1-AMP三种底物都有水解活力,其活力比例为1:1:0.1.在pH4.8条件下,为了保持竹笋DNase活力需疏基化合物存在;但是在pH8.0条件下,疏基化合物对这种酶活力具有明显的抑制效应。除低浓度(1×10~(-5)M)的KH_2PO_4和NaF对酶活力分别抑制45%和42%。一价金属离子K~+、Na~+和NH_4+(10~(-2)M)对酶活力有一些刺激效应外,高浓度(1×10~(-3)M)的KH_2PO_2对竹笋DNase活力有10%~20%的刺激作用;在二价金属离子中,除Ca~(++)和Mg~(++)具有微弱刺激作用外,其它离子都有抑制效应。金属螯合剂EDTA(10~(-3)M)对竹笋DNase活力有明显抑制暗示,这种酶含有对活力所需要的金属离子。

Abstract: The deoxyribonuclease (DNase) from Phyllostachys pubescebs shoots was purified by heat treatment, ammonium sulfate fractionation, Sephadex G-100 and DEAE-cellulose column chromatography. The data show that final purification obtained was 27.2 fold, photographs of SDS-polyacrylamide dise gels stained with Coomassie blue R-250 after electrophoresis of this DNase. preparation showed single band. The MW of the enzyme was 5.4×104. The enzyme gave pH optima of 5.0-5.2 and temperature optima of 65℃ . The purified enzyme preparation hydrolyzes denatured DNA and the 5’-AME at 1 : 1 : 0.1 activities. The absolute requirement for a sulfhydryl compound for maintenance of activity at pH 4. 8 and the marked inhibition of the enzyme activity by the same sulfhydryl compounds at pH 8.0 Except for KH2po4at lower concentration (1 x 10~5M) , there several ative effects on the enzymeactivity, approximately 45% and 42% inhibition and sodium fluoride of 1 x 10~3M. The addition of 10~2M one valent cation K+, Na+and NH+4 to the incubative mixture resulted in a 10%-20% stimulation of the DNase activity, Of a number cf two valent Cations tested, only ca++and Mg++produce a slightly stimulating effect on activity (5%-8% stimulation) , other cation found to exert markedly inhibiting effect on the activity includes Co++, Cu++, Zn++, Hg++, and Mn++. Metal chelating agent EDTA (10~3M) resulted in marked inhibition of the DNase activity, which implies that this DNase contains metal ions for maintained activity.