摘要
以巴西橡胶树高产无性系热研7-33-97的新鲜胶乳为材料,分离纯化HMG-CoA还原酶的实验结果表明,低温下收集胶乳,在49000g,15℃下离心60min,离心胶乳底层部分经2％BrijW-1表面活性剂处理,可将其中结合在黄色体膜上的HMG-CoA还原酶洗脱下来,经40％(NH_4)_2SO_4盐析、Blue-dextron-agarose亲和层析、Mono Q(Pharmacia)离子交换层析,可达到HMG-CoA还原酶的纯化。用C_(?) Ultrapore TM反相柱(Beckman高效液相色谱)鉴定其纯度,用DU-70酶动力学分析系统(Beckman)检测底物NADPH在340nm处的消减,测定其HMG-CoA还原酶的活性。酶促反应动力学曲线的分析结果,前20min酶的反应初速度呈线性关系;以吸收光谱曲线分析发现,HMG-CoA还原酶在280nm处有最大吸收峰;用SDS-page测定酶亚基分子量为42K道尔顿;用IEF测定该酶的等电点为pH5.8。
This paper on isolation and purification of HMG-CoA reductase in fresh Hevea latex of high yielding clone Reyan 7-33-97 reported that HMG-CoA reductase conju-gated with lutoid membrane in bottom fraction of centrifuged latex, which was collected at low temperature and centrifuged for 63 min at 49000g, 15℃, was eluted out by the surface-active agent (2% Brij W—1), and HMG-CoA reductase itself was purified by salt preciptation with 40% (NH_4)_2SO_4, affinity chromatography (blue—dextron—agarose) and Mono Q ion-exchange chromatography. The purity and activity of HMG-CoA reductase were determined by C_8 Ultrapore TM reverse phase column and by assaying the extinction of the substrate NADPH at 340nm with the DU—70 analytic system of enzyme kinetics respectively. The initial velocity of enzymatic reaction of HMG-CoA reductase was linear on the kinetic curve of enzymic reaction in the first 20 min. The reductase showed the maximum absorption peak on the curve of absorption spectrum,and its subunit molecular weight was 42K daltons and its isoelectic point pH5.8 determined by SDS—page and IEF respectively.
出处
《热带作物学报》
CSCD
1992年第2期1-8,共8页
Chinese Journal of Tropical Crops
基金
国家自然科学基金
