摘要
从意蜂Apismellifera工蜂体内分离提纯酸性磷酸酶 (ACPase,EC3 1 3 2 ) ,并对其性质进行了研究。将工蜂酸性磷酸酶的初提物经分段盐析、DEAE SepharoseFF离子交换层析及SephadexG 2 0 0凝胶过滤等纯化步骤 ,得到经聚丙烯酰胺凝胶电泳为单一蛋白区带的酶液。提纯倍数为 77 2 4 ,酶液比活力为 16 2 2U mg(对硝基苯磷酸二钠作底物 )。利用凝胶过滤法测定酶的相对分子质量为 135kD ,SDS PAGE测定酶的亚基相对分子质量为 6 3 1kD。酶的等电点为 4 4 6和 4 79。非还原 还原 (NR R)单向、双向SDS PAGE显示酶分子含有链内二硫键。对二级结构圆二色谱分析显示 ,酶分子中α 螺旋占 13 84 % ,β 折叠占2 5 6 8% ,无规则卷曲占 5 6 34%。氨基酸组成分析结果表明 ,酸性磷酸酶约含有 5 0 7个氨基酸残基 ,富含门冬氨酸残基。
Acid phosphatase (ACPase, EC3.1.3.2) was isolated and purified from the Italian honeybee, Apis mellifera L., and properties of the enzyme had been studied. Acid phosphatase was partially obtained from A. mellifera by homogenate, ammunium sulfate fractionation, chromatography with DEAE-sepharose FF and gel filtration with Sephadex G-200. The purified enzyme moved as a single electrophoretic band in PAGE. The purification multiple was 77.24, and the specific activity 16.22 U/mg with pNPP as its substrate. The molecular weight of ACPase was 135 kD determined with gel filtration and its subunit weight was 63.1 kD determined with SDS-PAGE. Isoelectric focusing study showed that pI values of the enzyme were 4.46 and 4.79. NR/R single and two dimensions SDS-PAGE indicated that the enzyme contained intrachain disulfide bond. Circular dichroism spectrum was investigated, and it was found that the proportion of α-helix, β-sheet and random coil in the enzyme was 13.84%, 25.68% and 56.34% respectively. Amino acid composition analysis showed that there were about 507 amino acids in the ACPase, with plenty of Asp.
出处
《昆虫学报》
CAS
CSCD
北大核心
2004年第3期310-315,共6页
Acta Entomologica Sinica
关键词
意蜂工蜂
酸性磷酸酶
分离纯化
性质
Apis mellifera
acid phosphatase
isolation and purification
properties
