摘要
白蜡虫Ericeruspela雌成虫经匀浆 ,正丁醇抽提 ,硫酸铵分段盐析 ,SephadexG 150凝胶过滤等步骤 ,得到比活力为 136 65U mg蛋白酶制品。用苯甲基磺酰氟、N 溴代琥珀酰亚胺、三硝基苯磺酸、二巯基苏糖醇、对氯汞苯甲酸、琥珀酸酐、溴乙酸、碘乙酸等化学修饰剂在一定条件下选择修饰白蜡虫碱性磷酸酶的几种氨基酸残基 ,并测定酶活力变化。结果表明 :苯甲基磺酰氟、N 溴代琥珀酰亚胺、三硝基苯磺酸、琥珀酸酐、二巯基苏糖醇的修饰能显著抑制酶的活力 ,活力的降低与修饰剂的浓度有关。氯汞苯甲酸、溴乙酸、碘乙酸的修饰对酶的抑制作用影响较小。初步认为 :丝氨酸、赖氨酸和色氨酸残基是白蜡虫碱性磷酸酶的必需功能基团 。
The alkaline phosphatase was purified from the female adults of Ericerus pela (Chavannes) through homogenation, n butanol extraction, ammonium sulfate fractionation and Sephadex G 150 column gel filtration. The purification attained to 16 83 folds and the specific activity was 136 65 U/mg. The optimum pH value for the enzyme was 8 5 and temperature at 37℃. The K m was 2 08 mmol/L with disodium phenyl phosphate as its substrate. The enzyme was selectively modified by PMSF, NBS, DTT, PCMB, TNBS, SUAN, BrAc and IAc, and changes in the enzyme activity were detected. The reactions of AKP to PMSF, NBS, TNBS, SUAN and DTT resulted in obvious deline of the enzyme activity, showing the doze response curves within certain dosages of the modifiers. The bromoacetic acid, iodoacetic acid and p chloromercuribenzoate were found to have little inhibition effect on AKP activity. All of these suggested that Ser, Lys and Trp residues should considered as indispensable functional groups of AKP, and partial disulfide bonds as essential for the function of the enzyme.
出处
《昆虫学报》
CAS
CSCD
北大核心
2001年第3期257-262,共6页
Acta Entomologica Sinica
基金
四川大学青年科学基金资助项目
关键词
白蜡虫
碱性磷酸酶
必需基团
化学修饰
Ericerus pela
alkaline phosphatase
essential groups
chemical modification
