瘦肉型猪(PIC344)精液碱性磷酸酶的分离纯化及部分性质研究

Isolation,Purification and Properties of Alkaline Phosphatase from Sperm of PIC Pigs(PIC344)

用正丁醇抽提、硫酸铵分级沉淀、DEAE SepharoseFF和SephacrylS 200柱层系,从瘦肉型猪(PIC344)精液中提取出碱性磷酸酶。纯化倍数11 46,比活为81 23U/mg蛋白。提取液经PAGE和SDS PAGE检测,呈现一条带。该酶相对分子质量为90 12KD,亚基分子质量为48 41KD,该酶为两个相同亚基组成。等电点为8 19,最适pH值9 5,最适温度为40℃,以磷酸苯二钠为底物测得Km值为3 98×10-4mol/L。经分析,Cu2+、Cd2+为酶的抑制剂,Ni2+、Ca2+、Mg2+为该酶的激活剂。选用CuSO4、Cd(NO3)2作酶的抑制类型判断,结果表明,CuSO4为非竞争性抑制,抑制常数为1 32×10-3mol/L,Cd(NO3)2为竞争性抑制,抑制常数为3 33×10-4mol/L。

Alkaline phosphatase(AKP) was isolated and purified from the sperm of PIC pigs(PIC344) and its kinetic property was examined.The partially purified AKP was purified by salting-out,DEAE-Sepharose FF ion-exchange column,and gel filtrition with sephacryl S-200.The purified enzyme moved as a single electrophoretic band in PAGE.The specific activity was 81.23U/mg.It's subunit weight was 48.41 KD with SDS-PAGE,moleculer weight was 90.12 KD with gel filtrition on Sephacryl S-300(AKTA FPLC).The optimun pH value for the enzyme was 9.5.The optimun temperature for the enzyme was about 40℃.The Michealis-Menton constant(Km) was 3.98×10^(-4)mol/L with disodium phenyl phosphate as its substrate.Ni^(2+),Ca^(2+),Mg^(2+),Sn^(2+) could activate the enzyme determined from 1 mmol/L to 8 mmol/L,Cu^(2+),Cd^(2+) could inhibit the enzyme from 1mmol/L to 3 mmol/L.CuSO_4,Cd(NO_3)_2 was selected for determing types of inhibition,and the result showed that CuSO_4 was a noncompetitive inhibitor,the inhibition constant being 1.32×10^(-3)mol/L,Cd(NO_3)_2 was a competitive inhibitor,with inhibition constant 3.33×10^(-4)mol/L.