南方鲇碱性磷酸酶的分离纯化及部分性质的研究

用正丁醇抽提，硫酸铵分级沉淀，ＤＥＡＥ－纤维素和ＳｅｐｈａｃｒｙｌＳ－２００柱层析，从南方鲇（Ｓｉｌｕｒｕｓ　ｍｅｒｉｄｉｏｎａｌｉｓ　Ｃｈｅｎ）肠粘膜中提取出碱性磷酸酶（ＡＫＰ）。提纯倍数为３９．５０倍，比活为６８．３５μ／ｍｇ蛋白，提取酶液经ＰＡＧＥ和ＳＤＳ－ＰＡＧＥ只呈现一条区带。该酶的分子量为１３２１４０，Ｎ末端氨基酸为门冬氨酸，最适ｐＨ为１０．１０，７．５＞ｐＨ＞１１．５时不稳定，最适温度为４０℃左右，对热不很稳定，以磷酸苯二钠为底物其Ｋ_ｍ值为１．７２×１０~（－３）ｍｏｌ／Ｌ。Ｍｇ~（２＋）、Ｍｎ~（２＋）为该酶的激活剂，ＫＨ_２ＰＯ_４、Ｌ－ＣｙＳ、ＭＥ、ＤＦＰ、ＥＤＴＡ－Ｎａ_２为抑制剂。选用ＫＨ_２ＰＯ_４和ＤＦＰ作抑制类型的判断，结果表明，ＫＨ_２ＰＯ_４属竞争性掏剂，其抑制常数为２．３ｍｍｏｌ／Ｌ；ＤＦＰ为非竞争性抑制剂，抑制常数为１．０５ｍｍｏｌ／Ｌ。

Studies on the Purification and Prorerties of Alkaline Phosphatase from Silurus meridionalis Chen

Alkaline phosphatase (AKP) was purified from the intestinal mucosa of silurus meridionalis by a procedure involving extraction with butaanol, ammonium sulfate fractionation and chromatography on DEAE-cellulose (DE-32) and Sephacryl S-200 column. The purification multipe was 39. 50 and the specific activity was 68. 35 unit/mg protein, In PAGE and SDS-PAGE, the intestinal mucosa of silures meridionalis AKP formed a single band.The molecular weight for the enzyme was 132 140, N-terminal amino acid was Asp and the optimum PH was 10. 10 ; when 7. 5>pH>11. 5 ,it was unstable. The optimum temperature was about 40℃, it was unstable against heat. The Km value was 1. 72×10￣(-3)M with disodium phenyl phosphate as its substrate. The activator of enzyme was;Mn￣(2+) , while the inhibitors were KH_2PO_4 ,L-Cys ,ME,DEP,and EDTA-Na, KH_2PO_4 and DFP were selected for determining types of inhibition and the results showed that KH_2PO_4 was a competitive inhibitor, the inhibition constant being 2. 3mM, and that DFP was a noncompetitive inhibitor,the inhibition constant being 1. 05mM.