猪肾碱性磷酸酶的提纯及性质研究

Purification and properties of alkaline phosphatase from porcine kidney

目的 :从猪肾中提取碱性磷酸酶 (ALP) ,并对其能否作为参考品进行研究。 方法 :从猪肾皮质中分离刷状缘微绒毛膜 ,用正丁醇溶解膜结合的酶 ,经硫酸铵沉淀 ,DEAE Sephacel、ConA Sepharose、SephadexG 2 0 0及抗γ 谷酰基转移酶 (GGT)免疫亲和层析提纯ALP。 结果 :提纯的ALP比活达 4 0 2kU/g蛋白 ,几乎不含杂酶 (未检出ALT、AST、ChE、AMY、CK、LDH ,GGT仅含 2U/780UALP)。其对底物 4 硝基磷酸酚 (4 NPP)的Km值为 1.35mmol/L ,最适pH值为 10 .4 0。 结论 :提纯的猪肾ALP与人血清中的ALP酶动力学特性非常相似 ,为进一步研制ALP参考品奠定了基础。

Objective: For preparing the reference material of alkaline phosphatase(ALP), the purification and properties of ALP from porcine kidney were studied. Methods: The ALP purification procedure included isolation microvillus of porcine kidney cortices by solubilization the membrance-binding enzymes with 1-butanol, precipitation with ammonium sulfate, chromatography on DEAE-Sephacel,ConA-Sepharose,Sephadex G-200 and a immuno-affinity column of anti-GGT antibodies. Results: The purified enzyme had a specific activity of 402 kU/g protein at 37℃and was almost free of contaminating enzymes. The apparent Michaelis constants was 1.35 mmol/L, and the optimum pH was 10.40. Conclusion: The kinetic properties of the preparation were very close to those of the enzyme present in the human serum, The product was suitable as enzyme preparation for producing the enzyme reference material.