猪铜蓝蛋白的分离纯化及对垂体细胞分泌生长激素的影响

Purification of swine serum ceruloplasmin and effect of ceruloplasmin on growth hormone secretion of swine pituitary cells in vitro

采用硫酸铵分级沉淀分离,DEAE-sepharose离子交换,羟基磷灰石吸附层析,Superdex-200凝胶过滤纯化,从猪血清中分离纯化出电泳纯的一种蛋白质。进一步研究,其相对分子质量为123 500,等电点为5.08,分子含铜原子数为6个,含糖量为7.8%,亚基数为1,经分析为猪血清铜蓝蛋白。用含不同质量浓度铜蓝蛋白(0、0.1、0.2、0.4、0.81、.2 g/L)的无血清培养液培养已培养48 h的新生仔猪脑垂体细胞12 h,于2、4、81、2 h分别收集培养液,用放免法测定培养液中GH浓度,结果表明铜蓝蛋白质量浓度为0.2、0.4、0.8、1.2 g/L组垂体细胞生长激素的分泌显著高于0 g/L组,其中以0.4 g/L组、换液培养8 h,垂体细胞生长激素分泌最为明显。

By means of the ammonium sulfate graded precipitation,DEAE-sepharose ion exchange, hydroxyapatite absorption chromatography and Superdex-200 gel filtration,a kind of protein was purified to electrophoretic hom- ogeneity from swine serum. The protein was characterized as follow：the molecular mass was approximately 123 500, isoelectric point was 5.08,and the copper content was estimated to be 6 atoms per molecule,and it contains 7.8% of hydronate,also it was a single subunit protein. According to the results as mentiones above,the protein was identified as porcine serum ceruloplasmin. Swine pituitary cell was isolated form three-day new born piglet and was incubated for 48 h in presence of 10% fetal calf serum in DMEM,then the cells were treated for 12 h with various concentrations of ceruloplasmin（0,0.1,0. 2,0. 4,0. 8,1. 2 g/L）in serum-free DMEM. Culture medium was collected in 2,4,8, 12 h thereafter for growth hormone measurement with the radioimmunoassay. The results showed that the growth hormone concentration was evidently higher in treatments of 0.2,0.4,0. 8,1.2 g/L than that of 0 g/L, and was highest in treatment of 0.4 g/L for 8 h incubation.