Imai N, Higuchi M, Kawamura A, Tomonoh K, Oh-Eda M, Fujiwara M, Shimonaka Y, Ochi N
Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co. Ltd, Shizuoka, Japan.
Eur J Biochem. 1990 Dec 12;194(2):457-62. doi: 10.1111/j.1432-1033.1990.tb15639.x.
Various partially or fully desialylated human erythropoietins were obtained by neuraminidase digestion of the hormone, without non-specific proteolysis and degradation of carbohydrates. Asialoerythropoietin showed a specific activity of 220-IU/mg protein in vivo, although that of the intact erythropoietin was 2.2 x 10(5) IU/mg. A linear relationship was found between the logarithm of the specific activity in vivo and the number of sialic acids. The asialoerythropoietin showed a four-times-higher specific activity in vitro compared with intact erythropoietin using mouse bone marrow cells. It also showed an approximately six-times-higher specific activity in a colony-forming assay for the erythroid colony-forming unit and the erythroid burst-forming unit. Partially or fully de-N-glycosylated erythropoietin derivatives also showed lower in vivo activity but higher in vitro activity than the intact erythropoietin, dependent on the number of sialic acids. To clarify the reason for the enhanced biological activity of asialoerythropoietin in vitro, the binding of intact 125I-erythropoietin or 125I-asialoerythropoietin to cells containing specific receptors for the hormone was analyzed. 125I-asialoerythropoietin bound to spleen cells from anemic mice approximately five times faster than did intact 125I-erythropoietin. The amount of 125I-asialoerythropoietin internalized by target cells, measured in the absence of NaN3, was four times higher than that of intact erythropoietin. These results demonstrate that asialoerythropoietin binds to its receptor faster than the intact form. This may be the main reason for the increased activity of asialoerythropoietin in vitro.
通过用神经氨酸酶消化激素获得了各种部分或完全去唾液酸化的人促红细胞生成素，未发生非特异性蛋白水解和碳水化合物降解。去唾液酸促红细胞生成素在体内的比活性为220 IU/mg蛋白质，而完整促红细胞生成素的比活性为2.2×10⁵ IU/mg。发现体内比活性的对数与唾液酸数量之间存在线性关系。与使用小鼠骨髓细胞的完整促红细胞生成素相比，去唾液酸促红细胞生成素在体外的比活性高四倍。在红系集落形成单位和红系爆式集落形成单位的集落形成试验中，它的比活性也高约六倍。部分或完全去N-糖基化的促红细胞生成素衍生物在体内的活性也低于完整促红细胞生成素，但在体外的活性高于完整促红细胞生成素，这取决于唾液酸的数量。为了阐明去唾液酸促红细胞生成素在体外生物活性增强的原因，分析了完整的¹²⁵I-促红细胞生成素或¹²⁵I-去唾液酸促红细胞生成素与含有该激素特异性受体的细胞的结合情况。¹²⁵I-去唾液酸促红细胞生成素与贫血小鼠脾细胞的结合速度比完整的¹²⁵I-促红细胞生成素快约五倍。在不存在叠氮化钠的情况下测量，靶细胞内化的¹²⁵I-去唾液酸促红细胞生成素的量比完整促红细胞生成素高四倍。这些结果表明，去唾液酸促红细胞生成素与受体的结合比完整形式更快。这可能是去唾液酸促红细胞生成素在体外活性增加的主要原因。
