Extraction of erythropoietin from normal kidneys.
Extraction of erythropoietin from normal kidneys.
复制标题
从正常肾脏中提取促红细胞生成素。
作者：
J. Sherwood;E. Goldwasser
通过在0.1 M磷酸盐缓冲液中匀浆器官，从正常大鼠、牛、狗和兔的肾脏中提取显著量的活性促红细胞生成素。平均促红细胞生成素活性的提取物，测定饥饿大鼠试验，分别为0.26 U/g牛肾，0.41 U/g狗肾，和0.11 U/g大鼠肾。通过刺激培养的骨髓细胞中的血红蛋白合成（体外试验）测定，犬肾脏提取物的平均活性为0.35 U/g，并且在红细胞增多症小鼠中进行试验时，对59 Fe掺入循环红细胞产生剂量依赖性刺激。兔肾皮质提取物的平均活性为2.12 U/g，通过刺激培养的骨髓细胞中的血红蛋白合成来测量。当狗肾匀浆在DEAE-纤维素上分级时，在0.01 M醋酸盐缓冲液（pH 4.5）的存在下，所有促红细胞生成素活性均被吸附到交换剂上，并被0.1 M Na 2 HPO 4 -0.5 M NaCl（pH 8）完全洗脱。针对人尿促红细胞生成素的抗体完全灭活了狗肾提取物中的促红细胞生成因子。当在饥饿大鼠中测定时，来自供体犬的血清没有促红细胞生成素活性，表明提取物中的因子是细胞内促红细胞生成素，而不是被困在肾血管系统中的血浆中所含的促红细胞生成素。在这些肾匀浆中的促红细胞生成因子的完全失活的抗红细胞生成素和其行为的DEAE-纤维素表明，该因子在结构上类似于天然血浆促红细胞生成素。浸提液在不存在血清的情况下孵育时具有完全活性。
Significant amounts of active erythropoietin were extracted from the kidneys of normal rats, cattle, dogs, and rabbits by homogenization of the organs in 0.1 M phosphate buffer. The mean erythropoietin activities of the extracts, as determined by the starved-rat assay, were 0.26 U/g beef kidney, 0.41 U/g dog kidney, and 0.11 U/g rat kidney. The dog kidney extracts had a mean activity of 0.35 U/g, as measured by stimulation of hemoglobin synthesis in cultured bone marrow cells (in vitro assay) and produced a dose-dependent stimulation of 59Fe incorporation into circulating red cells when assayed in polycythemic mice. Extracts of rabbit kidney cortices had a mean activity of 2.12 U/g, as measured by stimulation of hemoglobin synthesis in cultured bone marrow cells. When the dog kidney homogenate was fractionated on DEAE-cellulose, all of the erythropoietin activity was adsorbed to the exchanger in the presence of 0.01 M acetate buffer, pH 4.5, and was completely eluted by 0.1 M Na2HPO4-0.5 M NaCl, pH 8. An antibody made against human urinary erythropoietin completely inactivated the erythropoietic factor in the dog kidney extract. Serum from a donor dog had no erythropoietin activity when assayed in the starved rat, suggesting that the factor in the extracts is intracellular erythropoietin rather than that contained in plasma trapped in the renal vasculature. The complete inactivation of the erythropoietic factor in these kidney homogenates by antierythropoietin and its behavior on DEAE-cellulose indicate that this factor is structurally similar to native plasma erythropoietin. The extracts are completely active without being incubated in the presence of serum.