Luben R A, Mundy G R, Trummel C L, Raisz L G
J Clin Invest. 1974 May;53(5):1473-80. doi: 10.1172/JCI107696.
Osteoclast-activating factor (OAF) is a soluble mediator found in supernates of human peripheral leukocytes which have been cultured with antigens or phytomitogens. OAF is a potent stimulator of osteoclastic resorption of fetal bone in organ culture. The present studies were designed to characterize OAF chemically. Bone resorbing activity from supernates of leukocytes cultured without added plasma was not lost on dialysis using a membrane with a molecular weight cutoff of 3,500, but was lost when heated to 60 degrees C for 30 min. The activity was lost after treatment with trypsin or pronase but not after treatment with ribonuclease or neuraminidase. Papain, which inactivated parathyroid hormone at a concentration of 25 mug/ml, did not inactivate OAF at 250 mug/ml. OAF did not react with an antibody to bovine parathyroid hormone which cross-reacts with human parathyroid hormone. OAF was also distinguished from active metabolites of vitamin D and from prostaglandin by extraction procedures and immunoassay for prostaglandin E(2). When the medium from activated leukocytes cultured with autologous plasma was fractionated by gel filtration on Sephadex, bone resorbing activity eluated both with plasma proteins and in lower molecular weight fractions. However, when medium from leukocytes cultured without added plasma was chromatographed, all the OAF activity was eluted in a sharp low molecular weight peak located between chymotrypsinogen (25,000 molecular weight) and ribonuclease A (13,700 molecular weight). This peak contained about 4% of the total protein originally present in the supernate. Its activity was destroyed by overnight incubation at 37 degrees C at pH 6 or 8, but not at pH 7.2. After incubation at 4 degrees C, the activity was lost at pH 3 or 10, but not at pH 4-9. The active fraction from Sephadex G-100 was therefore chromatographed at pH 7.2 on DEAE cellulose and carboxymethyl cellulose. The active material was not adsorbed; however, about sevenfold further purification was achieved by removal of contaminants. The material obtained after sequential Sephadex, DEAE and, carboxymethyl cellulose chromatography stimulated resorption of fetal rat bone in culture at concentrations of 0.75-3 mug protein/ml, indicating that this preparation of OAF was nearly as potent as bovine parathyroid hormone in this system.
破骨细胞激活因子（OAF）是一种可溶性介质，存在于用抗原或植物促细胞分裂剂培养的人外周血白细胞的上清液中。OAF是器官培养中胎儿骨破骨细胞吸收的有效刺激物。本研究旨在对OAF进行化学特性分析。未添加血浆培养的白细胞上清液中的骨吸收活性，在使用截留分子量为3500的膜进行透析时不会丧失，但在60℃加热30分钟后会丧失。用胰蛋白酶或链霉蛋白酶处理后活性丧失，但用核糖核酸酶或神经氨酸酶处理后活性不丧失。木瓜蛋白酶在浓度为25μg/ml时可使甲状旁腺激素失活，但在250μg/ml时不会使OAF失活。OAF与能与人甲状旁腺激素发生交叉反应的抗牛甲状旁腺激素抗体不发生反应。通过提取程序和前列腺素E2免疫测定，OAF也与维生素D的活性代谢产物以及前列腺素区分开来。当用葡聚糖凝胶对用自体血浆培养的活化白细胞的培养基进行凝胶过滤分级分离时，骨吸收活性在血浆蛋白部分以及较低分子量部分中洗脱出来。然而，当对未添加血浆培养的白细胞的培养基进行色谱分析时，所有OAF活性都在位于胰凝乳蛋白酶原（分子量25000）和核糖核酸酶A（分子量13700）之间的一个尖锐的低分子量峰中洗脱出来。该峰含有上清液中最初存在的总蛋白的约4%。其活性在pH 6或8、37℃过夜孵育后被破坏，但在pH 7.2时不会。在4℃孵育后，活性在pH 3或10时丧失，但在pH 4 - 9时不会。因此，将葡聚糖凝胶G - 100的活性部分在pH 7.2条件下在二乙氨基乙基纤维素和羧甲基纤维素上进行色谱分析。活性物质未被吸附；然而，通过去除污染物实现了约7倍的进一步纯化。经过依次的葡聚糖凝胶、二乙氨基乙基纤维素和羧甲基纤维素色谱分析后得到的物质，在浓度为0.75 - 3μg蛋白/ml时可刺激培养中的胎鼠骨吸收，表明这种OAF制剂在该系统中的效力几乎与牛甲状旁腺激素一样强。
