Partial Purification of Osteoclast-Activating Factor from Phytohemagglutinin-Stimulated Human Leukocytes

Richard A. Luben; Gregory R. Mundy; Clarence L. Trummel; Lawrence G. Raisz

doi:10.1172/JCI107696

Partial Purification of Osteoclast-Activating Factor from Phytohemagglutinin-Stimulated Human Leukocytes

Richard A. Luben, Gregory R. Mundy, Clarence L. Trummel, and Lawrence G. Raisz

Published May 1, 1974 - More info

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Abstract

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°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 μg/ml, did not inactivate OAF at 250 μg/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 E₂.

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°C at pH 6 or 8, but not at pH 7.2. After incubation at 4°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 μg protein/ml, indicating that this preparation of OAF was nearly as potent as bovine parathyroid hormone in this system.