Purification and partial sequence of human osteoclast-activating factor: identity with interleukin 1 beta.

FE Dewhirst, PP Stashenko, JE Mole… - Journal of immunology …, 1985 - journals.aai.org
FE Dewhirst, PP Stashenko, JE Mole, T Tsurumachi
Journal of immunology (Baltimore, Md.: 1950), 1985journals.aai.org
The lymphokine osteoclast-activating factor (OAF) was purified to homogeneity. OAF was
produced by human peripheral blood mononuclear cells stimulated with concanavalin A and
phorbol myristate acetate under serum-free culture conditions. OAF was purified by
sequential gel filtration, ion-exchange, and reverse-phase HPLC by following bone
resorptive activity. Homogeneity was indicated by the criteria of a single 17,800-dalton band
on silver-stained polyacrylamide gels, a single pI 6.8 band on isoelectric focusing, and a …
Abstract
The lymphokine osteoclast-activating factor (OAF) was purified to homogeneity. OAF was produced by human peripheral blood mononuclear cells stimulated with concanavalin A and phorbol myristate acetate under serum-free culture conditions. OAF was purified by sequential gel filtration, ion-exchange, and reverse-phase HPLC by following bone resorptive activity. Homogeneity was indicated by the criteria of a single 17,800-dalton band on silver-stained polyacrylamide gels, a single pI 6.8 band on isoelectric focusing, and a single aminoterminal sequence. Purified OAF stimulated half-maximal release of calcium from fetal rat long bones at a concentration of approximately 0.66 ng/ml. The amino-terminal sequence of OAF was determined and found to be identical to that of interleukin 1 beta. Homogeneous OAF possessed an activity of 8.2 X 10(6) U/mg in the thymocyte proliferation assay. Because the m.w., isoelectric point, amino-terminal sequence, and specific activity in the thymocyte proliferation assay are the same for homogeneous OAF and interleukin 1 beta, we conclude that they are the same molecule, and that interleukin 1 beta is the major protein with OAF activity produced by lectin-stimulated peripheral blood mononuclear cells.
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