Properties and purification of a colony‐stimulating factor of granulocytes and macrophages produced by mouse spleen cells

Mouse splenocytes are induced by pokeweed mitogen to secrete a factor that stimulates mouse hemopoetic (spelling per Nomina Histologica in the Nomina Anatomica, 5th edition, 1983, Williams and Wilkins, Baltimore) progenitor cells to undergo proliferation and differentiation into granulocytes and macrophages in a semi‐solid culture system. The granulocyte and macrophage colony‐stimulating factor (GM‐CSF) was purified with a four‐step procedure that includes ultrafiltration, chromatography on DEAE‐agarose, Sephacryl S‐200, and chromatofocusing gel. The isoelectric point (pI) of 4.2 of the GM‐CSF was determined by analytical isoelectrofocusing gel electrophoresis. The sensitivity of the biological activity of GM‐CSF to digestion by trypsin and neuraminidase suggests that GM‐CSF is a glycoprotein with its sugar moieties at the active site. The GM‐CSF is also sensitive to heat denaturation at 60°C or higher suggesting that a three‐dimensional conformation is required for its biological activity. The molecular w eight of GM‐CSF is approximately 57,000 Daltons as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.