Purification and properties of L cell-derived colony-stimulating factor.
CSF was prepared by the growth of L cells in serum-free culture medium. This conditioned medium was subjected to a six-step purification schedule which included ultrafiltration, alcohol precipitation, and separation by DEAE-cellulose, Con A-Sepharose, Sephadex G-150, and sucrose density-gradient centrifugation. The resultant CSF was 1000-fold purified with 50% to 70% recovery of the starting activity. Granulocyte and macrophage colony formation was detected with 5 x 10(-12M CSF; maximum colonies were obtained with 3 ng per marrow culture. Two major peaks of activity were obtained; one was nonadherent to Con A, whereas the other was bound and specifically eluted with alpha-methylglucoside. Both fractions contained carbohydrate residues as they stained avidly with PAS, were inactivated by periodate, and showed altered electrophoretic mobility after treatment with neuraminidase. Following iodination, each purified fraction migrated in a single band in SDS-acrylamide gels. The molecular weight was estimated as 65,000 to 70,000 daltons. Following reduction with mercaptoethanol, the CSF fractions were reduced into subunits with molecular weights of approximately 35,000. These studies confirm the glycoprotein nature and subunit composition of L cell CSF. The methods described herein are useful for the purification of both the Con A-adherent and con A-nonadherent forms of CSF.
D055598:Chemical Phenomena; D002621:Chemistry; D003115:Colony-Stimulating Factors / Q000302:isolation & purification*; D007739:L Cells / Q000032:analysis*; D008722:Methods; D008970:Molecular Weight
D003115:Colony-Stimulating Factors