Purification and characterization of human colony-stimulating factor 1 from human pancreatic carcinoma (MIA PaCa-2) cells.
Colony-stimulating factor 1 (CSF-1) was purified from the serum-free conditioned medium of a human pancreatic carcinoma cell line (MIA PaCa-2) by a combination of conventional chromatography and high-performance liquid chromatography. The purity of human CSF-1 was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a diffuse single band of Mr 42,000-50,000 and by N-terminal amino acid analysis of glutamate residue. The CSF-1 was stable at 50 degrees C for 30 min. It is sensitive to treatment with trypsin, chymotrypsin, and subtilisin but less sensitive to papain digestion. Treatment of CSF-1 with different glycosidases did not affect the biological activity. Sulfhydryl reagents such as dithiothreitol (DTT), iodoacetic acid, and N-ethylmaleimide did not affect the biological activity at the concentration of 1 mM. However, CSF-1 activity was inhibited totally by the combination of 10 mM DTT and 1 mM SDS. Under denaturing and reducing conditions, CSF-1 appeared on SDS-PAGE as a single protein band of Mr 21,000-25,000 and concurrently lost its activity, indicating that human CSF-1 possibly consists of two similar subunits and that the intact quaternary structure is essential for the biological activity. When treated with neuraminidase and endo-beta-D-N-acetylglucosaminidase D, the molecular weight of CSF-1 was reduced to 36,000-40,000, and to 18,000-20,000 in the presence of mercaptoethanol. Because of the specificity of endo-beta-D-N-acetylglucosaminidase D, it is suggested that the carbohydrate moieties are Asn-linked "complex-type" units.
D001681:Biological Assay; D002277:Carcinoma / Q000032:analysis*; D002460:Cell Line; D002845:Chromatography; D003115:Colony-Stimulating Factors / Q000302:isolation & purification*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D007526:Isoelectric Point; D046911:Macromolecular Substances; D010190:Pancreatic Neoplasms / Q000032:analysis*; D010447:Peptide Hydrolases / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature
D003115:Colony-Stimulating Factors; D046911:Macromolecular Substances; D013439:Sulfhydryl Reagents; D006026:Glycoside Hydrolases; D010447:Peptide Hydrolases