Purification and characterization of human colony-stimulating factor 1 from human pancreatic carcinoma (MIA PaCa-2) cells

Jae Hung Shieh, John K. Cini, Ming Chi Wu, Adel A. Yunis

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

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 °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-β-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-β-d-N-acetylglucosaminidase D, it is suggested that the carbohydrate moieties are Asn-linked "complex-type" units.

Original languageEnglish (US)
Pages (from-to)205-213
Number of pages9
JournalArchives of Biochemistry and Biophysics
Volume253
Issue number1
DOIs
StatePublished - Feb 15 1987

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Fingerprint

Dive into the research topics of 'Purification and characterization of human colony-stimulating factor 1 from human pancreatic carcinoma (MIA PaCa-2) cells'. Together they form a unique fingerprint.

Cite this