Purification of two forms of colony-stimulating factor from mouse L-cell-conditioned medium

A. W. Burgess, D. Metcalf, I. J. Kozka, R. J. Simpson, G. Vairo, J. A. Hamilton, E. C. Nice

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Abstract

A modified procedure for the purification of the colony-stimulating factors (CSFs) in mouse L-cell-conditioned medium is used to isolate two forms of CSF, which are separable by reversed-phase high performance liquid chromatography with 300-Å pore size supports. The specific biological activity of these CSFs (2 x 109 colonies/mg) was considerably higher than has been achieved by other methods. Even at high concentration (200 pM) both molecules stimulated predominantly more macrophage than granulocyte colonies; however, the less hydrophobic form appeared to stimulate the formation of more pure granulocytic colonies. Almost twice as much of the less hydrophobic CSF was recovered from L-cell-conditioned medium. Analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both forms of L-cell CSF had apparent molecular masses of approximately 70,000 daltons. However, on reduction with 2-mercaptoethanol, while both forms generated a 39,000-dalton subunit, the less hydrophobic form also yielded a 32,000-dalton subunit. Storage of either form of L-cell CSF at pH 2.1, in the presence of acetonitrile or isopropanol, destroyed the biological activity. Electrophoretic analysis of the L-cell CSFs stored under these conditions indicated that this was associated with a spontaneous dissociation of the CSF dimer into the inactive subunits. There was some charge heterogeneity (pI 3.5-4.7) indicating different degrees of glycosylation. The unique N-terminal amino acid sequences of both forms of CSF were the same: (Lys-Glu-Val-Ser-Glu-His-X-Ser-His-Met-Ile-Gly-Asn). Thus, the polypeptide chains appear to be identical for the subunits of both forms of L-cell CSF.

Original languageEnglish
Pages (from-to)16004-16011
Number of pages8
JournalJournal of Biological Chemistry
Volume260
Issue number29
Publication statusPublished - 1 Dec 1985
Externally publishedYes

Cite this

Burgess, A. W., Metcalf, D., Kozka, I. J., Simpson, R. J., Vairo, G., Hamilton, J. A., & Nice, E. C. (1985). Purification of two forms of colony-stimulating factor from mouse L-cell-conditioned medium. Journal of Biological Chemistry, 260(29), 16004-16011.
Burgess, A. W. ; Metcalf, D. ; Kozka, I. J. ; Simpson, R. J. ; Vairo, G. ; Hamilton, J. A. ; Nice, E. C. / Purification of two forms of colony-stimulating factor from mouse L-cell-conditioned medium. In: Journal of Biological Chemistry. 1985 ; Vol. 260, No. 29. pp. 16004-16011.
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abstract = "A modified procedure for the purification of the colony-stimulating factors (CSFs) in mouse L-cell-conditioned medium is used to isolate two forms of CSF, which are separable by reversed-phase high performance liquid chromatography with 300-{\AA} pore size supports. The specific biological activity of these CSFs (2 x 109 colonies/mg) was considerably higher than has been achieved by other methods. Even at high concentration (200 pM) both molecules stimulated predominantly more macrophage than granulocyte colonies; however, the less hydrophobic form appeared to stimulate the formation of more pure granulocytic colonies. Almost twice as much of the less hydrophobic CSF was recovered from L-cell-conditioned medium. Analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both forms of L-cell CSF had apparent molecular masses of approximately 70,000 daltons. However, on reduction with 2-mercaptoethanol, while both forms generated a 39,000-dalton subunit, the less hydrophobic form also yielded a 32,000-dalton subunit. Storage of either form of L-cell CSF at pH 2.1, in the presence of acetonitrile or isopropanol, destroyed the biological activity. Electrophoretic analysis of the L-cell CSFs stored under these conditions indicated that this was associated with a spontaneous dissociation of the CSF dimer into the inactive subunits. There was some charge heterogeneity (pI 3.5-4.7) indicating different degrees of glycosylation. The unique N-terminal amino acid sequences of both forms of CSF were the same: (Lys-Glu-Val-Ser-Glu-His-X-Ser-His-Met-Ile-Gly-Asn). Thus, the polypeptide chains appear to be identical for the subunits of both forms of L-cell CSF.",
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Burgess, AW, Metcalf, D, Kozka, IJ, Simpson, RJ, Vairo, G, Hamilton, JA & Nice, EC 1985, 'Purification of two forms of colony-stimulating factor from mouse L-cell-conditioned medium', Journal of Biological Chemistry, vol. 260, no. 29, pp. 16004-16011.

Purification of two forms of colony-stimulating factor from mouse L-cell-conditioned medium. / Burgess, A. W.; Metcalf, D.; Kozka, I. J.; Simpson, R. J.; Vairo, G.; Hamilton, J. A.; Nice, E. C.

In: Journal of Biological Chemistry, Vol. 260, No. 29, 01.12.1985, p. 16004-16011.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - Purification of two forms of colony-stimulating factor from mouse L-cell-conditioned medium

AU - Burgess, A. W.

AU - Metcalf, D.

AU - Kozka, I. J.

AU - Simpson, R. J.

AU - Vairo, G.

AU - Hamilton, J. A.

AU - Nice, E. C.

PY - 1985/12/1

Y1 - 1985/12/1

N2 - A modified procedure for the purification of the colony-stimulating factors (CSFs) in mouse L-cell-conditioned medium is used to isolate two forms of CSF, which are separable by reversed-phase high performance liquid chromatography with 300-Å pore size supports. The specific biological activity of these CSFs (2 x 109 colonies/mg) was considerably higher than has been achieved by other methods. Even at high concentration (200 pM) both molecules stimulated predominantly more macrophage than granulocyte colonies; however, the less hydrophobic form appeared to stimulate the formation of more pure granulocytic colonies. Almost twice as much of the less hydrophobic CSF was recovered from L-cell-conditioned medium. Analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both forms of L-cell CSF had apparent molecular masses of approximately 70,000 daltons. However, on reduction with 2-mercaptoethanol, while both forms generated a 39,000-dalton subunit, the less hydrophobic form also yielded a 32,000-dalton subunit. Storage of either form of L-cell CSF at pH 2.1, in the presence of acetonitrile or isopropanol, destroyed the biological activity. Electrophoretic analysis of the L-cell CSFs stored under these conditions indicated that this was associated with a spontaneous dissociation of the CSF dimer into the inactive subunits. There was some charge heterogeneity (pI 3.5-4.7) indicating different degrees of glycosylation. The unique N-terminal amino acid sequences of both forms of CSF were the same: (Lys-Glu-Val-Ser-Glu-His-X-Ser-His-Met-Ile-Gly-Asn). Thus, the polypeptide chains appear to be identical for the subunits of both forms of L-cell CSF.

AB - A modified procedure for the purification of the colony-stimulating factors (CSFs) in mouse L-cell-conditioned medium is used to isolate two forms of CSF, which are separable by reversed-phase high performance liquid chromatography with 300-Å pore size supports. The specific biological activity of these CSFs (2 x 109 colonies/mg) was considerably higher than has been achieved by other methods. Even at high concentration (200 pM) both molecules stimulated predominantly more macrophage than granulocyte colonies; however, the less hydrophobic form appeared to stimulate the formation of more pure granulocytic colonies. Almost twice as much of the less hydrophobic CSF was recovered from L-cell-conditioned medium. Analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both forms of L-cell CSF had apparent molecular masses of approximately 70,000 daltons. However, on reduction with 2-mercaptoethanol, while both forms generated a 39,000-dalton subunit, the less hydrophobic form also yielded a 32,000-dalton subunit. Storage of either form of L-cell CSF at pH 2.1, in the presence of acetonitrile or isopropanol, destroyed the biological activity. Electrophoretic analysis of the L-cell CSFs stored under these conditions indicated that this was associated with a spontaneous dissociation of the CSF dimer into the inactive subunits. There was some charge heterogeneity (pI 3.5-4.7) indicating different degrees of glycosylation. The unique N-terminal amino acid sequences of both forms of CSF were the same: (Lys-Glu-Val-Ser-Glu-His-X-Ser-His-Met-Ile-Gly-Asn). Thus, the polypeptide chains appear to be identical for the subunits of both forms of L-cell CSF.

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Burgess AW, Metcalf D, Kozka IJ, Simpson RJ, Vairo G, Hamilton JA et al. Purification of two forms of colony-stimulating factor from mouse L-cell-conditioned medium. Journal of Biological Chemistry. 1985 Dec 1;260(29):16004-16011.