Purification and amino-terminal sequence of an insulin-like growth factor-binding protein secreted by rat liver BRL-3A cells

C. Mottola, Richard G MacDonald, J. L. Brackett, J. E. Mole, J. K. Anderson, M. P. Czech

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Abstract

A protein preparation that specifically binds insulin-like growth factors (IGFs) I and II was purified from medium conditioned by rat liver BRL-3A cells using molecular sieve chromatography in 1 M acetic acid followed by affinity chromatography on IGF-II-agarose. The affinity-purified IGF-binding protein exhibits a single major band with apparent M(r) = 36,300 under reducing conditons on sodium dodecyl sulfate-polyacrylamide gels. The IGF-binding protein is efficiently and specifically cross-linked to either 125I-IGF-I (human) or 125I-IGF-II (rat) using disuccinimidyl suberate. An IGF-binding protein of similar apparent molecular weight was also affinity purified from rat hepatoma H-35 cell conditioned medium and found to differ from the BRL-3A protein such that potent polyclonal antisera prepared in rabbits against the purified BRL-3A IGF-binding protein exhibited a much lower titer for the H-35 protein in an enzyme-linked immunosorbent assay and upon immunoblotting. In order to determine whether a single BRL-3A IGF-binding protein is present in the affinity-purified preparation, the protein was prepared for sequencing on a Sephacryl S-300 column in 6 M guanidine HCl after reduction and alkylation. The amino acid composition (expressed in percentages) of this IGF-binding protein was determined to be: Cys = 5.5, Lys = 4.8, His = 2.8, Arg = 7.8, Asx = 10.2, Thr = 5.1, Ser = 3.9, Glx = 15.7, Gly = 17.4, Ala = 7.3, Val = 4.6, Met = 1.4, Ile = 2.4, Leu = 8.3, Tyr = 1.0, Phe = 1.9. Sequencing of the NH2-terminal portion of this protein led to the identification of 31 amino acids in the following order: Phe-Arg-Cys-Pro-Pro-Cys-Thr-Pro-Glu-Arg-Leu-Ala-Ala-Cys-Gly- Pro-Pro-Pro-Asp-Ala-Pro-Cys-Ala-Glu-Leu-Val-Arg-Glu-Pro-Gly- Cys. We conclude that rat liver BRL-3A cells secrete a single major IGF-binding protein capable of binding both IGF-I and IGF-II.

Original languageEnglish (US)
Pages (from-to)11180-11188
Number of pages9
JournalJournal of Biological Chemistry
Volume261
Issue number24
StatePublished - Dec 1 1986

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Insulin-Like Growth Factor Binding Proteins
Liver
Purification
Rats
Insulin-Like Growth Factor II
Insulin-Like Growth Factor I
Conditioned Culture Medium
Proteins
Affinity chromatography
Amino Acids
Immunosorbents
Guanidine
Molecular sieves
Alkylation
Chromatography
Affinity Chromatography
Immunoblotting
Protein Binding
Sodium Dodecyl Sulfate
Acetic Acid

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Purification and amino-terminal sequence of an insulin-like growth factor-binding protein secreted by rat liver BRL-3A cells. / Mottola, C.; MacDonald, Richard G; Brackett, J. L.; Mole, J. E.; Anderson, J. K.; Czech, M. P.

In: Journal of Biological Chemistry, Vol. 261, No. 24, 01.12.1986, p. 11180-11188.

Research output: Contribution to journalArticle

Mottola, C. ; MacDonald, Richard G ; Brackett, J. L. ; Mole, J. E. ; Anderson, J. K. ; Czech, M. P. / Purification and amino-terminal sequence of an insulin-like growth factor-binding protein secreted by rat liver BRL-3A cells. In: Journal of Biological Chemistry. 1986 ; Vol. 261, No. 24. pp. 11180-11188.
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N2 - A protein preparation that specifically binds insulin-like growth factors (IGFs) I and II was purified from medium conditioned by rat liver BRL-3A cells using molecular sieve chromatography in 1 M acetic acid followed by affinity chromatography on IGF-II-agarose. The affinity-purified IGF-binding protein exhibits a single major band with apparent M(r) = 36,300 under reducing conditons on sodium dodecyl sulfate-polyacrylamide gels. The IGF-binding protein is efficiently and specifically cross-linked to either 125I-IGF-I (human) or 125I-IGF-II (rat) using disuccinimidyl suberate. An IGF-binding protein of similar apparent molecular weight was also affinity purified from rat hepatoma H-35 cell conditioned medium and found to differ from the BRL-3A protein such that potent polyclonal antisera prepared in rabbits against the purified BRL-3A IGF-binding protein exhibited a much lower titer for the H-35 protein in an enzyme-linked immunosorbent assay and upon immunoblotting. In order to determine whether a single BRL-3A IGF-binding protein is present in the affinity-purified preparation, the protein was prepared for sequencing on a Sephacryl S-300 column in 6 M guanidine HCl after reduction and alkylation. The amino acid composition (expressed in percentages) of this IGF-binding protein was determined to be: Cys = 5.5, Lys = 4.8, His = 2.8, Arg = 7.8, Asx = 10.2, Thr = 5.1, Ser = 3.9, Glx = 15.7, Gly = 17.4, Ala = 7.3, Val = 4.6, Met = 1.4, Ile = 2.4, Leu = 8.3, Tyr = 1.0, Phe = 1.9. Sequencing of the NH2-terminal portion of this protein led to the identification of 31 amino acids in the following order: Phe-Arg-Cys-Pro-Pro-Cys-Thr-Pro-Glu-Arg-Leu-Ala-Ala-Cys-Gly- Pro-Pro-Pro-Asp-Ala-Pro-Cys-Ala-Glu-Leu-Val-Arg-Glu-Pro-Gly- Cys. We conclude that rat liver BRL-3A cells secrete a single major IGF-binding protein capable of binding both IGF-I and IGF-II.

AB - A protein preparation that specifically binds insulin-like growth factors (IGFs) I and II was purified from medium conditioned by rat liver BRL-3A cells using molecular sieve chromatography in 1 M acetic acid followed by affinity chromatography on IGF-II-agarose. The affinity-purified IGF-binding protein exhibits a single major band with apparent M(r) = 36,300 under reducing conditons on sodium dodecyl sulfate-polyacrylamide gels. The IGF-binding protein is efficiently and specifically cross-linked to either 125I-IGF-I (human) or 125I-IGF-II (rat) using disuccinimidyl suberate. An IGF-binding protein of similar apparent molecular weight was also affinity purified from rat hepatoma H-35 cell conditioned medium and found to differ from the BRL-3A protein such that potent polyclonal antisera prepared in rabbits against the purified BRL-3A IGF-binding protein exhibited a much lower titer for the H-35 protein in an enzyme-linked immunosorbent assay and upon immunoblotting. In order to determine whether a single BRL-3A IGF-binding protein is present in the affinity-purified preparation, the protein was prepared for sequencing on a Sephacryl S-300 column in 6 M guanidine HCl after reduction and alkylation. The amino acid composition (expressed in percentages) of this IGF-binding protein was determined to be: Cys = 5.5, Lys = 4.8, His = 2.8, Arg = 7.8, Asx = 10.2, Thr = 5.1, Ser = 3.9, Glx = 15.7, Gly = 17.4, Ala = 7.3, Val = 4.6, Met = 1.4, Ile = 2.4, Leu = 8.3, Tyr = 1.0, Phe = 1.9. Sequencing of the NH2-terminal portion of this protein led to the identification of 31 amino acids in the following order: Phe-Arg-Cys-Pro-Pro-Cys-Thr-Pro-Glu-Arg-Leu-Ala-Ala-Cys-Gly- Pro-Pro-Pro-Asp-Ala-Pro-Cys-Ala-Glu-Leu-Val-Arg-Glu-Pro-Gly- Cys. We conclude that rat liver BRL-3A cells secrete a single major IGF-binding protein capable of binding both IGF-I and IGF-II.

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