Characterization and partial purification of the insulin-like growth factor (IGF)-dependent IGF binding protein-4-specific protease from human fibroblast conditioned media

James B. Lawrence, Laurie K. Bale, Tufia C Haddad, Jay T. Clarkson

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Insulin-like growth factors (IGFs) are one of the most potent stimulators of cell growth. IGFs are modulated by six high affinity binding proteins (IGFBPs) which are, in turn, regulated through post-translational modifications such as proteolysis. In the conditioned media of human fibroblasts, IGFBP-4 is cleaved by an apparently novel IGFBP-4-specific protease that requires IGF for functional activity. We have used several biochemical manipulations, including size exclusion chromatography, native gel electrophoresis, chaotropic salt precipitation, hydrophobic interaction chromatography, ion exchange chromatography, isoelectric focusing, lectin affinity chromatography, and metal chelating affinity chromatography to both characterize and partially purify the IGF-dependent IGFBP-4 protease. Our results indicate that this protease is a highly glycosylated, Zn+2 binding metalloprotease with a native molecular weight greater than 200 kDa.

Original languageEnglish (US)
Pages (from-to)25-34
Number of pages10
JournalGrowth Hormone and IGF Research
Volume9
Issue number1
DOIs
StatePublished - 1999

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Pregnancy-Associated Plasma Protein-A
Somatomedins
Conditioned Culture Medium
Fibroblasts
Affinity Chromatography
Insulin-Like Growth Factor Binding Protein 4
Insulin-Like Growth Factor Binding Proteins
Ion Exchange Chromatography
Isoelectric Focusing
Metalloproteases
Post Translational Protein Processing
Hydrophobic and Hydrophilic Interactions
Lectins
Proteolysis
Gel Chromatography
Electrophoresis
Chromatography
Carrier Proteins
Peptide Hydrolases
Salts

Keywords

  • Human fibroblasts
  • IGF-II
  • IGFBP-4
  • Metalloprotease

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

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abstract = "Insulin-like growth factors (IGFs) are one of the most potent stimulators of cell growth. IGFs are modulated by six high affinity binding proteins (IGFBPs) which are, in turn, regulated through post-translational modifications such as proteolysis. In the conditioned media of human fibroblasts, IGFBP-4 is cleaved by an apparently novel IGFBP-4-specific protease that requires IGF for functional activity. We have used several biochemical manipulations, including size exclusion chromatography, native gel electrophoresis, chaotropic salt precipitation, hydrophobic interaction chromatography, ion exchange chromatography, isoelectric focusing, lectin affinity chromatography, and metal chelating affinity chromatography to both characterize and partially purify the IGF-dependent IGFBP-4 protease. Our results indicate that this protease is a highly glycosylated, Zn+2 binding metalloprotease with a native molecular weight greater than 200 kDa.",
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author = "Lawrence, {James B.} and Bale, {Laurie K.} and Haddad, {Tufia C} and Clarkson, {Jay T.}",
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T1 - Characterization and partial purification of the insulin-like growth factor (IGF)-dependent IGF binding protein-4-specific protease from human fibroblast conditioned media

AU - Lawrence, James B.

AU - Bale, Laurie K.

AU - Haddad, Tufia C

AU - Clarkson, Jay T.

PY - 1999

Y1 - 1999

N2 - Insulin-like growth factors (IGFs) are one of the most potent stimulators of cell growth. IGFs are modulated by six high affinity binding proteins (IGFBPs) which are, in turn, regulated through post-translational modifications such as proteolysis. In the conditioned media of human fibroblasts, IGFBP-4 is cleaved by an apparently novel IGFBP-4-specific protease that requires IGF for functional activity. We have used several biochemical manipulations, including size exclusion chromatography, native gel electrophoresis, chaotropic salt precipitation, hydrophobic interaction chromatography, ion exchange chromatography, isoelectric focusing, lectin affinity chromatography, and metal chelating affinity chromatography to both characterize and partially purify the IGF-dependent IGFBP-4 protease. Our results indicate that this protease is a highly glycosylated, Zn+2 binding metalloprotease with a native molecular weight greater than 200 kDa.

AB - Insulin-like growth factors (IGFs) are one of the most potent stimulators of cell growth. IGFs are modulated by six high affinity binding proteins (IGFBPs) which are, in turn, regulated through post-translational modifications such as proteolysis. In the conditioned media of human fibroblasts, IGFBP-4 is cleaved by an apparently novel IGFBP-4-specific protease that requires IGF for functional activity. We have used several biochemical manipulations, including size exclusion chromatography, native gel electrophoresis, chaotropic salt precipitation, hydrophobic interaction chromatography, ion exchange chromatography, isoelectric focusing, lectin affinity chromatography, and metal chelating affinity chromatography to both characterize and partially purify the IGF-dependent IGFBP-4 protease. Our results indicate that this protease is a highly glycosylated, Zn+2 binding metalloprotease with a native molecular weight greater than 200 kDa.

KW - Human fibroblasts

KW - IGF-II

KW - IGFBP-4

KW - Metalloprotease

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