Purification and sequence analysis of two rat tissue inhibitors of metalloproteinases

William T. Roswit, David W. McCourt, Nicola Partridge, John J. Jeffrey

Research output: Contribution to journalArticle

Abstract

Two protein inhibitors of metalloproteinases (TIMP) were isolated from medium conditioned by the clonal rat osteosarcoma line UMR 106-01. Initial purification of both a 30-kDa inhibitor and a 20-kDa inhibitor was accomplished using heparin-Sepharose chromatography with dextran sulfate elution followed by DEAE3-Sepharose and CM-Sepharose chromatography. Purification of the 20-kDa inhibitor to homogeneity was completed with reverse-phase high-performance liquid chromatography. The 20-kDa inhibitor was identified as rat TIMP-2. The 30-kDa inhibitor, although not purified to homogeneity, was identified as rat TIMP-1. Amino terminal amino acid sequence analysis of the 30-kDa inhibitor demonstrated 86% identity to human TIMP-1 for the first 22 amino acids while the sequence of the 20-kDa inhibitor was identical to that of human TIMP-2 for the first 22 residues. Treatment with peptide:N-glycosidase F indicated that the 30-kDa rat inhibitor is glycosylated while the 20-kDa inhibitor is apparently unglycosylated. Inhibition of both rat and human interstitial collagenase by rat TIMP-2 was stoichiometric, with a 1:1 molar ratio required for complete inhibition. Exposure of UMR 106-01 cells to 10-7 m parathyroid hormone resulted in approximately a 40% increase in total inhibitor production over basal levels.

Original languageEnglish (US)
Pages (from-to)402-410
Number of pages9
JournalArchives of Biochemistry and Biophysics
Volume292
Issue number2
DOIs
StatePublished - Feb 1 1992

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Tissue Inhibitor of Metalloproteinases
Metalloproteases
Purification
Sequence Analysis
Rats
Agarose Chromatography
Proteins
Chromatography
Sepharose
Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
Amino Acids
Dextran Sulfate
Matrix Metalloproteinase 1
Protein Sequence Analysis
High performance liquid chromatography
Osteosarcoma
Reverse-Phase Chromatography
Conditioned Culture Medium
Parathyroid Hormone
Amino Acid Sequence

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Purification and sequence analysis of two rat tissue inhibitors of metalloproteinases. / Roswit, William T.; McCourt, David W.; Partridge, Nicola; Jeffrey, John J.

In: Archives of Biochemistry and Biophysics, Vol. 292, No. 2, 01.02.1992, p. 402-410.

Research output: Contribution to journalArticle

Roswit, William T. ; McCourt, David W. ; Partridge, Nicola ; Jeffrey, John J. / Purification and sequence analysis of two rat tissue inhibitors of metalloproteinases. In: Archives of Biochemistry and Biophysics. 1992 ; Vol. 292, No. 2. pp. 402-410.
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