Purification and sequence analysis of two rat tissue inhibitors of metalloproteinases

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

Research output: Contribution to journalArticlepeer-review


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
Issue number2
StatePublished - Feb 1 1992

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology


Dive into the research topics of 'Purification and sequence analysis of two rat tissue inhibitors of metalloproteinases'. Together they form a unique fingerprint.

Cite this