The Carboxyl Fragment Released by Bacterial Collagenase from Human Type I Procollagen: Antibodies to the Propeptide Determinants

Burton D. Goldberg*, Robert G. Phelps, Efrat Kessler, Michael J. Klein, Mark B. Taubman

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

Abstract

A protocol is offered for the isolation of the carboxyterminal propeptides of human type I procollagen and the development of an antibody specific for these propeptides. Type I procollagen was harvested from the media of cultured human fibroblasts. Digestion with bacterial collagenase released a carboxyterminal fragment that was isolated by ion-exchange chromatography. The fragment contained telopeptides joined to propeptides and could be cleaved by a carboxyl procollagen peptidase. Rabbit antibodies raised to the colt agenase-generated fragment were sequentially adsorbed on affinity columns of the reference antigen and human type I collagen. The antibody obtained was shown by sensitive radioimmunoassays to recognize conformational carboxyl propeptide determinants and not to react with triple helical and telopeptide determinants of human type I collagen. Indirect immunofluorescence and indirect immunoperoxidase staining of cultured fibroblasts localized the antigen in the cytoplasm, at the cell surface, and in the extracellular matrix. A radioimmunoassay with the same antibody has reported altered concentrations of the antigen in the sera of patients with diseases affecting collagen metabolism (Taubman, et al., 1976; Savolainen et al., 1984; Carey et al., 1985).

Original languageEnglish
Pages (from-to)393-404
Number of pages12
JournalTopics in Catalysis
Volume5
Issue number5
DOIs
StatePublished - 1985

Keywords

  • EDTA
  • PMSF
  • SDS-PAGE
  • affinity-purified antibody
  • carboxyl propeptide antigen
  • disodium ethylenediamine -tetra acetate
  • immunocytochemistry
  • phenyl methyl -sulfon
  • radioimmunoassay for specificity
  • sodium lauryl sulfate-polyacrylamide gel electrophoresis

Cite this