Exocytosis of active cathepsin B enzyme activity at pH 7.0, inhibition and molecular mass

Eur J Biochem. 1999 Aug;264(1):100-9. doi: 10.1046/j.1432-1327.1999.00582.x.

Abstract

Lysosomal cathepsin B has been implicated in parasitic, inflammatory and neoplastic diseases. Most of these pathologies suggest a role for cathepsin B outside the cells, although the origin of extracellular active enzyme is not well defined. The activity of extracellular cathepsin B is difficult to assess because of the presence of inhibitors and inactivation of the enzyme by oxidizing agents. Therefore, we have developed a continuous assay for measurement of cathepsin B activity produced pericellularly by living cells. The kinetic rate of Z-Arg-Arg-NHMec conversion was monitored and the assay optimized for enzyme stability, cell viability and sensitivity. To validate the assay, we determined that human liver cathepsin B was stable and active under the conditions of the assay and its activity could be inhibited by the selective epoxide derivative CA-074. Via this assay, we were able to demonstrate that active cathepsin B was secreted pericellularly by viable cells. Both preneoplastic and malignant cells secreted active cathepsin B. Pretreatment of cells with the membrane-permeant proinhibitor CA-074Me completely abolished pericellular and total cathepsin B activity whereas pretreatment with the active drug CA-074 had no effect. Immunoprecipitation and immunoblotting experiments suggested that the active enzyme species was 31-kDa single-chain cathepsin B. Exocytosis of cathepsin B was not related to secretion of proenzyme or secretion from mature lysosomes. Our results suggest an alternative pathway for exocytosis of active cathepsin B.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cathepsin B / antagonists & inhibitors
  • Cathepsin B / chemistry
  • Cathepsin B / metabolism*
  • Cell Line, Transformed
  • Exocytosis*
  • Fluorescent Antibody Technique
  • Humans
  • Hydrogen-Ion Concentration
  • Kinetics
  • Molecular Weight
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Tumor Cells, Cultured

Substances

  • Cathepsin B