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

Lysosomal cathepsin B has been implicated in parasitic, inflammatory and ne oplastic diseases. Most of these pathologies suggest a role for cathepsin B outside the cells, although the origin of extracellular active enzyme is n ot 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 m easurement 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 validat e the assay, we determined that human liver cathepsin B was stable and acti ve 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 d emonstrate that active cathepsin B was secreted pericellularly by viable ce lls. Both preneoplastic and malignant cells secreted active cathepsin B. Pr etreatment of cells with the membrane-permeant proinhibitor CA-074Me comple tely abolished pericellular and total cathepsin B activity whereas pretreat ment with the active drug CA-074 had no effect. Immunoprecipitation and imm unoblotting experiments suggested that the active enzyme species was 31-kDa single-chain cathepsin B. Exocytosis of cathepsin B was not related to sec retion of proenzyme or secretion from mature lysosomes. Our results suggest an alternative pathway for exocytosis of active cathepsin B.