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

被引:128
作者
Linebaugh, BE
Sameni, M
Day, NA
Sloane, BF
Keppler, D
机构
[1] Wayne State Univ, Sch Med, Dept Pharmacol, Detroit, MI 48201 USA
[2] Wayne State Univ, Sch Med, Barbara Ann Karmanos Canc Inst, Detroit, MI 48201 USA
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1999年 / 264卷 / 01期
关键词
cathepsin B; cellular assay; cysteine peptidase; exocytosis; MCF-10A;
D O I
10.1046/j.1432-1327.1999.00582.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:100 / 109
页数:10
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