Comparison of Kinetic Properties of Native and Recombinant Human Cathepsin D

Abstract

The aspartic proteinase cathepsin D has been shown to function primarily in the degradation of proteins in the lysosomes of most higher eukaryotic cells and, recently, has been evaluated as a prognostic factor in node-negative breast cancer. Synthesized as a preproenzyme and cleaved cotranslationally to proenzyme, procathepsin D is then further processed to the single-chain and finally two-chain active forms of cathepsin D which reside in the lysosome. The rapidity of the initial cleavage of procathepsin D makes it difficult to isolate the zymogen form. In order that the processing¹ and active site requirements of cathepsin D may be examined, human fibroblast procathepsin D has been expressed in E. coli, refolded from solubilized inclusion bodies, and purified using a pepstatin affinity column. To confirm that the recombinant enzyme is an acceptable model for studying substrate specificity and the processing mechanism in vitro, kinetic properties of recombinant cathepsin D were compared to those of native cathepsin D isolated from human placenta and spleen. Kinetic parameters were determined for a series of synthetic chromogenic peptide substrates derived from a parent substrate with the following sequence.