Abstracts

Abstract

Procathepsin B undergoes autocatalytic processing to its active form after exposure to acidic pH. In the present study the effect of pH, proenzyme concentration and dextran sulphate on the processing of recombinant human procathepsin B was investigated in order to determine the mechanism of this process. The processing was followed fluorimetricaly, using Z-Arg-Arg-AMC as a substrate for cathepsin B. We obtained sigmoidal curves, which were concentration dependent, indicating that the processing of cathepsin B is a bimolecular process. The K, and kat values of 4.1pM and 2 . 4~1 0 "~ respectively, were obtained by simultaneus nonlinear regression analysis of 15 data sets. Processing of procathepsin B was also followed by SDS-PAGE. Activation was completed within 30 minutes, in agreement with simulation experiments at high procathepsin B concentration (1 8-8OnM). Changes in tertiary and secondary structures during the processing were followed by circular dichroism measurements of procathepsin B, cathepsin B and mutant inactive procathepsin B(C25S).