Avtokatalitsko procesiranje rekombinantnega človeškega prokatepsina B : magistrsko delo
Rozman Pungerčar, Jerica
In vitro experiments showed that procathepsin B can be activated by different proteinases, such as pepsin and cathepsin B, as well as by autocatalytic processing at acidic pH. Procathepsin B ...autoprocessing was followed fluorimetrically by discontinuous metods. Incubation of procathepsin B at pH 4.5 and 37 degree C led to sigmoidal increase of activity versus time, which, together with the concentration dependence of procathepsin B autoprocessing, indicate that autocatalytic processing of cathepsin B is a bimolecular process. Ks and k values of 2.1 microM in 0.12 s-1, respectively, were obtained by simultaneous non-linear regression analysis of experimental data. Procathepsin B autoprocessing was substantially slower in the presence of 10% (vžv) and 20% (vžv) glycerol, providing further evidence for the bimolecularity of the process. Autoprocessing of cathepsin B was notably accelerated in the presence of active cathepsin B giving additional support for this idea. To find out if a conformational change preceeding activation isa necessary step in autocatalytic processing of procathepsin B, circular dichroism experiments on procathepsin B, cathepsin B and inactive mutant procathepsin B (Cys29Ser) were performed at pH 4.3 at the beginning of processing and after prolonged exposure to pH 4.3. Slight changes in the secondary structure were observed only in the case of procathepsin B, suggesting that autoprocessing of procathepsin B is not accompanied by a conformational rearrangement of the protein and that propeptide unfolds after activation of the enzyme. Autoprocessing of procathepsin B was found to be optimal at pH r 4.5 (t1ž2 = 75 min at 37 degree C). The processing rate decreased towards acidic and neutral pH and was negligible already at pH 6.0. In the presence of 5 microM dextran sulphate processing was completed within 190 min even at pH 6.0 - 7. (Abstract truncated at 2000 characters)
Type of material - master's thesis
Publication and manufacture - Ljubljana : [J. Rozman], 1999
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