Alzheimer's disease (AD) brain accumulates beta-protein (A beta) a peptide proteolytically derived from the beta-amyloid precursor protein (APP). The abnormal production and aggregation of A beta have been implicated in the pathogenesis of the disease. The mechanism of production of A beta in vivo is not yet clear; but endoproteases capable of degrading APP are likely to be involved in the process. We have isolated a protease from AD brain by following its activity in digesting a synthetic peptide of 10 amino acids derived from the APP sequence flanking the N-terminus of A beta. The protease was purified by a fractionation scheme including ammonium sulfate precipitation and column chromatography using hydrophobic interaction, anion exchange, affinity, hydroxyapatite and size exclusion gels. The purity of the final product was assessed on a silver stained SDS gel by the presence of a single band. Microsequencing was performed following trypsin digestion of the sample. Internal peptide sequences were found to have sequence homology to cysteine proteases in the database. The enzyme requires DTT for activity and can be inhibited by specific inhibitors of cysteine but not serine proteases. The purified enzyme has a pI of 5.0 and a native tetrameric structure with subunits of 48 kD each. The enzyme is capable of digesting APP and generating a short peptide recognizable by antibodies specific to the C-terminus of APP. Interestingly, the purified protease also forms heat- and SDS-stable complexes with APP.
The lysyl ester analogue p-nitrophenyl alpha-amino-p-toluate hydrobromide was synthesized, and its reactions with thrombin, trypsin, and plasmin were studied by stopped-flow and conventional methods. Kinetic parameters were compared with those determined for the arginyl ester analogue, p-nitrophenyl p-guanidinobenzoate hydrochloride, with these enzymes. By following nitrophenol release or proflavin absorption changes in the stopped-flow spectrophotometer, the constants Ks (enzyme-substrate binding), k2 (acylation), and k3 (deacylation) were determined. The major findings were: (1) Ks values were similar regardless of the substrate or the enzyme; (2) k3 was approximately the same for the reaction of the lysyl ester analogue with any enzyme; (3) k2 for the lysyl ester analogue was 1100 times greater with trypsin than with thrombin; and (4) k2 with thrombin was 60 times greater for the arginyl than for the lysyl ester analogue. The results suggest that the limited cleavage of lysyl bonds by thrombin is due in part to restricted acylation rather than substrate binding. The active site of thrombin, compared with that of trypsin, appears to have a more stringent requirement for the spatial relationship between the cationic group and the bond cleaved in substrates.
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