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A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl
Published on: November 28, 2014
Activation profiles of the zymogen of aspergilloglutamic peptidase
Xiang-Ping Huang1, Yutaka Yabuki, Masaki Kojima
1Laboratory of Molecular Biochemistry, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Abstract:
Aspergilloglutamic peptidase produced by Aspergillus niger var. macrosporus belongs to the novel glutamic peptidase family. Its zymogen is autocatalytically activated under acidic conditions to the mature enzyme with a two-chain structure. Analyses by SDS-PAGE and mass spectrometry of the activation products of the recombinant zymogen showed that the major pathway of activation includes initial fast cleavage at Glu12-Ala13, followed by stepwise cleavages in the N-terminal and intervening propeptide regions. Essentially the same activation profile was obtained with the recombinant zymogen lacking the N-terminal 12-aa sequence. The missing region includes the most prominent cluster of basic residues of the propeptide, indicating low importance of this cluster for activation and refolding of the zymogen.
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