Figure 1
From: Mammalian plasma fetuin-B is a selective inhibitor of ovastacin and meprin metalloproteinases
Activation of pro-ovastacin by human plasmin or by the combination of plasminogen and tissue-type plasminogen activator. (a,b) Ponceau S staining of pro-ovastacin (5.4 µM; 18 µg/lane) following activation with plasmin, separation by SDS-PAGE (12% acrylamide) and transfer onto PVDF membrane. The 54 kDa fragment (#lane 1) comprises the full length pro-ovastacin starting with the N-terminal sequence 24APSA. The 46 kDa fragment (##lane 1) is C-terminally truncated and has the same N-terminus. Edman degradation of the 28 kDa fragment (*lane 3) yielded the N-terminal sequence 86LLSV. (c) Casein-zymography showing plasmin activity (60 kDa) and plasmin-activated ovastacin (28 kDa lane 7), and absence of activity in pro-ovastacin (lane 6). (d) Cartoon showing the composition of pro-ovastacin (#, ##) and ovastacin (*, **) variants produced by proteolytic processing; top line, full-length Strep-tagged pro-ovastacin. (e) Amino acid sequence of pro-ovastacin with C-terminal Strep-tag (italics); catalytic domain in bold face. Protein mass spectrometry of the 28 kDa and the 31 kDa fragments (* and ** in a, lane 3) yielded underlined and orange colored peptides, respectively. (f) 600 nM pro-ovastacin was treated with t-PA (6:1), PLG (10:1) or PLG/t-PA (10:1:1) for 30 min at 37 °C before addition of 10 mM Pefabloc®. PL = activation by active plasmin. 100% ovastacin activity corresponds to a turnover rate of 4.6 nM/s Ac-RE(Edans)-DR-Nle-VGDDPY-K(Dabcyl)-NH2. The error bars indicate the standard deviation of duplicate measurements.
