Additional file 4.

M, L, and ML variants are resistant to furin, revealed by an in vitro protease digestion assay. Ten-amino-acid polypeptides that contained the cleavage site of proBDNF with the indicated amino acid residues were conjugated with MOCAc and DNP at the N- and C-termini, respectively [60]: the latter quenches the fluorescence in the absence of peptide cleavage. The peptides were incubated with the widely distributed prohormone convertase, furin (10 units/ml) [2]. Cleavage of wild-type BDNF (BDNF_RR) peptide by furin would separate the fluorogenic group from the quencher and would thus generate a measurable fluorescent signal. A time-course study revealed a gradual increase in fluorescence after the incubation of the BDNFRR peptide with furin [2], which reached a maximum at 4 h (black circles). In contrast, R125M-BDNF (BDNF_M), R127L-BDNF (BDNF_L), and R125M/R127L-BDNF (BDNF_ML) peptides did not elicit a significant increase in fluorescence (red, blue, and purple circles, respectively). Mass spectrometric analysis confirmed that the BDNF_RR peptide was precisely cleaved at the processing site (data not shown).

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Koshimizu et al. Molecular Brain 2009 2:27   doi:10.1186/1756-6606-2-27