In vitro cleavage of APPswe by pure recombinant ASP-2; differential cleavage occurs at acidic or alkaline pHPanel A: anti-CT15 IgG immunoprecipitated (35S)-labelled APPswe (2 μM APPswe) was incubated with either just buffer (lane 1) or with pure ASP-2 at 6 nM (lane 2). The total reaction was analysed on a16.5% Tris-Tricine gel, stained and fixed, and amplified as described in the methods. The gel was then dried and exposed on a Kodak-X-Omat film. Panel B: Immunoprecipitated APPswe samples as above were incubated with or without ASP-2 as described above at pH 5 or ph 8.5 as indicated in the figure. The blots were developed with anti-CT15 IgG that is specific for the C-terminal of APP. The bottom panels are the same reaction probed with the 6E10 clone antibody, which was raised specifically against the first residues of Aβ and thus only recognises β-secretase cleaved fragments that produce Aβ. Panel C: A typical gel calibration standard curve for the low molecular weight standards (carbonic anhydrase, 34.3 kDa, soybean trypsin inhibitor, 26 kDa, Lysozyme, 17.9, kDa, Aprotinin, 8 kDa, insulin, 4 kDa) plotted as the Log10 of molecular weight against Rf, for size estimation of peptides. For all panels α- and β-secretase cleavage are designated by arrows and the letters α or β for the respective secretase cleavage. β1 and β2 indicate cleavage at the Aspartate (1) and the Glutamate (11) sites of Aβ, producing the C-100 and a C-90 fragment respectively.
Sidera et al. BMC Biochemistry 2002 3:25 doi:10.1186/1471-2091-3-25