Fig. 1. Identification of BNIP-H and KGA interaction. (A) Bacterially expressed GST-BNIP-H immobilised on glutathione-Sepharose beads was incubated either with lysis buffer or rat brain lysate. As a control, glutathione-Sepharose beads coated with GST-BNIP-H were mixed with lysis buffer and processed the same way. After elution, bound proteins and brain lysate were resolved by SDS-PAGE and visualised by silver staining (lanes 1-5). A unique band around 70 kDa (labelled with asterisks) was subjected to trypsin-digestion followed by MALDI-TOF analysis (see Materials and Methods) and identified as kidney-type glutaminase (KGA). Comparison of eluate and brain lysate indicated an enrichment of KGA by BNIP-H (lanes 4 and 5). The identification was confirmed by western blotting with an anti-glutaminase antibody (lanes 6 and 7). (B,C) The GST pull-down and its analysis as described in A were repeated with whole-cell lysates prepared from PC12 (B) and Neuro2A cells (C). (D) The unique band, as indicated in A, was subjected to MALDI-TOF analyses as described in Materials and Methods. The tryptic peptides that correspond to and cover 27% of the protein sequence of rat KGA are underlined.