Pulification and identification of substrate proteins for selective ubquitin-dependent proteolysis during NGF-induced neurite outgrowth of PC12h cells.

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Optimization of purification protocols.Using K562 cells we established methods for purifying cellular ubiquitinated proteins. The first step is an immunoaffinity chromatography using a monoclonal antibody FK2 to ubiquitin. One ml of FK2-Sepharose gel was able to adsorb abount 0.2 mg of ubiquitin equivalent, and the adsorbed materials were completely eluted with 3..5 M MgClィイD22ィエD2. Then the materials were fractionated by gel filtration using Superdex 75, and two characteristic fractions having high molecular weights (HMW) (>30 kDa) and low molecular weights (LMW) (<30 kDa) were obtained. The former was mainly composed of multi-ubiquitinated proteins. In the latter we found ubiquitin thioesters with ubiquitin-conjugating enzymes (E2) as well as ubiquitinated proteins.Ubiquitinated protein variety during neurite elongation.By the combination of immunoprecipitation using FK2 antibody and SDS-PAGE, we visualized ubiquitinated proteins in PC12h cells cultivated in the presence of nerve gro … More wth factor (NGF). Three bands, 18, 19, and 36-kDa, newly appeared, and intensities of six bands, 20, 23, 26, 51, 68, and 93-kDa, increased.Purification of ubiquitinated proteins.Ubiquitinated protein mixtures (0.56 mg protein) were obtained from NGF-treated PC12h cells (2 x 10ィイD19ィエD1 cells) by the immunoaffinity chromatography. The mixtures were further separated by the gel filtration, and resulted in the two distinctive fractions of HMW and LMW. Several protein bands were isolated from the latter by SDS-PAGE, and amino acid sequencing of their peptide fragments obtained by lysyl-endopeptidase is ongoing in turns. The analysis of the 26-kDa band resulted in two amino acid sequences of ubiquitin digests and five unknown sequences.A monoclonal antibody against ubiquitin C-terminal tag peptide.This antibody is required for identification of the target proteins. Because of antigen insolubility, we had to change the peptide design, and finally established the desired antibody, which will be applied to the analysis of HMW ubiquitinated proteins. Less