FIG. 7.

Gel shift experiments to demonstrate interaction of AE and CP (recombinant and reconstituted). (A) Native PAGE of rAE-rCP mixtures. The purified components (20 pmol each of rAE [lane 6] or rCP [lane 7]) or rAE-rCP mixtures with AE/CP ratios (pmol:pmol) of 100:10 (lane 1), 50:10 (lane 2), 10:10 (lane 3), 10:50 (lane 4), and 10:100 (lane 5) were separated by native PAGE (8%) after preincubation (2 h, 10°C) under anoxic conditions. The gel was silver stained. Band a, AE-CP complex; band b, AE; band c, CP. The molecular masses of the marker proteins used are indicated on the left. (B) Influence of the corrinoid cofactor on the interaction. Mixtures of rAE (2 pmol) and rCP (100 pmol; without [−B₁₂] and with [+B₁₂] the corrinoid cofactor hydroxocobalamin) were separated by native PAGE (8%) after preincubation (2 h, 10°C) under anoxic conditions. For protein detection, specific antibodies against AE were used.