Fig. 1.

Purified Bet3 has self-palmitoylating activity. (A) Bet3 purified from E. coli (E; 19 μg) or from yeast (Y; 8 μg) was incubated with [³H]Pal-CoA for 30 min at 30°C. Samples were subjected to SDS/PAGE under nonreducing conditions and fluorography for 4 h. (B) Bet3 purified from yeast was incubated with [³H]Pal-CoA in the absence (−) or presence of SDS (0.1%), urea (Ur; 6.75 M), or Triton X-100 (TX; 0.1%) or heated to 80°C for 5 min before the assay was performed. (C) Increasing amounts of Br-Pal were added to the reaction with Bet3 purified from yeast. Palmitate alone had no effect on the reaction up to a concentration of 1 mM. (D) Bet3 purified from yeast was incubated with [³H]Pal-CoA for the times (in minutes) indicated. (Inset) The resulting fluorogram was quantified, and relative palmitoylation is plotted against time of incubation. (E) Bet3 purified from yeast was incubated with [³H]Pal-CoA at different pH values as indicated. (Inset) The resulting fluorogram was quantified, and relative palmitoylation is plotted against the pH.