Figure 9.

The AF-FOB conjugate does not reach its target in vivo.A) The IC₅₀ values of free AF (○), DFOB-AF (•) and FOB-AF (▪) for protopor. oxidase activity were determined in vitro on whole cell extracts. B) Wild-type S. cerevisiae cells were plated as a lawn on YPGly agar. Wells were created in the agar with a hole punch, into which 50 μL of 1 mm FOB-AF or 25 μL FOB 2 mm + 25 μL AF 2 mm was dispensed. Cell growth on the plate was examined after 3 days at 30 seconds. It was inhibited only with FOB + AF (inhibition visible as a halo around the bottom well). C) Accumulation of FOB and FOB conjugates in cells overexpressing SIT1. Sit1Δ cells transformed with pGAL-SIT1-GFP were cultured overnight in raffinose-containing medium to midexponential growth phase. We then added glucose (2%) or galactose (2%) and cultured the cells for a further 4 h. ⁵⁵Fe-labelled FOB, FOB-NBD or FOB-AF was then added, and the amount of ⁵⁵Fe accumulated by the cells was determined after 15 min of incubation. Results are expressed as means ± standard error of the mean for six experiments. D) Accumulated FOB-AF inhibits protopor. oxidase in vitro. Sit1Δ cells transformed with pGAL-SIT1-GFP were cultured overnight in galactose-containing medium in the presence of 100 μm FOB (□) or 100 μm FOB-AF (▪). The cells were then washed and disrupted with glass beads. The activity of protopor. oxidase was determined on whole cell extracts at various protein concentrations. Protopor., protoporphyrinogen.