Comparison of AGT activity (A), BG resistance
(B), and protein stability (C) in the
mutants AGT-2, -3, -5, and -11 and wt AGT. (A) AGT
activity was determined in extracts from retrovirally transduced K562.
Activity was normalized to protein expression. Percent values represent
mutant AGT activity relative to wt AGT activity. Data represent the
mean of five experiments. Bars = SD. (B) BG
resistance was determined by preincubation of AGT-2 (□),
AGT-3 (⋄), AGT-5 (○), AGT-11 (▵), and wt AGT
(▿) transduced cell extracts with 0–2,000 μM BG before
quantitation of activity. Data are presented as the percent activity
remaining after exposure to BG relative to the activity in
non-BG-treated controls. Data represent the mean of four experiments.
(C) Protein stability was measured by Western blot
comparisons of AGT expression levels in transduced K562 treated with 50
μg/ml cycloheximide for 12 h vs. steady-state AGT levels
in untreated cells. Protein levels were determined by densitometric
quantitation of signal intensity. Data represent the mean of four
experiments.