those from iron-starved cells at 26°C (stationary and exponential phase, respectively; Table 4). Table 4 Reaction rates for four Y. pestis enzyme classes comparing -Fe vs. +Fe conditions Reaction ratea) (nmol min-1 mL-1); (U mL-1)b) Reaction ratea) (nmol min-1 mL-1); (U mL-1)b) Enzyme +Fe, exp, n = 4 e) -Fe, early, n = 5 e) p-value f) +Fe, stat, n = 4 e) -Fe, late, n = 5 e) p-value f) Aconitase c) 2.31 1.14 0.019 4.98 1.82 0.008 Pyruvate oxidase
c) 167.5 1307 0.0001 463.0 2405 click here 0.0001 Catalase d) 82.5 31.8 0.0001 93.4 29.0 0.0001 Superoxide dismutase d) 887.8 426.9 0.002 448.5 312.5 0.234 a) Spectrophotometric assays in 96-well microtiter plates were used for the determination of enzyme reaction rates. Total protein concentrations
in crude cell lysates were the same for all samples used in a given enzyme assay: aconitase, 0.5 mg/mL; pyruvate oxidase, 0.4 mg/mL; catalase, 0.15 mg/mL; superoxide dismutase, 1.1 μg/mL. b) Units ml-1 was the definition for the superoxide dismutase reaction rate. All assays were performed in duplicate. c) Reaction rates from the linear part of the slope of the absorbance change over time. d) Reaction rates from endpoint assays. e) Number of biological replicates of cell lysates (n); exp: abbreviation for exponential, early: early growth phase equivalent to exp. phase (-Fe); average OD600 = 0.66 (+Fe) and OD600 = 0.47 (-Fe); stat: abbreviation for stationary growth phase, late: late growth phase equivalent Nutlin-3a datasheet to stat. phase (-Fe); average OD600 = 2.0 (+Fe) and OD600 = 0.81 (-Fe). True exponential and stationary growth phases were not observed for cell cultures in iron-free media. f) p-values were calculated from to comparison of reaction
rates (+ Fe vs. -Fe) using a two-tailed t-test method. The question STK38 arose whether iron-starved Y. pestis cells activated a different metabolic route of pyruvate degradation able to produce reducing equivalents (NADH and UQH2) for the electron transport chain. Pyruvate oxidase (PoxB) degrades pyruvate to acetate and is a flavin-dependent, iron-independent enzyme that generates UQH2 . The pyruvate oxidase pathway indeed appeared to be important, as judged by the strong abundance increase of PoxB#39 (Figure 4) under -Fe conditions. The flavin cofactor may be recruited from redox activities of two flavodoxins. FldA3#44 was quite abundant and moderately increased in iron-depleted cells (Figure 4). FldA was identified in faint 2D spots and not reproducibly quantitated. PoxB activity measurements revealed excellent correlation between enhanced abundances and increased reaction rates in iron-starved cells. PoxB activities were 5.3-fold and 7.8-fold higher in lysates of iron-starved cells than in lysates of iron-replete cells at 26°C (stationary and exponential phase, respectively; Table 4). Electron transport chains are localized in the IM, a fact that compromised the analysis of subunits of these IM protein complexes in 2D gels.