1% (v/v) TFA. External mass calibration was performed with low-mass peptide standards (PerSeptive Biosystems). For the characterization of products of cell wall breakdown, postsource decay (PSD) fragment ion spectra were obtained after isolation of the DNA Damage inhibitor appropriate precursor using timed ion selection. Fragment ions were
refocused onto the final detector by stepping the voltage applied to the reflector and individual segments combined using perseptive biosystems software (De Simone et al., 2009). CHCA was used in this study according to Boneca et al., 2000. The sample (1 μL, in water) was loaded on the target, dried, and re-dissolved in CHCA (1 μL, 10 mg mL−1 in 0.1% TFA in 50% aqueous acetonitrile). For http://www.selleckchem.com/products/sorafenib.html each sample, 200 laser pulses were accumulated. Concentration of purified sakacin A was calculated by assuming ε280 = 14 105
(mol−1 cm−1; http://web.expasy.org/protparam/; Kelly et al., 2005). The bacteriocin titer was determined by a serial dilution assay, activity being defined as the reciprocal of the last serial dilution that exhibited a clear zone of inhibition and being expressed as activity units (AU; De Kwaadsteniet et al., 2005). Changes in the cell transmembrane electrical potential were measured by quenching of the potential-sensitive fluorescent probe 3, 3-dipropylthiadicarbocyanine iodide (diSC3; Molecular Probes Inc., Eugene, OR; Deraz et al., 2005). Cells were suspended in 50 mM potassium-HEPES, pH 7, containing 0.2% glucose (final OD600 nm = 0.4), to give glucose-energized cells. The probe (5 μM) and nigericin (1.5 μM) were mixed with the
glucose-energized cell suspension, and sakacin A (80 AU mL−1) or valinomycin (1.5 μM) was added as appropriate. Fluorescence was measured at 30 °C in a spectrofluorometer (Model LS 50; PerkinElmer, Milan, Italy), with excitation at 643 nm and emission at 666 nm (Suzuki et al., 2005). Changes in the transmembrane pH gradient were measured with the pH-sensitive fluorescent probe 5 (and 6) carboxyfluorescein diacetate succinimidyl ester (cFDASE; Molecular Probes Inc.; McAuliffe et al., 1998). The cells were concentrated threefold in 1.5 mL of 50 mM potassium-HEPES Epothilone B (EPO906, Patupilone) buffer, pH 8, and then incubated at 30 °C for 10 min with the probe (1 μM). Nonconjugated probe was eliminated by incubating the cells with 10 mM lactose at 30 °C for 30 min. The cells were washed twice, suspended in 50 mM potassium phosphate buffer at pH 7 and placed on ice until used. The intracellular pH was determined by diluting the lactose-loaded cells to a concentration of 107 CFU mL−1 in a 3-mL glass cuvette. Fluorescence was measured as reported earlier. Bacterial cell walls were isolated according to Simelyte et al. (2000).