We have established a fluorescence polarization assay system by which degradation of sigma(32), a physiological substrate, by FtsH can be monitored spectrometrically. Using the system, it was found that an FtsH hexamer degrades similar to0.5 molecules of Cy3-sigma(32) per min at 42degreesC and hydrolyzes similar to140 ATP molecules during the degradation of a single molecule of Cy3-sigma(32). Evidence also suggests that degradation of sigma(32) proceeds from the N-terminus to the C-terminus. Although FtsH does not have a robust enough unfoldase activity to unfold a tightly folded proteins such as green fluorescent protein, it can unfold proteins with lower T(m)s such as glutathione S-transferase (T-m = 52degreesC). (C) 2003 Elsevier Inc. All rights reserved.