Spectrometric analysis of degradation of a physiological substrate sigma32 by Escherichia coli AAA protease FtsH.
We have established a fluorescence polarization assay system by which degradation of sigma32, a physiological substrate, by FtsH can be monitored spectrometrically. Using the system, it was found that an FtsH hexamer degrades approximately 0.5 molecules of Cy3-sigma32 per min at 42 degrees C and hydrolyzes approximately 140 ATP molecules during the degradation of a single molecule of Cy3-sigma32. Evidence also suggests that degradation of sigma32 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) = 52 degrees C).
Okuno
Takashi
T
Division of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 862-0976, Japan.
Yamada-Inagawa
Tomoko
T
Karata
Kiyonobu
K
Yamanaka
Kunitoshi
K
Ogura
Teru
T
D049069:ATP-Dependent Proteases; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002232:Carbocyanines; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005454:Fluorescence Polarization; D005982:Glutathione Transferase; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D020125:Mutation, Missense; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods
D001426:Bacterial Proteins; D002232:Carbocyanines; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; C089187:cyanine dye 3; C069774:heat-shock sigma factor 32; D005982:Glutathione Transferase; D049069:ATP-Dependent Proteases; C513383:FtsH protein, E coli