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Kotlarz, Agnieszka; Szalewska-Pałasz, Agnieszka; Wgrzyn, Grzegorz; Lipíska, Barbara

doi:10.1023/a:1008853209547

Cited by 2 publications

(2 citation statements)

References 8 publications

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“…Induction with IPTG produced a high level of expression of each protein. σ 32 was easily purified by a modification of existing protocols ( , ). Attempts to extract soluble FtsH from a crude E. coli membrane fraction were unsuccessful; even high concentrations (5% v/v) of the nonionic detergent NP-40 failed to solubilize FtsH protein.…”

Section: Resultsmentioning

confidence: 99%

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Coupled Kinetics of ATP and Peptide Hydrolysis by Escherichia coli FtsH Protease

2003

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FtsH from Escherichia coli is an ATP- and Zn(2+)-dependent integral membrane protease that is involved in the degradation of regulatory proteins such as sigma(32) and uncomplexed subunits of membrane protein complexes such as secY of the protein translocase. We describe a protocol for solubilizing the recombinant enzyme from inclusion bodies and its subsequent refolding and purification to near homogeneity. This is a high-yield protocol and produces in excess of 20 mg of purified FtsH per liter of E. coli culture. We found that refolded FtsH has biochemical properties similar to detergent extracted overexpressed protein described previously. FtsH forms a large complex with an apparent mass of 1200 kDa as determined by gel filtration. Both ATPase and protease activities are coincident with this large complex; smaller forms of FtsH do not exhibit either activity. While FtsH-catalyzed hydrolysis of ATP can occur in the absence of protein substrate (k(c) = 22 min(-1); K(m) = 23 microM), proteolysis shows an absolute dependence on nucleoside-5'-triphosphates, including ATP, CTP, and various analogues. In the presence of 5 mM ATP, FtsH catalyzes the hydrolysis of sigma(32) with the following observed kinetic parameters: k(c) = 0.18 min(-1) and K(m) = 8.5 microM. Significantly, this reaction is processive and generates no intermediate species, but rather, approximately 10 peptide products, all of MW <3 kDa. FtsH protease also efficiently hydrolyzes the peptide Phe-Gly-His-(NO)2Phe-Phe-Ala-Phe-OMe. Hydrolysis occurs exclusively at the (NO)2Phe-Phe bond (k(c) = 2.1 min(-1); K(m) = 12 microM), and like proteolysis, shows an absolute dependence on NTPs. We propose a mechanism for the coupled hydrolytic activities of FtsH toward ATP and peptide substrates that is consistent with a recently proposed structural model for FtsH.

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Section: Resultsmentioning

confidence: 99%

“…Purification of σ 32 . The purification protocol is a modification of existing protocols ( , ). Cells containing the σ 32 expression construct were grown at 28 °C in 350 mL of LB media plus 100 μg/mL ampicillin.…”

Section: Methodsmentioning

confidence: 99%

Coupled Kinetics of ATP and Peptide Hydrolysis by Escherichia coli FtsH Protease

2003

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The DnaK chaperones from the archaeon Methanosarcina mazei and the bacterium Escherichia coli have different substrate specificities.

Żmijewski

Skórko-Glonek²,

Tanfani³

et al. 2007

Acta Biochim Pol

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Hsp70 (DnaK) is a highly conserved molecular chaperone present in bacteria, eukaryotes, and some archaea. In a previous work we demonstrated that DnaK from the archaeon Methanosarcina mazei (DnaK(Mm)) and the DnaK from the bacterium Escherichia coli (DnaK(Ec)) were functionally similar when assayed in vitro but DnaK(Mm) failed to substitute for DnaK(Ec) in vivo. Searching for the molecular basis of the observed DnaK species specificity we compared substrate binding by DnaK(Mm) and DnaK(Ec). DnaK(Mm) showed a lower affinity for the model peptide (a-CALLQSRLLS) compared to DnaK(Ec). Furthermore, it was unable to negatively regulate the E. coli sigma32 transcription factor level under heat shock conditions and poorly bound purified sigma32, which is a native substrate of DnaK(Ec). These observations taken together indicate differences in substrate specificity of archaeal and bacterial DnaKs. Structural modeling of DnaK(Mm) showed some structural differences in the substrate-binding domains of DnaK(Mm) and DnaK(Ec), which may be responsible, at least partially, for the differences in peptide binding. Size-exclusion chromatography and native gel electrophoresis revealed that DnaK(Mm) was found preferably in high molecular mass oligomeric forms, contrary to DnaK(Ec). Oligomers of DnaK(Mm) could be dissociated in the presence of ATP and a substrate (peptide) but not ADP, which may suggest that monomer is the active form of DnaK(Mm).

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Cited by 2 publications

References 8 publications

Coupled Kinetics of ATP and Peptide Hydrolysis by Escherichia coli FtsH Protease

Coupled Kinetics of ATP and Peptide Hydrolysis by Escherichia coli FtsH Protease

The DnaK chaperones from the archaeon Methanosarcina mazei and the bacterium Escherichia coli have different substrate specificities.

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