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The ATPase complex of Escherichia coli : studies on the DCCD-binding protein

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Title: The ATPase complex of Escherichia coli : studies on the DCCD-binding protein
Author: Loo, Tip Wah
Degree: Doctor of Philosophy - PhD
Program: Biochemistry and Molecular Biology
Copyright Date: 1983
Subject Keywords Carbodiimides;Adenosine triphosphatase
Issue Date: 2010-05-02
Publisher University of British Columbia
Series/Report no. UBC Retrospective Theses Digitization Project [http://www.library.ubc.ca/archives/retro_theses/]
Abstract: The ATPase complex of E. coll consists of two functional units. ECF[sub=1] is an extrinsic membrane protein having the active site(s) for ATP synthesis and hydrolysis. F₀ is intrinsic and catalyzes the reversible transfer of protons across the membrane. ECF[sub=1] consists of five polypeptides (α- ε) ranging in molecular weight from 13 000 - 57 000. F₀ has three polypeptides (9 000, 18 000, 24 000), the smallest of which is the dicyclohexylcarbodiimide (DCCD)-binding protein postulated to be a transmembrane pathway for proton translocation. An ECF₁F₀ complex was solubilized from the membranes of E. coli with N-lauroyl sarcosine and purified by chromatography on Phenyl-Sepharose CL-4B followed by sedimentation of the enzyme at 250 000 xg for 16-17 h. The purified ECF₁F₀ complex consisted of the eight polypeptides described above, as well as associated polypeptides of molecular weights 30 000, 28 000 and 14 000. Removal of ECF₁ from the membranes of the wild-type E. coli resulted in the membranes becoming leaky to protons so that they could not be energized. The unc mutants, E. coli AN382, CBT-302 and N₁₄₄ could maintain a proton gradient across the membrane in the absence of ECF₁. A normal DCCD-binding protein was present in the F₀ complex of each mutant. However, the 18 000 dalton polypeptide of F₀ was absent in the membranes of E. coli N₁₄₄, suggesting that it was required for a functional F₀. The involvement of the 18 000 dalton polypeptide in the proton-translocating activity was also suggested by the observation that this polypeptide was absent in the ECF₁F₀ complex immunoprecipitated from trypsin-treated "stripped" vesicles, which had been reconstituted with ECF₁. Although these trypsin-treated "stripped" vesicles could rebind ECF₁, the membranes could not be energized during ATP hydrolysis. Leakiness of the membranes to protons could be repaired by the reaction of the ECF₁ stripped membranes with DCCD or ECF₁. Similarly, antibody raised against the DCCD-binding protein prevented this leakage of protons. The antibody also inhibited the rebinding of ECF₁ to the "stripped" everted membrane vesicles. These results indicated that the DCCD-binding protein was exposed on the cytoplasmic surface of the cell. Attempts to show whether the DCCD-binding protein was transmembranous were not successful. Radioimmunoassay techniques were used to show In vitro, the involvement of the arginyl residue(s) of the DCCD-binding protein in the binding of ECF₁. Binding of ECF₁ to the DCCD-binding protein appeared to involve the α and/or β subunits of ECF₁. Chemical modification of the methionyl residue(s) of the DCCD-binding protein did not alter its capacity to bind ECF₁, but destroyed the antigenic site(s) of the polypeptide. In summary, these results are consistent with the proposed "loop" arrangement of the DCCD-binding protein in which the polar central region of this molecule is at the cytoplasmic surface of the cell membrane.
Affiliation: Science, Faculty of
URI: http://hdl.handle.net/2429/24313
Scholarly Level: Graduate

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