Go to  Advanced Search

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

Show full item record

Files in this item

Files Size Format Description   View
UBC_1983_A1 L65_8.pdf 14.63Mb Adobe Portable Document Format   View/Open
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
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.
URI: http://hdl.handle.net/2429/24313
Series/Report no. UBC Retrospective Theses Digitization Project [http://www.library.ubc.ca/archives/retro_theses/]
Scholarly Level: Graduate

This item appears in the following Collection(s)

Show full item record

All items in cIRcle are protected by copyright, with all rights reserved.

UBC Library
1961 East Mall
Vancouver, B.C.
Canada V6T 1Z1
Tel: 604-822-6375
Fax: 604-822-3893