Date of Award
1987
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Abstract
Twenty-one monoclonal antibodies to Escherichia coli F{dollar}\sb 1{dollar}-ATPase were characterized and used in structural studies of the enzyme. Solid phase competition assays placed antibodies recognizing the same or overlapping epitopes into competition subgroups. Immunoprecipitation and membrane-binding studies identified those antibodies recognizing external epitopes on soluble and membrane-bound forms of the holoenzyme and demonstrated that the {dollar}\delta{dollar} and {dollar}\epsilon{dollar} subunits are substantially exposed in F{dollar}\sb 1{dollar}F{dollar}\sb 0{dollar}.;Analysis of partially cleaved {dollar}\alpha{dollar} subunit using Western blots mapped antibody epitopes to its carboxyl-half. Analysis of CNBr/weak acid cleaved {dollar}\beta{dollar} located the non-inhibitory B-II antibody epitopes between Asp-345 and Met-380 and the inhibitory B-I antibody epitopes between Asp-381 and the carboxyl terminus. The {dollar}\epsilon{dollar}-binding site on {dollar}\beta{dollar} was mapped to the same region as the B-I antibodies. The Fab fragment of a B-I antibody and {dollar}\epsilon{dollar} were cross-linked indicating the proximity of their binding sites in native F{dollar}\sb 1{dollar}.;Cross-linking studies revealed neither differences in quaternary structure between soluble and membrane-bound F{dollar}\sb 1{dollar} nor any association between {dollar}\delta{dollar} or {dollar}\epsilon{dollar} and any F{dollar}\sb 0{dollar} subunit. Cross-linking studies of F{dollar}\sb 1{dollar} in the presence of the ATPase activators ethylene glycol and antibody {dollar}\epsilon{dollar}-4 examined the mechanism of {dollar}\epsilon{dollar}-inhibition. Ethylene glycol displaced {dollar}\epsilon{dollar} only from its site of 1-ethyl-3- (3-(dimethylamino)propyl) carbodiimide (EDC) cross-linkage to {dollar}\beta{dollar} demonstrating that {dollar}\epsilon{dollar}-inhibition is mediated through {dollar}\beta{dollar}. In contrast, antibody {dollar}\epsilon{dollar}-4 did not alter EDC cross-linking of {dollar}\epsilon{dollar} to {dollar}\beta{dollar} showing that this interaction is not always inhibitory. {dollar}\epsilon{dollar} could also be cross-linked to {dollar}\beta{dollar} by EDC in F{dollar}\sb 1{dollar}F{dollar}\sb 0{dollar} where it is not inhibitory. The inhibitory effect of {dollar}\epsilon{dollar} and the B-I antibodies binding to the carboxyl-terminal region of {dollar}\beta{dollar} region suggested a function for this region.;The antibodies were used to characterize an {dollar}\alpha{dollar}-{dollar}\delta{dollar} cross-link produced during column centrifugation of F{dollar}\sb 1{dollar} by oxidation of sulfhydryls. This cross-link affected neither the enzyme's activity nor susceptibility to {dollar}\epsilon{dollar}-inhibition, casting doubt on rotational models of F{dollar}\sb 1{dollar} catalysis. The {dollar}\alpha{dollar}-{dollar}\delta{dollar} cross-link also resulted in a loss of binding affinity between F{dollar}\sb 1{dollar} and F{dollar}\sb 0{dollar}.
Recommended Citation
Tozer, Richard Gerard, "Applications Of Monoclonal Antibodies To Studies Of Structure/function Relationships In Escherichia Coli F(1)-atpase" (1987). Digitized Theses. 1671.
https://ir.lib.uwo.ca/digitizedtheses/1671