An investigation of structure-function relationships of (Ca2-Mg2+)- ATPase with monoclonal antibodies
An investigation of structure-function relationships of (Ca2-Mg2+)- ATPase with monoclonal antibodies
Thirty seven monoclonal antibodies (mAbs) were generated against (Ca2+-Mg2+)-ATPase from rabbit skeletal muscle sarcoplasmic reticulum for the purpose of studying structure-function relationships. No conformation specific (E1, E2) antibodies were found, indicating the limited extent of structural alteration of the antibody binding sites (epitopes) during calcium transport, which may reflect the magnitude of change accompanying E1-E2 transitions. Five distinct binding regions were identified on the ATPase by competitive binding of labelled and unlabelled mAbs. Limited proteolysis of the ATPase enabled description of the epitope location of 14 mAbs in the primary sequence. Following digestion of ATPase, anti-peptide antibodies specific for the N- and C-termini of the ATPase were used to identify the origin of fragments recognised by individual mABs. Antibody epitopes were defined to an apparent resolution of 11-16 residues, although these limits were extended to 75-94 residues in the absence of the precise molecular weight of proteolytic fragments. Ten mAbs (6 of which bind in the same spatial region, as defined in competitive displacement assays) produce inhibition of ATPase activity (30-70% inhibition). Inhibition was generally more marked at low ATPase concentrations (1-10μM) but a return to control levels of activity was not exhibited at high ATP (1-10mM). Release of enzyme-bound calcium at steady state was used as a measure of the ratio of E1_total/E2_total. This ratio was reduced by one inhibitory mAb, consistent with a reduction in the rate of dephosphorylation. Three other inhibitory mAbs increased this ratio, without stabilising E1. One of these (the only one tested) also decreased the rate of phosphoenzyme formation by ATP, and the concentration of phosphoenzyme at steady state. Potential mechanisms of inhibition of ATPase activity compatible with the data in the presence of mAb are discussed.
University of Southampton
1988
Colyer, John
(1988)
An investigation of structure-function relationships of (Ca2-Mg2+)- ATPase with monoclonal antibodies.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Thirty seven monoclonal antibodies (mAbs) were generated against (Ca2+-Mg2+)-ATPase from rabbit skeletal muscle sarcoplasmic reticulum for the purpose of studying structure-function relationships. No conformation specific (E1, E2) antibodies were found, indicating the limited extent of structural alteration of the antibody binding sites (epitopes) during calcium transport, which may reflect the magnitude of change accompanying E1-E2 transitions. Five distinct binding regions were identified on the ATPase by competitive binding of labelled and unlabelled mAbs. Limited proteolysis of the ATPase enabled description of the epitope location of 14 mAbs in the primary sequence. Following digestion of ATPase, anti-peptide antibodies specific for the N- and C-termini of the ATPase were used to identify the origin of fragments recognised by individual mABs. Antibody epitopes were defined to an apparent resolution of 11-16 residues, although these limits were extended to 75-94 residues in the absence of the precise molecular weight of proteolytic fragments. Ten mAbs (6 of which bind in the same spatial region, as defined in competitive displacement assays) produce inhibition of ATPase activity (30-70% inhibition). Inhibition was generally more marked at low ATPase concentrations (1-10μM) but a return to control levels of activity was not exhibited at high ATP (1-10mM). Release of enzyme-bound calcium at steady state was used as a measure of the ratio of E1_total/E2_total. This ratio was reduced by one inhibitory mAb, consistent with a reduction in the rate of dephosphorylation. Three other inhibitory mAbs increased this ratio, without stabilising E1. One of these (the only one tested) also decreased the rate of phosphoenzyme formation by ATP, and the concentration of phosphoenzyme at steady state. Potential mechanisms of inhibition of ATPase activity compatible with the data in the presence of mAb are discussed.
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Published date: 1988
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Local EPrints ID: 461002
URI: http://eprints.soton.ac.uk/id/eprint/461002
PURE UUID: 29a1e477-19f6-46e9-827e-3ff3c2705c24
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Date deposited: 04 Jul 2022 18:33
Last modified: 04 Jul 2022 18:33
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Author:
John Colyer
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