Immunological studies of a calcium pump
Immunological studies of a calcium pump
The surface exposed epitopes for a number of monoclonal antibodies (mAb) binding to the (Ca2+-Mg2+)-ATPasc purified from skeletal muscle sarcoplasmic reticulum have been defined by studying the binding to fusion proteins generated from cDNA fragment libraries and to sets of hexameric peptides synthesised on plastic pins. These epitopes have been confirmed by showing that the mAbs bind to individual peptides containing the epitopes and that antipeptide antibodies raised to these peptides bind to the native ATPase. Three surface exposed epitopes have been defined in the nucleotide binding domain of the ATPase, suggesting considerable surface exposure of this region on the top surface of the protein. Another surface exposed mAb epitope has been located in the proposed fourth stalk region.
Binding of mAbs and an antipeptide antibody to two of the epitopes located in the nucleotide binding domain was shown to inhibit ATPase activity and Ca2+ uptake by the protein. Evidence is presented to suggest that the two activities remain coupled when mAbs bind and that the reduction in both can be attributed to the inhibition of ATPase activity.
A method is described for the definition of surface exposed epitopes using polyclonal antibodies. Five surface exposed epitopes on the ATPase were defined by this method and were found to contain or overlap either the epitopes of mAbs or the two main tryptic sites. The surface exposure of these and the mAb epitopes was confirmed by competitive binding studies and epitope mapping of antipeptide antisera raised to peptides containing the sequences of the epitopes.
By comparing the sequences of these epitopes with the aligned sequences of other P-type cation pumps it is shown that most are located in non-conserved regions with no predicted consensus secondary structure.
University of Southampton
Tunwell, Richard Edward Alistair
1991
Tunwell, Richard Edward Alistair
Tunwell, Richard Edward Alistair
(1991)
Immunological studies of a calcium pump.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The surface exposed epitopes for a number of monoclonal antibodies (mAb) binding to the (Ca2+-Mg2+)-ATPasc purified from skeletal muscle sarcoplasmic reticulum have been defined by studying the binding to fusion proteins generated from cDNA fragment libraries and to sets of hexameric peptides synthesised on plastic pins. These epitopes have been confirmed by showing that the mAbs bind to individual peptides containing the epitopes and that antipeptide antibodies raised to these peptides bind to the native ATPase. Three surface exposed epitopes have been defined in the nucleotide binding domain of the ATPase, suggesting considerable surface exposure of this region on the top surface of the protein. Another surface exposed mAb epitope has been located in the proposed fourth stalk region.
Binding of mAbs and an antipeptide antibody to two of the epitopes located in the nucleotide binding domain was shown to inhibit ATPase activity and Ca2+ uptake by the protein. Evidence is presented to suggest that the two activities remain coupled when mAbs bind and that the reduction in both can be attributed to the inhibition of ATPase activity.
A method is described for the definition of surface exposed epitopes using polyclonal antibodies. Five surface exposed epitopes on the ATPase were defined by this method and were found to contain or overlap either the epitopes of mAbs or the two main tryptic sites. The surface exposure of these and the mAb epitopes was confirmed by competitive binding studies and epitope mapping of antipeptide antisera raised to peptides containing the sequences of the epitopes.
By comparing the sequences of these epitopes with the aligned sequences of other P-type cation pumps it is shown that most are located in non-conserved regions with no predicted consensus secondary structure.
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Published date: 1991
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Local EPrints ID: 460483
URI: http://eprints.soton.ac.uk/id/eprint/460483
PURE UUID: ed0c5f94-cf7c-4f6f-9ec0-6e80e53c14cb
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Date deposited: 04 Jul 2022 18:23
Last modified: 04 Jul 2022 18:23
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Author:
Richard Edward Alistair Tunwell
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