The use of antibodies to determine the structure and function of the (Ca2+-Mg2+)-ATPase and the 53kDa glycoprotein of sarcoplasmic reticulum
The use of antibodies to determine the structure and function of the (Ca2+-Mg2+)-ATPase and the 53kDa glycoprotein of sarcoplasmic reticulum
Seven monoclonal antibodies were raised against the 53kDa glycoprotein of sarcoplasmic reticulum (SR). Epitope mapping revealed 3 different locations for the antibodies and localisation studies using chemical labelling, proteolytic enzyme digestions and competitive binding assays suggest an entirely lumenal location for this glycoprotein. No evidence was found for any association of this glycoprotein with the (Ca2+-Mg2+)-ATPase. No effects on the calcium transport function of SR vesicles were found on the binding of these antibodies to the glycoprotein. These data show that the postulated role of the 53kDa glycoprotein as a modulator of ATPase activity is unproven.
Thirty one peptides were synthesised from the primary sequence of rabbit skeletal muscle SR (Ca2+-Mg2+-ATPase. All peptides, except one, elicited an immune response with the antibodies recognising their respective peptides and the denatured ATPase. Of those antibodies against peptides corresponding to regions of the phosphorylation and nucleotide-binding domains, 4 of the epitopes were found to be unexposed in the native structure (residues 493-503, 539-553, 177-189 and 303-314) with the latter 2 requiring high concentrations of denaturing detergent (SDS) for exposure. All other epitopes in these regions were exposed but none of the antibodies directed against these epitopes affected the steady-state activity of the ATPase.
Antibodies raised against peptides corresponding to the inter-membranous loops of the ATPase were used to define transmembrane organisation of the ATPase. Both N- and C-termini were located in the cytoplasm and epitope 877-888 located in the lumen of SR vesicles. Epitope 808-818 was also located to the cytoplasm but only after removal of large, overlaying protein domains by proteinase K. The other inter-membranous loops (residues 277-288, 780-791, 949-958 and 915-924) were only detected by antibodies on denaturation of the ATPase with SDS, indicating a close association with the membrane. The evidence presented in this project supports a 10 transmembranous model for the ATPase with very short inter-membranous loops, with one exception, on the lumenal side of the SR membrane.
University of Southampton
Matthews, Ian
3cc6e23b-692f-4153-b67a-db41dde454cb
1993
Matthews, Ian
3cc6e23b-692f-4153-b67a-db41dde454cb
Matthews, Ian
(1993)
The use of antibodies to determine the structure and function of the (Ca2+-Mg2+)-ATPase and the 53kDa glycoprotein of sarcoplasmic reticulum.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Seven monoclonal antibodies were raised against the 53kDa glycoprotein of sarcoplasmic reticulum (SR). Epitope mapping revealed 3 different locations for the antibodies and localisation studies using chemical labelling, proteolytic enzyme digestions and competitive binding assays suggest an entirely lumenal location for this glycoprotein. No evidence was found for any association of this glycoprotein with the (Ca2+-Mg2+)-ATPase. No effects on the calcium transport function of SR vesicles were found on the binding of these antibodies to the glycoprotein. These data show that the postulated role of the 53kDa glycoprotein as a modulator of ATPase activity is unproven.
Thirty one peptides were synthesised from the primary sequence of rabbit skeletal muscle SR (Ca2+-Mg2+-ATPase. All peptides, except one, elicited an immune response with the antibodies recognising their respective peptides and the denatured ATPase. Of those antibodies against peptides corresponding to regions of the phosphorylation and nucleotide-binding domains, 4 of the epitopes were found to be unexposed in the native structure (residues 493-503, 539-553, 177-189 and 303-314) with the latter 2 requiring high concentrations of denaturing detergent (SDS) for exposure. All other epitopes in these regions were exposed but none of the antibodies directed against these epitopes affected the steady-state activity of the ATPase.
Antibodies raised against peptides corresponding to the inter-membranous loops of the ATPase were used to define transmembrane organisation of the ATPase. Both N- and C-termini were located in the cytoplasm and epitope 877-888 located in the lumen of SR vesicles. Epitope 808-818 was also located to the cytoplasm but only after removal of large, overlaying protein domains by proteinase K. The other inter-membranous loops (residues 277-288, 780-791, 949-958 and 915-924) were only detected by antibodies on denaturation of the ATPase with SDS, indicating a close association with the membrane. The evidence presented in this project supports a 10 transmembranous model for the ATPase with very short inter-membranous loops, with one exception, on the lumenal side of the SR membrane.
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Published date: 1993
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Local EPrints ID: 462260
URI: http://eprints.soton.ac.uk/id/eprint/462260
PURE UUID: 34edcd3f-959d-4edc-9f34-a1310b5a530b
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Date deposited: 04 Jul 2022 19:04
Last modified: 04 Jul 2022 19:04
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Author:
Ian Matthews
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