Affinity labeling of calmodulin-binding proteins in skeletal muscle sarcoplasmic reticulum
Autor(a) principal: | |
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Data de Publicação: | 1988 |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10316/11639 |
Resumo: | 125I-Calmodulin (125I-CaM) binding to sarcoplasmic reticulum (SR) membranes isolated from skeletal muscle cells was investigated, and the CaM receptors associated with the membrane were identified by using the photoaffinity cross-linker methyl-4-azidobenzimidate or the chemical cross-linker dithiobis-N-hydroxysuccinimidyl propionate. Exogenous CaM binds to CaM-depleted membranes in a Ca2+- or Mg2+-dependent way. When both cations are added together to the reaction medium, the stimulatory effects appear to be additive, suggesting that Ca2+ and Mg2+ act by two distinct mechanisms. The Ca2+/Mg2+-dependent binding of CaM is specific since it is inhibited by unlabeled CaM or by trifluoperazine. Furthermore, it is saturable and shows one class of high affinity binding sites with a KD of about 52 nM and a beta max of about 5 pmol/mg of protein. The sensitivity of Ca2+ is expressed in two steps reaching half-saturation at free Ca2+ concentrations of about 1.6 x 10(-7) and 3 x 10(-5) M, respectively. On the other hand, the sensitivity to Mg2+ is expressed in one step with a half-saturation Mg2+ concentration of about 2 x 10(-3) M. Electrophoretic analysis in a polyacrylamide gradient and subsequent autoradiography demonstrated a major CaM-binding protein of about 60 kDa and five minor CaM receptors of about 148, 125, 41, 33, and 23 kDa, respectively. The major labeled protein (60 kDa) probably represents the CaM-dependent component involved in Ca2+ release from SR, whereas the others represent a previously unrecognized class of CaM receptors in skeletal SR. |
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Affinity labeling of calmodulin-binding proteins in skeletal muscle sarcoplasmic reticulum125I-Calmodulin (125I-CaM) binding to sarcoplasmic reticulum (SR) membranes isolated from skeletal muscle cells was investigated, and the CaM receptors associated with the membrane were identified by using the photoaffinity cross-linker methyl-4-azidobenzimidate or the chemical cross-linker dithiobis-N-hydroxysuccinimidyl propionate. Exogenous CaM binds to CaM-depleted membranes in a Ca2+- or Mg2+-dependent way. When both cations are added together to the reaction medium, the stimulatory effects appear to be additive, suggesting that Ca2+ and Mg2+ act by two distinct mechanisms. The Ca2+/Mg2+-dependent binding of CaM is specific since it is inhibited by unlabeled CaM or by trifluoperazine. Furthermore, it is saturable and shows one class of high affinity binding sites with a KD of about 52 nM and a beta max of about 5 pmol/mg of protein. The sensitivity of Ca2+ is expressed in two steps reaching half-saturation at free Ca2+ concentrations of about 1.6 x 10(-7) and 3 x 10(-5) M, respectively. On the other hand, the sensitivity to Mg2+ is expressed in one step with a half-saturation Mg2+ concentration of about 2 x 10(-3) M. Electrophoretic analysis in a polyacrylamide gradient and subsequent autoradiography demonstrated a major CaM-binding protein of about 60 kDa and five minor CaM receptors of about 148, 125, 41, 33, and 23 kDa, respectively. The major labeled protein (60 kDa) probably represents the CaM-dependent component involved in Ca2+ release from SR, whereas the others represent a previously unrecognized class of CaM receptors in skeletal SR.The American Society for Biochemistry and Molecular Biology1988-09-15info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articlehttp://hdl.handle.net/10316/11639http://hdl.handle.net/10316/11639engJournal of Biological Chemistry. 263:26 (1988) 12872-128770021-9258Vale, M. Graça P.info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2020-05-25T07:06:31Zoai:estudogeral.uc.pt:10316/11639Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T20:55:39.906261Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Affinity labeling of calmodulin-binding proteins in skeletal muscle sarcoplasmic reticulum |
title |
Affinity labeling of calmodulin-binding proteins in skeletal muscle sarcoplasmic reticulum |
spellingShingle |
Affinity labeling of calmodulin-binding proteins in skeletal muscle sarcoplasmic reticulum Vale, M. Graça P. |
title_short |
Affinity labeling of calmodulin-binding proteins in skeletal muscle sarcoplasmic reticulum |
title_full |
Affinity labeling of calmodulin-binding proteins in skeletal muscle sarcoplasmic reticulum |
title_fullStr |
Affinity labeling of calmodulin-binding proteins in skeletal muscle sarcoplasmic reticulum |
title_full_unstemmed |
Affinity labeling of calmodulin-binding proteins in skeletal muscle sarcoplasmic reticulum |
title_sort |
Affinity labeling of calmodulin-binding proteins in skeletal muscle sarcoplasmic reticulum |
author |
Vale, M. Graça P. |
author_facet |
Vale, M. Graça P. |
author_role |
author |
dc.contributor.author.fl_str_mv |
Vale, M. Graça P. |
description |
125I-Calmodulin (125I-CaM) binding to sarcoplasmic reticulum (SR) membranes isolated from skeletal muscle cells was investigated, and the CaM receptors associated with the membrane were identified by using the photoaffinity cross-linker methyl-4-azidobenzimidate or the chemical cross-linker dithiobis-N-hydroxysuccinimidyl propionate. Exogenous CaM binds to CaM-depleted membranes in a Ca2+- or Mg2+-dependent way. When both cations are added together to the reaction medium, the stimulatory effects appear to be additive, suggesting that Ca2+ and Mg2+ act by two distinct mechanisms. The Ca2+/Mg2+-dependent binding of CaM is specific since it is inhibited by unlabeled CaM or by trifluoperazine. Furthermore, it is saturable and shows one class of high affinity binding sites with a KD of about 52 nM and a beta max of about 5 pmol/mg of protein. The sensitivity of Ca2+ is expressed in two steps reaching half-saturation at free Ca2+ concentrations of about 1.6 x 10(-7) and 3 x 10(-5) M, respectively. On the other hand, the sensitivity to Mg2+ is expressed in one step with a half-saturation Mg2+ concentration of about 2 x 10(-3) M. Electrophoretic analysis in a polyacrylamide gradient and subsequent autoradiography demonstrated a major CaM-binding protein of about 60 kDa and five minor CaM receptors of about 148, 125, 41, 33, and 23 kDa, respectively. The major labeled protein (60 kDa) probably represents the CaM-dependent component involved in Ca2+ release from SR, whereas the others represent a previously unrecognized class of CaM receptors in skeletal SR. |
publishDate |
1988 |
dc.date.none.fl_str_mv |
1988-09-15 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10316/11639 http://hdl.handle.net/10316/11639 |
url |
http://hdl.handle.net/10316/11639 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Journal of Biological Chemistry. 263:26 (1988) 12872-12877 0021-9258 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.publisher.none.fl_str_mv |
The American Society for Biochemistry and Molecular Biology |
publisher.none.fl_str_mv |
The American Society for Biochemistry and Molecular Biology |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
instname_str |
Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
instacron_str |
RCAAP |
institution |
RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
collection |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository.name.fl_str_mv |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
repository.mail.fl_str_mv |
|
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1799133843242876928 |