Purification, calcium binding properties, and ultrastructural localization of the 53,000- and 160,000 (Sarcalumenin)-Dalton glycoproteins of the sarcoplasmic reticulum

Ekkehard Leberer; Barry G Timms; Kevin P Campbell; David H MacLennan

doi:10.1016/S0021-9258(19)38787-3

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Purification, calcium binding properties, and ultrastructural localization of the 53,000- and 160,000 (Sarcalumenin)-Dalton glycoproteins of the sarcoplasmic reticulum

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Purification, calcium binding properties, and ultrastructural localization of the 53,000- and 160,000 (Sarcalumenin)-Dalton glycoproteins of the sarcoplasmic reticulum

Ekkehard Leberer, Barry G Timms, Kevin P Campbell and David H MacLennan

The Journal of biological chemistry, Vol.265(17), pp.10118-10124

1990

DOI: 10.1016/S0021-9258(19)38787-3

PMID: 2112542

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Abstract

The 53-kDa glycoprotein and sarcalumenin (160-kDa glycoprotein) were extracted from rabbit skeletal muscle sarcoplasmic reticulum with EGTA and purified by fractionation on DEAE-Sephadex A-25 and lentil lectin-Sepharose 4B. Sarcalumenin was shown to bind up to 400 nmol of Ca2+/mg of protein at pH 7.5, which is equivalent to binding of approximately 35 mol of Ca2+/mol of protein. The apparent dissociation constant was 300 microM in the presence of 20 mM KCl and 600 microM in 150 mM KCl. The 53-kDa glycoprotein did not bind any Ca2+ under the conditions examined. Immunoblot analysis of isolated sarcoplasmic reticulum subfractions demonstrated the presence of the two glycoproteins in both the longitudinal sarcoplasmic reticulum and the terminal cisternae. Their concentrations were higher, however, in the longitudinal sarcoplasmic reticulum vesicles. Comparative immunoelectron microscopic studies using monoclonal antibodies revealed a codistribution of the 53-kDa glycoprotein with the Ca2(+)-ATPase in all regions of the free sarcoplasmic reticulum. A similar distribution was found for sarcalumenin, although immunolabeling was much weaker. The colocalization of the 53-kDa glycoprotein and sarcalumenin with the Ca2(+)-ATPase and the Ca2+ binding properties of sarcalumenin suggest that the glycoproteins may be involved in the sequestration of Ca2+ in the nonjunctional regions of the sarcoplasmic reticulum.

Fundamental and applied biological sciences. Psychology

Proteins

Biological and medical sciences

Glycoproteins

Analytical, structural and metabolic biochemistry

Details

Title: Subtitle: Purification, calcium binding properties, and ultrastructural localization of the 53,000- and 160,000 (Sarcalumenin)-Dalton glycoproteins of the sarcoplasmic reticulum
Creators: Ekkehard Leberer - Univ. Toronto, Charles H. Best inst., banting best dep. medical res., Toronto ON M5G 1L6, Canada
Barry G Timms - Univ. Toronto, Charles H. Best inst., banting best dep. medical res., Toronto ON M5G 1L6, Canada
Kevin P Campbell - Univ. Toronto, Charles H. Best inst., banting best dep. medical res., Toronto ON M5G 1L6, Canada
David H MacLennan - Univ. Toronto, Charles H. Best inst., banting best dep. medical res., Toronto ON M5G 1L6, Canada
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.265(17), pp.10118-10124
DOI: 10.1016/S0021-9258(19)38787-3
PMID: 2112542
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: American Society for Biochemistry and Molecular Biology; Bethesda, MD
Language: English
Date published: 1990
Academic Unit: Neurology; Molecular Physiology and Biophysics; Iowa Neuroscience Institute
Record Identifier: 9984068367102771

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