Low-affinity Ca(2+)-binding sites versus Zn(2+)-binding sites in histidine-rich Ca(2+)-binding protein of skeletal muscle sarcoplasmic reticulum.
Histidine-rich Ca(2+)-binding protein (HRC) is a 170 kDa protein that can be identified in the isolated sarcoplasmic reticulum from rabbit skeletal muscle by its ability to bind [125I]low-density lipoprotein on blots after SDS-PAGE and that appears to be bound to the junctional membrane through calcium bridges. Molecular cDNA cloning of this protein predicts the existence of a Ca(2+)-binding domain and of a distinct heavy-metal binding domain at the cystein-rich COOH-terminus. Here we demonstrate, using radioactive ligand blot techniques, that HRC protein binds 45Ca at low affinity, as well as being able to bind 65Zn, but at different sites, that are largely inhibitable by prior reductive alkylation of the protein. In contrast to Ca(2+)-binding protein calsequestrin not having detectable 65Zn-binding sites, HRC protein bound selectively to immobilized Zn2+ on IDA-agarose affinity columns. Our results also indicate that rabbit and human 140 kDa HRC protein have common properties.
Picello
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Dipartimento di Scienze, Biomediche e Sperimentali, Università di Padova, Italy.
Damiani
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Margreth
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D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D002135:Calcium-Binding Proteins; C489419:HRC protein, Oryctolagus cuniculus; D008077:Lipoproteins, LDL; C068969:HRC protein, human; D015032:Zinc; D002118:Calcium