Characterization of high-capacity low-affinity calcium binding protein of liver endoplasmic reticulum: calsequestrin-like and divergent properties.
It had been previously demonstrated that endoplasmic reticulum membranes from rat hepatocytes contain a major calsequestrin-like protein, on account of electrophoretic and Stains All-staining properties (Damiani et al., J. Biol. Chem. 263, 340-343). Here we show that a Ca2+-binding protein sharing characteristics in size and biochemical properties with this protein is likewise present in the isolated endoplasmic reticulum from human liver. Human calsequestrin-like protein was characterized as 62 kDa, highly acidic protein (pl 4.5), using an extraction procedure from whole tissue, followed by DEAE-Cellulose chromatography, that was originally developed for purification of skeletal muscle and cardiac calsequestrin. Liver calsequestrin-like protein bound Ca2+ at low affinity (Kd = 4 mM) and in high amounts (Bmax = 1600 nmol Ca2+/mg of protein), as determined by equilibrium dialysis, but differed strikingly from skeletal muscle calsequestrin for the lack of binding to phenyl-Sepharose resin in the absence of Ca2+, and of changes in intrinsic fluorescence upon binding of Ca2+. Thus, these results suggest that liver 62 kDa protein, in spite of its calsequestrin-like Ca2+-binding properties, does not contain a Ca2+-regulated hydrophobic site, which is a specific structural feature of the calsequestrin-class of Ca2+-binding proteins.
Damiani
E
E
CNR, Centro di Studio per la Biologia e la Fisiopatologia Muscolare, Universita di Padova, Italy.
Heilmann
C
C
Salvatori
S
S
Margreth
A
A
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D002155:Calsequestrin / Q000032:analysis* / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000032:analysis*; D006801:Humans; D007526:Isoelectric Point; D008099:Liver / Q000648:ultrastructure*; D008970:Molecular Weight; D009124:Muscle Proteins / Q000032:analysis*; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D002135:Calcium-Binding Proteins; D002155:Calsequestrin; D009124:Muscle Proteins; D010446:Peptide Fragments; D010447:Peptide Hydrolases; D002118:Calcium