title: Purification of a 90-kDa protein (Band VII) from cardiac sarcoplasmic reticulum. Identification as calnexin and localization of casein kinase II phosphorylation sites.
abstract: Purification and sequencing of proteins from cardiac sarcoplasmic reticulum (SR) vesicles have provided a framework for the study of SR function. Included among the SR proteins so far investigated are a collection of intralumenal proteins that stain blue with the protein dye Stains-All in sodium dodecyl sulfate-polyacrylamide gels. A single prominent blue staining SR protein of apparent M(r) = 90,000 (Band VII), however, has remained uncharacterized. In the work described here, purification of Band VII from dog cardiac SR vesicles, along with amino acid sequencing and cDNA cloning, identified this protein as calnexin, a homologue of calreticulin recently described in dog pancreatic microsomes. Using ryanodine-mediated calcium oxalate loading of SR vesicles followed by density gradient centrifugation, we have shown that calnexin is a bona fide SR protein and an integral constituent of both junctional and free SR vesicles. Calnexin was found to be a substrate for casein kinase II and was phosphorylated at two distinct sites localized to the carboxyl- and amino-terminal ends of the molecule. Enrichment of calnexin in cardiac SR vesicles indicates a role for calnexin involving the specialized function of the SR membrane.
authors.affiliation: Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.
authors.forename: S E
authors.initials: SE
authors.lastname: Cala
authors.forename: C
authors.initials: C
authors.lastname: Ulbright
authors.forename: J S
authors.initials: JS
authors.lastname: Kelley
authors.forename: L R
authors.initials: LR
authors.lastname: Jones
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D037281:Calnexin; D047390:Casein Kinase II; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009206:Myocardium / Q000737:chemistry*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000737:chemistry*; D014357:Trypsin / Q000494:pharmacology
chemical_list: D002135:Calcium-Binding Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D010769:Phosphothreonine; D037281:Calnexin; D010768:Phosphoserine; D004247:DNA; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D014357:Trypsin