title: Purification of a sarcoplasmic reticulum protein that binds Ca2+ and plasma lipoproteins.
abstract: A protein in the sarcoplasmic reticulum of rabbit skeletal and cardiac muscle was identified because of its ability to bind 125I-labeled low density lipoprotein (LDL) with high affinity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein, referred to as the 165-kDa protein, is restricted to striated muscle. It was not detected in 14 other tissues, including several that contain smooth muscle, but it appears in rat L6 myoblasts when they differentiate into myocytes. Immunofluorescence and immunoelectron microscopic studies revealed that the protein is present throughout the sarcoplasmic reticulum and the terminal cisternae. It binds 45Ca2+ on nitrocellulose blots and stains metachromatically with Stains-all, a cationic dye that stains Ca2+-binding proteins. It does not appear to be a glycoprotein, and it appears slightly larger than the 160-kDa glycoprotein previously described in sarcoplasmic reticulum. The 165-kDa protein binds LDL, beta-migrating very low density lipoprotein, and a cholesterol-induced high density lipoprotein particle that contains apoprotein E as its sole apoprotein with much higher affinity than it binds high density lipoprotein. The protein is stable to boiling and to treatment with sodium dodecyl sulfate, but it becomes sensitive to these treatments when its cystine residues are reduced and alkylated. The protein was purified 1300-fold to apparent homogeneity from rabbit skeletal muscle membranes. It differs from the cell surface LDL receptor in that 1) its apparent molecular weight is not changed by reduction and alkylation; 2) it is present in Watanabe-heritable hyperlipidemic rabbits, which lack functional LDL receptors; 3) binding of lipoproteins is not inhibited by EDTA; and 4) it is located within the lumen of the sarcoplasmic reticulum where it has no access to plasma lipoproteins. It is unlikely that this protein ever binds lipoproteins in vivo; however, its lipoprotein binding activity has facilitated its purification to homogeneity and suggests that this protein has unusual structural features. The role of the 165-kDa protein in Ca2+ homeostasis in the sarcoplasmic reticulum, if any, remains to be determined.
authors.affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235.
authors.forename: S L
authors.initials: SL
authors.lastname: Hofmann
authors.forename: M S
authors.initials: MS
authors.lastname: Brown
authors.forename: E
authors.initials: E
authors.lastname: Lee
authors.forename: R K
authors.initials: RK
authors.lastname: Pathak
authors.forename: R G
authors.initials: RG
authors.lastname: Anderson
authors.forename: J L
authors.initials: JL
authors.lastname: Goldstein
mesh_terms: D000311:Adrenal Glands / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002132:Calcium Radioisotopes; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D007150:Immunohistochemistry; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008099:Liver / Q000032:analysis; D051379:Mice; D008810:Mice, Inbred C57BL; D008854:Microscopy, Electron; D008970:Molecular Weight; D009132:Muscles / Q000032:analysis*; D009206:Myocardium / Q000032:analysis*; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D011973:Receptors, LDL / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000032:analysis*
chemical_list: D002132:Calcium Radioisotopes; D008077:Lipoproteins, LDL; D011506:Proteins; D011973:Receptors, LDL; D004492:Edetic Acid; D002118:Calcium