title: Antibodies against the 53 kDa glycoprotein inhibit the rotational dynamics of both the 53 kDa glycoprotein and the Ca(2+)-ATPase in the sarcoplasmic reticulum membrane.
abstract: The purpose of this study is to better define the relationship of the 53 kDa glycoprotein (GP-53) of the sarcoplasmic reticulum (SR) to other SR proteins. Towards that end the effects of antibodies against GP-53 on the rotational dynamics of maleimide spin-labeled proteins of SR of rabbit skeletal muscle were investigated. The labeling protocol used in this study provided 1.6 +/- 0.3 moles spin label incorporated per 10(5) g SR protein. Labeling specificity studies indicated that nearly 70% of the label bound specifically to the Ca(2+)-ATPase, with the remainder bound to GP-53. Using saturation-transfer electron paramagnetic resonance (ST-EPR), it was determined that the rotational mobility (i.e., the rate of rotation) of the spin-labeled SR proteins decreased greater than 5-fold upon preincubation of MSL-SR with an antiserum against the GP-53, while preincubation of MSL-SR with preimmune serum had no effect. Preincubation of MSL-SR with a monoclonal antibody against the GP-53 produced a 4-fold decrease in the rotational mobility of the MSL-SR proteins compared to control measurements. Further, these effects showed a marked calcium dependence: the decrease in the rotational mobility of the MSL-SR proteins preincubated with anti-GP-53 antibodies in 500 microM Ca2+ was 3-6-fold greater than that of MSL-SR preincubated with antibodies in 5 mM EGTA. While MSL was bound to both Ca(2+)-ATPase and GP-53, model calculations indicated that the decreases observed in the rotational mobility of the MSL-SR proteins caused by the anti-GP-53 monoclonal antibodies were too large to be accounted for by effects on GP-53 alone. The calculations suggest that the rotational rate of Ca(2+)-ATPase was also diminished by anti-GP-53 monoclonal antibodies, indicating an interaction between GP-53 and Ca(2+)-ATPase in the SR membrane.
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville.
authors.forename: J E
authors.initials: JE
authors.lastname: Mahaney
authors.forename: C P
authors.initials: CP
authors.lastname: Weis
authors.forename: C M
authors.initials: CM
authors.lastname: Grisham
authors.forename: H
authors.initials: H
authors.lastname: Kutchai
mesh_terms: D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000276:immunology / Q000378:metabolism*; D003902:Detergents; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D008301:Maleimides; D008562:Membrane Glycoproteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D012399:Rotation; D012519:Sarcoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism*; D013113:Spin Labels
chemical_list: D000911:Antibodies, Monoclonal; D003902:Detergents; D008301:Maleimides; D008562:Membrane Glycoproteins; D013113:Spin Labels; D000252:Calcium-Transporting ATPases