Characterization of the molecular form of cardiac phospholamban.
The native form of phospholamban is not known and it is presently under debate whether this protein exists as a monomer or an oligomer in cardiac sarcoplasmic reticulum. The currently accepted model for phospholamban is pentameric, based primarily on its behavior in SDS-polyacrylamide gel electrophoresis. In this study, sucrose density gradient centrifugation and gel filtration chromatography were used to determine the form of phospholamban under nondenaturing conditions. Purified phospholamban or phospholamban present in solubilized cardiac sarcoplasmic reticulum was centrifuged through 5-20% sucrose density gradients in the absence or presence of n-octylgucoside. The sucrose density gradient fractions were assayed for acid precipitable 32P-incorporation in the presence of [gamma-32P]ATP and cAMP-dependent protein kinase catalytic subunit. 32P-containing peak fractions were subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis, using a phospholamban-polyclonal antibody, to confirm the presence of phosopholamban. Purified phospholamban migrated with an apparent molecular weight of 25,000 daltons in the sucrose gradients in either the absence or presence of detergent. Phospholamban present in solubilized cardiac sarcoplasmic reticulum migrated with a similar apparent molecular weight when detergent was included in the sucrose gradients. In addition, solubilized cardiac sarcoplasmic reticulum was subjected to gel filtration chromatography in the presence of deoxycholate. Under these conditions phospholamban migrated with an apparent molecular weight of 24,500 daltons. These data suggest that phospholamban prefers an oligomeric assembly and this may be the form present in cardiac sarcoplasmic reticulum membranes.
Harrer
J M
JM
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0575.
Kranias
E G
EG
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000302:isolation & purification; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003902:Detergents; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides; D046911:Macromolecular Substances; D008970:Molecular Weight; D009206:Myocardium / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D000099211:Phospholamban
D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D002135:Calcium-Binding Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D003902:Detergents; D005960:Glucosides; D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; D000099211:Phospholamban; C018619:octyl-beta-D-glucoside