CHARACTERIZATION OF THE MOLECULAR-FORM OF CARDIAC PHOSPHOLAMBAN

The native form of phospholamban is not known and it is presently unde r debate whether this protein exists as a monomer or an oligomer in ca rdiac sarcoplasmic reticulum. The currently accepted model for phospho lamban is pentameric, based primarily on its behavior in SDS-polyacryl amide gel electrophoresis. In this study, sucrose density gradient cen trifugation and gel filtration chromatography were used to determine t he form of phospholamban under nondenaturing conditions. Purified phos pholamban 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 gradie nt fractions were assayed for acid precipitable P-32-incorporation in the presence of [gamma-P-32]ATP and cAMP-dependent protein kinase cata lytic subunit. P-32-containing peak fractions were subjected to SDS-po lyacrylamide gel electrophoresis and immunoblot analysis, using a phos pholamban-polyclonal antibody, to confirm the presence of phospholamba n. Purified phospholamban migrated with an apparent molecular weight o f 25,000 daltons in the sucrose gradients in either the absence or pre sence of detergent. Phospholamban present in solubilized cardiac sarco plasmic reticulum migrated with a similar apparent molecular weight wh en detergent was included in the sucrose gradients. In addition, solub ilized 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 da ltons. These data suggest that phospholamban prefers an oligomeric ass embly and this may be the form present in cardiac sarcoplasmic reticul um membranes.