Phospholamban (PLB) was rapidly isolated from canine cardiac sarcoplasmic reticulum using immunoaffinity chromatography and prepared by solid phase peptide synthesis. The two proteins are indistinguishable when analyzed by SDS-polyacrylamide gel electrophoresis and exhibit pentameric oligomeric states. They are similarly detected on Western blots, are phosphorylation substrates, have identical amino acid compositions that directly reflect their predicted values, yield the same internal amino acid sequences upon CNBr digestion, and have molecular mass values agreeing with the expected value (ϳ6123 Da). Native and synthetic PLB reduced the calcium sensitivity of Ca
2؉ATPase, which is reversed by anti-PLB antibody. A Cys-to-Ser PLB analog, where the cysteines (36, 41, and 46) were substituted by serines, is monomeric on SDS-polyacrylamide gel electrophoresis, can be phosphorylated, and is recognized by polyclonal antisera. PLB migrates with a sedimentation coefficient of 4.8 S in sedimentation velocity ultracentrifugation experiments, whereas Cys-toSer PLB does not sediment, consistent with a monomeric state. Circular dichroism spectral analysis of PLB indicates about 70% ␣-helical structure, whereas Cys-toSer PLB manifests only about 30%. Because the physiochemical properties of native and synthetic PLB appear identical, the more readily available synthetic protein should be suitable for more extensive structural studies.
Phospholamban (PLB)1 is an oligomeric membrane protein present in stoichiometric amounts associated with the Ca 2ϩ ATPase of cardiac sarcoplasmic reticulum (SR). PLB in its unphosphorylated state attenuates the catalytic activity of the Ca 2ϩ ATPase by reducing its apparent calcium sensitivity. Phosphorylation of PLB (1-3), treatment with antibodies directed against PLB (4 -7), or mild trypsin proteolysis of PLB (8) ATPase activity. Data supporting reversible and direct regulation of the calcium pump by PLB are abundant, yet the detailed mechanistic and structural requirements for PLB inhibition remain to be described.The primary structure of PLB has been deduced from its cDNA (15). It is highly conserved among species, has an acetylated amino terminus, and consists of 52 amino acids with a predicted molecular mass of 6123 Da. Several secondary structure models (an example is shown in Fig. 1) have been proposed (13,14,16) with the following general features: (i) PLB is an amphipathic peptide with a hydrophilic NH 2 terminus, part of which is predicted to form an ␣-helix whose exact length and position are uncertain, (ii) serine 16 and threonine 17 are the sites for protein phosphorylation by cAMP-dependent protein kinase (PKA) and calcium-calmodulin-dependent protein kinase, respectively, and (iii) the hydrophobic COOH-terminal amino acids (31-52) form a transmembrane ␣-helical segment and are involved in protein oligomerization. The quaternary structure of PLB under nondenaturing conditions is assumed to be pentameric. PLB migrates as a homopentamer with an apparent M r of 28,000 in SDS-PAGE, w...
