BIOCHEMICAL AND BIOPHYSICAL COMPARISON OF NATIVE AND CHEMICALLY SYNTHESIZED PHOSPHOLAMBAN AND A MONOMERIC PHOSPHOLAMBAN ANALOG

Phospholamban (PLB) was rapidly isolated from canine cardiac sarcoplas mic reticulum using immunoaffinity chromatography and prepared by soli d phase peptide synthesis, The two proteins are indistinguishable when analyzed by SDS-polyacrylamide gel electrophoresis and exhibit pentam eric 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 val ues agreeing with the expected value (similar to 6123 Da). Native and synthetic PLB reduced the calcium sensitivity of Ca2+ ATPase, which is reversed by anti-PLB antibody, A Cys-to-Ser PLB analog, where the cys teines (36, 41, and 46) were substituted by serines, is monomeric on S DS polyacrylamide gel electrophoresis, can be phosphorylated, and is r ecognized by polyclonal antisera, PLB migrates with a sedimentation co efficient of 4.8 S in sedimentation velocity ultracentrifugation exper iments, whereas Cys-to-Ser PLB does not sediment, consistent with a mo nomeric state, Circular dichroism spectral analysis of PLB indicates a bout 70% alpha-helical structure, whereas Cys-to-Ser PLB manifests onl y about 30%. Because the physiochemical properties of native and synth etic PLB appear identical, the more readily available synthetic protei n should be suitable for more extensive structural studies.