Biochemical and biophysical comparison of native and chemically synthesized phospholamban and a monomeric phospholamban analog.
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 (approximately 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 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-to-Ser PLB does not sediment, consistent with a monomeric state. Circular dichroism spectral analysis of PLB indicates about 70% alpha-helical structure, whereas Cys-to-Ser 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.
Mayer
E J
EJ
Department of Pharmacology, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
McKenna
E
E
Garsky
V M
VM
Burke
C J
CJ
Mach
H
H
Middaugh
C R
CR
Sardana
M
M
Smith
J S
JS
Johnson
R G
RG
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003488:Cyanogen Bromide; D003545:Cysteine; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D012694:Serine; D013329:Structure-Activity Relationship; D000099211:Phospholamban
D000251:Adenosine Triphosphatases; D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D003488:Cyanogen Bromide; D003545:Cysteine; D046911:Macromolecular Substances; D010446:Peptide Fragments; D012694:Serine; D000099211:Phospholamban