Total synthesis and functional properties of the membrane-intrinsic protein phospholamban.
The membrane-intrinsic protein phospholamban (PLN), the regulatory protein of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, was chemically synthesized. The synthesis was accomplished by double couplings and efficient capping procedures, thus eliminating hydrophobic failure sequences. The crude peptide was purified by high-performance liquid chromatographic ion exchange and gel permeation chromatography in chloroform-methanol mixtures. Ion spray mass spectroscopy showed that the product had the correct molecular mass. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis runs produced the typical monomer-pentamer structural pattern. A predominantly helical CD spectrum was obtained in 0.075% C12E8 (67.9% helix, 1.8% beta, 12.2% turn, 18.1% random coil). Synthetic PLN was phosphorylated in detergent solutions by protein kinase A with a stoichiometry close to 1:1 (Pi to PLN monomer). Reconstitution of the isolated skeletal muscle SR Ca2+ ATPase in phosphatidylcholine membranes in the presence of PLN using the freezing and thawing technique yielded a preparation with lower Ca(2+)-dependent ATPase activity. The inhibition was mainly due to a decrease in the affinity (Km(Ca)) of the ATPase for Ca2+ and was partially reversed by PLN phosphorylation with protein kinase A. By contrast, addition of PLN to diluted intact SR vesicles uncoupled the Ca(2+)-transport reaction, suggesting an ionophoric effect of PLN. Because this effect was observed at very high PLN-to-SR vesicle ratios and was not influenced by PLN phosphorylation, its biological function is doubtful.
Vorherr
T
T
Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), Zürich.
Wrzosek
A
A
Chiesi
M
M
Carafoli
E
E
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism / Q000502:physiology; D002942:Circular Dichroism; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D014461:Ultracentrifugation; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D000099211:Phospholamban