title: Accurate quantitation of phospholamban phosphorylation by immunoblot.
abstract: We have developed a quantitative immunoblot method to measure the mole fraction of phospholamban (PLB) phosphorylated at Ser16 (X(p)) in biological samples. In cardiomyocytes, PLB phosphorylation activates the sarcoplasmic reticulum calcium ATPase (SERCA), which reduces cytoplasmic Ca(2+) to relax the heart during diastole. Unphosphorylated PLB (uPLB) inhibits SERCA at low [Ca(2+)] but phosphorylated PLB (pPLB) is less inhibitory, so myocardial physiology and pathology depend critically on X(p). Current methods of X(p) determination by immunoblot provide moderate precision but poor accuracy. We have solved this problem using purified uPLB and pPLB standards produced by solid-phase peptide synthesis. In each assay, a pair of blots is performed with identical standards and unknowns using antibodies partially selective for uPLB and pPLB, respectively. When performed on mixtures of uPLB and pPLB, the assay measures both total PLB (tPLB) and X(p) with accuracy of 96% or better. We assayed pig cardiac sarcoplasmic reticulum (SR) and found that X(p) varied widely among four animals, from 0.08 to 0.38, but there was remarkably little variation in the ratios of X(p)/tPLB and uPLB/SERCA, suggesting that PLB phosphorylation is tuned to maintain homeostasis in SERCA regulation.
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, 55455, USA.
authors.forename: Naa-Adjeley
authors.initials: NA
authors.lastname: Ablorh
authors.forename: Tyler
authors.initials: T
authors.lastname: Miller
authors.forename: Florentin
authors.initials: F
authors.lastname: Nitu
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Gruber
authors.forename: Christine
authors.initials: C
authors.lastname: Karim
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
mesh_terms: D000818:Animals; D002135:Calcium-Binding Proteins / Q000032:analysis*; D015151:Immunoblotting / Q000379:methods*; D010766:Phosphorylation; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism; D013552:Swine; D000099211:Phospholamban
chemical_list: D002135:Calcium-Binding Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban