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Titolo:
Characterization and quantitation of phospholamban and its phosphorylationstate using antibodies
Autore:
Mayer, EJ; Huckle, W; Johnson, RG; McKenna, E;
Indirizzi:
Merck Res Labs, Dept Pharmacol, W Point, PA 19486 USA Merck Res Labs W Point PA USA 19486 Dept Pharmacol, W Point, PA 19486 USA
Titolo Testata:
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
fascicolo: 1, volume: 267, anno: 2000,
pagine: 40 - 48
SICI:
0006-291X(20000107)267:1<40:CAQOPA>2.0.ZU;2-J
Fonte:
ISI
Lingua:
ENG
Soggetto:
CARDIAC SARCOPLASMIC-RETICULUM; BETA-ADRENERGIC STIMULATION; SODIUM DODECYL-SULFATE; PROTEIN-KINASE; INTACT HEART; SITES; CA2+; DEPHOSPHORYLATION; ISOPROTERENOL; INHIBITORS;
Keywords:
phospholamban; phosphorylation; phosphorylation specific antibodies;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
31
Recensione:
Indirizzi per estratti:
Indirizzo: McKenna, E Merck Res Labs, Dept Pharmacol, WP26-265, W Point, PA 19486 USAMerck Res Labs WP26-265 W Point PA USA 19486 oint, PA 19486 USA
Citazione:
E.J. Mayer et al., "Characterization and quantitation of phospholamban and its phosphorylationstate using antibodies", BIOC BIOP R, 267(1), 2000, pp. 40-48

Abstract

Quantitative immunoassays to discriminate and quantitate phospholamban andits phosphorylation states in heart homogenates were developed using knownamounts of protein determined by amino acid analysis. Synthetic 1-52 phospholamban, the hydrophilic 1-25 peptide, and 1-25 phosphopeptides containingP-Ser(16), P-Thr(17), and dually phosphorylated (P-Ser(16), P-Thr(17)) were used to calibrate immunoblot systems. In addition, synthetic 1-52 peptidewas phosphorylated using cAMP-dependent protein kinase (PSer(16)) or Ca2+-calmodulin protein kinase (P-Thr(17)) and then separated from unphosphorylated 1-52 by HPLC prior to quantitation. Further, canine cardiac sarcoplasmic reticulum was phosphorylated in vitro using [gamma-P-32]-ATP with cAMP-dependent protein kinase and/or Ca2+-calmodulin-dependent protein kinase as well as sequential phosphorylation in both orders to assess the veracity of antibody recognition of phosphorylated forms. Western blots proved useful in characterizing the reactivity of the different antibodies to phospholamban and phosphorylated phospholamban, but were inefficient for accurate quantitation and problems with antibody recognition of dually phosphorylated phospholamban were found. mAb 1D11 recognized all forms of phospholamban, polyclonal antibodies 285 and PS-16 were highly selective for P-Ser(16) phospholamban but had diminished reactivity to diphosphorylated (P-Ser(16), P-Thr(17)) phospholamban, and polyclonal antibody PT-17, although selective for P-Thr(17) phospholamban, generated very weak signals on Western blots and reacted poorly with diphosphorylated phospholamban. Results in quantitative immunodot blot experiments were even more compelling. None of the phosphorylation specific antibodies reacted with the diphospho 1-25 phospholamban peptide, Transgenic mouse hearts expressing varying levels of PLB and ferret heart biopsy samples taken before and after isoproterenol perfusion were analyzed. In all samples containing phospholamban, a basal level of Ser(16) phosphorylation (about 4% of the total PLB population) and a lesser amount ofThr(17) phosphorylation was observed. Upon isoproterenol perfusion, Ser(16) phosphorylation increased only to 17% of the total phospholamban population with a similar change in Thr(17) phosphorylation. This suggests that phospholamban phosphorylation may serve as an electrostatic switch that dissociates inactive calcium pump complexes into catalytically active units. Thus, direct correlations between phospholamban phosphorylation state and contractile parameters may not be valid. (C) 2000 Academic Press.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 02/12/20 alle ore 05:32:41