Critical Evaluation of Cardiac Ca2+-ATPase Phosphorylation on Serine 38 Using a Phosphorylation Site-specific Antibody
| Autor: | Patricia Rodriguez, John Colyer, Wayne A. Jackson |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Male
Models Molecular Spectrometry Mass Electrospray Ionization Protein Conformation Blotting Western Immunoblotting Molecular Sequence Data Enzyme-Linked Immunosorbent Assay Calcium-Transporting ATPases Biology Biochemistry Protein Structure Secondary Sarcoplasmic Reticulum Calcium-Transporting ATPases Serine Cell membrane Epitopes Dogs Western blot medicine Animals Myocyte Amino Acid Sequence Phosphorylation Rats Wistar Molecular Biology Cells Cultured Muscle Cells Dose-Response Relationship Drug Sequence Homology Amino Acid medicine.diagnostic_test Endoplasmic reticulum Vesicle Cell Membrane Cardiac muscle Cell Biology Molecular biology Protein Structure Tertiary Rats Sarcoplasmic Reticulum medicine.anatomical_structure Calibration Rabbits Peptides |
| Zdroj: | Journal of Biological Chemistry. 279:17111-17119 |
| ISSN: | 0021-9258 |
| Popis: | The phosphorylation of the cardiac muscle isoform of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a) on serine 38 has been described as a regulatory event capable of very significant enhancement of enzyme activity (Hawkins, C., Xu, A., and Narayanan, N. (1994) J. Biol. Chem. 269, 31198-31206). Independent confirmation of these observations has not been forthcoming. This study has utilized a polyclonal antibody specific for the phosphorylated serine 38 epitope on the Ca(2+)-ATPase to evaluate the phosphorylation of SERCA2a in isolated sarcoplasmic reticulum vesicles and isolated rat ventricular myocytes. A quantitative Western blot approach failed to detect serine 38-phosphorylated Ca(2+)-ATPase in either kinase-treated sarcoplasmic reticulum vesicles or suitably stimulated cardiac myocytes. Calibration standards confirmed that the detection sensitivity of assays was adequate to detect Ser-38 phosphorylation if it occurred on at least 1% of Ca(2+)-ATPase molecules in SR vesicle experiments or on at least 0.1% of Ca(2+)-ATPase molecules in cardiac myocytes. The failure to detect a phosphorylated form of the Ca(2+)-ATPase in either preparation (isolated myocyte, purified sarcoplasmic reticulum vesicles) suggests that Ser-38 phosphorylation of the Ca(2+)-ATPase is not a significant regulatory feature of cardiac Ca(2+) homeostasis. |
| Databáze: | OpenAIRE |
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