Phospholamban Inhibits Ca-atpase Conformational Changes Involving the E2 Intermediate.
From: Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA.
Biochemistry
- Publish Date: Feb 2007
- ISSN: 0006-2960
- Volume: 46
- Issue: 7
- Pages: 1999-2009
- Medium: Print
- Language: English
- Citation (JAMA): Waggoner Jason R, Huffman Jamie, Froehlich Jeffrey P, et al. Phospholamban Inhibits Ca-atpase Conformational Changes Involving the E2 Intermediate.. Biochemistry Feb 2007;46:1999-2009
Abstract
We have used steady-state fluorescence spectroscopy in combination with enzyme kinetic assays to test the hypothesis that phospholamban (PLB) stabilizes the Ca-ATPase in the E2 intermediate state. The cardiac muscle Ca-ATPase (SERCA2a) isoform was expressed either alone or coexpressed with PLB in High-Five insect cells and was isolated as insect cell microsomes. Fluorescence studies of the Ca-ATPase covalently labeled with the probe 5-(2-((iodoacetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid showed that PLB decreased the amplitude of the Ca-ATPase E2 — > E1 conformational transition by 45 +/- 3% and shifted the [Ca2+] dependence of the transition to higher Ca2+ levels (DeltaKCa = 230 nM), similar to the effect of PLB on Ca-ATPase activity. Similarly, PLB decreased the amplitude of Ca-ATPase phosphorylation by inorganic phosphate (Pi) by 55 +/- 2% and decreased slightly the affinity for Pi (DeltaK0.5 = 70 microM). However, PLB did not affect the Ca2+-dependent inhibition of Ca-ATPase phosphorylation by Pi. Finally, PLB decreased Ca-ATPase sensitivity to vanadate, increasing the IC50 value by 300 nM. The results suggest that PLB binding to Ca-ATPase stabilizes the enzyme in a conformation distinct from E2, decreasing the number of enzymes in the E2 state capable of undergoing ligand-dependent conformational changes involving the Ca-free E2 intermediate. The inability of conformation-specific ligands to fully convert this E2-like state into E1 or E2 implies that these states are not in a simple equilibrium relationship.
Mesh Headings (Keywords): Animals, Antibodies, Monoclonal, Calcium-Binding Proteins, Cells, Cultured, Dogs, Fluorescent Dyes, Insects, Microsomes, Naphthalenesulfonates, Phosphoric Acids, Phosphorylation, Protein Conformation, Sarcoplasmic Reticulum Calcium-Transporting ATPases, Spectrometry, Fluorescence
Check for Full Text / PubMed Unique Identifier (PMID): 17261028
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