Back to Search
Start Over
Involvement of the L6–7 Loop in SERCA1a Ca2+-ATPase Activation by Ca2+ (or Sr2+) and ATP
- Source :
- Journal of Biological Chemistry, Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2004, 279 (31), pp.32125-32133. ⟨10.1074/jbc.M402934200⟩, Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2004, 279, pp.32125-32133, Journal of Biological Chemistry, 2004, 279 (31), pp.32125-32133. ⟨10.1074/jbc.M402934200⟩
- Publication Year :
- 2004
- Publisher :
- Elsevier BV, 2004.
-
Abstract
- International audience; Wild-type (WT) and the double mutant D813A,D818A (ADA) of the L6-7 loop of SERCA1a were expressed in yeast, purified, and reconstituted into lipids. This allowed us to functionally study these ATPases by both kinetic and spectroscopic means, and to solve previous discrepancies in the published literature about both experimental facts and interpretation concerning the role of this loop in P-type ATPases. We show that in a solubilized state, the ADA mutant experiences a dramatic decrease of its calcium-dependent ATPase activity. On the contrary, reconstituted in a lipid environment, it displays an almost unaltered maximal calcium-dependent ATPase activity at high (millimolar) ATP, with an apparent affinity for Ca(2+) altered only moderately (3-fold). In the absence of ATP, the true affinity of ADA for Ca(2+) is, however, more significantly reduced (20-30-fold) compared with WT, as judged from intrinsic (Trp) or extrinsic (fluorescence isothiocyanate) fluorescence experiments. At low ATP, transient kinetics experiments reveal an overshoot in the ADA phosphorylation level primarily arising from the slowing down of the transition between the nonphosphorylated "E2" and "Ca(2)E1" forms of ADA. At high ATP, this slowing down is only partially compensated for, as ADA turnover remains more sensitive to orthovanadate than WT turnover. ADA ATPase also proved to have a reduced affinity for ATP in studies performed under equilibrium conditions in the absence of Ca(2+), highlighting the long range interactions between L6-7 and the nucleotide-binding site. We propose that these mutations in L6-7 could affect protonation-dependent winding and unwinding events in the nearby M6 transmembrane segment.Wild-type (WT) and the double mutant D813A,D818A (ADA) of the L6-7 loop of SERCA1a were expressed in yeast, purified, and reconstituted into lipids. This allowed us to functionally study these ATPases by both kinetic and spectroscopic means, and to solve previous discrepancies in the published literature about both experimental facts and interpretation concerning the role of this loop in P-type ATPases. We show that in a solubilized state, the ADA mutant experiences a dramatic decrease of its calcium-dependent ATPase activity. On the contrary, reconstituted in a lipid environment, it displays an almost unaltered maximal calcium-dependent ATPase activity at high (millimolar) ATP, with an apparent affinity for Ca(2+) altered only moderately (3-fold). In the absence of ATP, the true affinity of ADA for Ca(2+) is, however, more significantly reduced (20-30-fold) compared with WT, as judged from intrinsic (Trp) or extrinsic (fluorescence isothiocyanate) fluorescence experiments. At low ATP, transient kinetics experiments reveal an overshoot in the ADA phosphorylation level primarily arising from the slowing down of the transition between the nonphosphorylated "E2" and "Ca(2)E1" forms of ADA. At high ATP, this slowing down is only partially compensated for, as ADA turnover remains more sensitive to orthovanadate than WT turnover. ADA ATPase also proved to have a reduced affinity for ATP in studies performed under equilibrium conditions in the absence of Ca(2+), highlighting the long range interactions between L6-7 and the nucleotide-binding site. We propose that these mutations in L6-7 could affect protonation-dependent winding and unwinding events in the nearby M6 transmembrane segment.
- Subjects :
- Time Factors
SERCA1a ATPase
ATPase
Mutant
L6-7 loop
Biochemistry
chemistry.chemical_compound
Adenosine Triphosphate
P-type ATPase
strontium
Phosphorylation
Adenosine Triphosphatases
0303 health sciences
biology
Transition (genetics)
030302 biochemistry & molecular biology
Tryptophan
Hydrogen-Ion Concentration
Lipids
Fluorescence
Sarcoplasmic Reticulum
Transmembrane domain
Electrophoresis, Polyacrylamide Gel
Rabbits
fluorescence
Detergents
Calcium-Transporting ATPases
Phosphates
Sarcoplasmic Reticulum Calcium-Transporting ATPases
03 medical and health sciences
[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology
Animals
[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology
Muscle, Skeletal
Molecular Biology
030304 developmental biology
Binding Sites
calcium
Dose-Response Relationship, Drug
Cell Biology
Lipid Metabolism
Yeast
Protein Structure, Tertiary
Kinetics
chemistry
Mutation
Isothiocyanate
biology.protein
Subjects
Details
- ISSN :
- 00219258 and 1083351X
- Volume :
- 279
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....3b78482b39f02896c3b8bf59c84bd76b