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The cytoplasmic loop between putative transmembrane segments 6 and 7 in sarcoplasmic reticulum Ca2+-ATPase binds Ca2+
1Département de Biologie Cellulaire et Moléculaire, Section de Biophysique des Protéines et des Membranes, Commissariat à l'Energie Atomique et CNRS URA 2096, Centre d'Etudes de Saclay, 91191 Gif sur Yvette, Cedex, France.
The Journal of Biological Chemistry
|July 11, 1997
Summary
Proteinase K digestion of SERCA1 Ca2+-ATPase revealed that the 808-818 region is crucial for Ca2+ binding and enzyme function. This region, located in a cytoplasmic loop, is essential for the Ca2+-ATPase activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzyme kinetics
Background:
- Limited proteolysis of rabbit SERCA1 Ca2+-ATPase by proteinase K yields identified fragments.
- Focus on two C-terminal fragments, p20C (starting Gly-808) and p19C (starting Asp-818).
Purpose of the Study:
- To investigate the role of the C-terminal region 808-818 in Ca2+ binding and Ca2+-ATPase activity.
- To determine the functional significance of specific amino acid residues within this region.
Main Methods:
- Limited proteolysis using proteinase K.
- SDS-polyacrylamide gel electrophoresis (PAGE) to analyze fragment migration.
- 45Ca2+ labeling in overlay experiments to assess Ca2+ binding.
- Site-directed mutagenesis to create cluster mutants (D813A/D818A, D813A/D815A/D818A).
- Expression of mutant Ca2+-ATPases in Saccharomyces cerevisiae.
- Measurement of Ca2+-ATPase activity in yeast expression system.
Main Results:
- The longer fragment p20C binds Ca2+, confirmed by altered migration in Ca2+-containing SDS-PAGE and 45Ca2+ overlay.
- The shorter fragment p19C does not bind Ca2+ under the same experimental conditions.
- Cluster mutants D813A/D818A and D813A/D815A/D818A exhibit significantly reduced Ca2+-ATPase activity.
- Proteinase K accessibility indicates transmembrane segment M6 terminates before residue 808.
- Critical residues for enzyme activity are located in a cytoplasmic loop beginning at Gly-808.
Conclusions:
- The region 808-818 of SERCA1 Ca2+-ATPase is essential for both Ca2+ binding and catalytic activity.
- These findings align with studies on homologous enzymes like gastric H+, K+-ATPase.
- Key functional residues are situated in a cytoplasmic loop, despite their proximity to transmembrane regions.