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Ca2+ concentration during binding determines the manner in which annexin V binds to membranes
P J Trotter1, M A Orchard, J H Walker
1Department of Biochemistry and Molecular Biology, University of Leeds, U.K.
The Biochemical Journal
|June 1, 1995
Summary
Annexin V relocates to platelet membranes via two calcium-dependent mechanisms. One form binds reversibly, while another binds tightly, suggesting interaction with non-phospholipid membrane components.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Annexins are calcium-binding proteins involved in various intracellular processes.
- Platelet activation by thrombin induces Annexin V membrane association in two distinct ways.
- These interactions differ in their sensitivity to EGTA and Triton X-100.
Purpose of the Study:
- To investigate the mechanisms of Annexin V membrane association in platelets.
- To determine the role of calcium concentration in Annexin V binding.
- To identify potential binding partners of Annexin V on the membrane.
Main Methods:
- Platelet stimulation with thrombin and calcium ionophore A23187.
- EGTA and Triton X-100 extraction to differentiate binding.
- Triton X-114 phase separation to analyze membrane association.
- Isoelectric focusing to assess protein modification.
- Binding assays with phospholipid vesicles.
- Bifunctional cross-linking to identify protein complexes.
Main Results:
- Calcium ionophore A23187 mimics thrombin-induced Annexin V relocation.
- EGTA-resistant Annexin V binding requires higher calcium concentrations (0.8 microM) than EGTA-elutable binding.
- Membrane-associated Annexin V partitions into the aqueous phase after Triton X-114 treatment.
- Annexin V isoelectric point remains unchanged, indicating no covalent modification.
- EGTA-resistant binding to phospholipids is reversible, suggesting interaction with other membrane components.
- Annexin V and protein kinase C relocation are not always parallel.
- An 85 kDa complex containing Annexin V and a ~50 kDa protein was identified.
Conclusions:
- Annexin V exhibits distinct calcium-dependent membrane association modes in platelets.
- The EGTA-resistant form likely binds to non-phospholipid membrane components.
- Covalent modification of Annexin V is not responsible for its membrane association.
- Annexin V may form complexes with other membrane proteins, potentially involved in platelet function.