Calcium-dependent protein-membrane interaction and prothrombinase reaction: A kinetic and mechanistic study

This thesis project was designed to study the kinetics and the mechanisms of the calcium-dependent protein-membrane interaction and the prothrombinase reaction. Kinetic study of the annexin-membrane interaction showed the EGTA-induced protein dissociation was dependent on the membrane composition and couple other factors, which also affected the calcium requirement far the protein-membrane interaction. A direct correlation between these two parameters was found, in supporting of the calcium-bridging model.;Kinetic study of the prothrombin-membrane interaction showed the importance of acidic phospholipid, PS, in this reaction. Raising the PS content 5 fold resulted in 100 fold higher affinity between the protein and the membrane. Comparison between the prothrombin-LUVs interaction vs. prothrombin-SUVs interaction only revealed a two-fold higher affinity with the SUVs. These results suggested that the electrostatic interaction rather than the hydrophobic insertion should be responsible for the protein-membrane interaction.;Prothrombinase reaction relies heavily on the calcium-dependent protein-membrane interaction. The dynamics of the prothrombin-membrane interaction provided an important base for the mechanistic study of this reaction. Depending on the membrane composition and vesicle size, several mechanisms may contribute to this enzyme reaction. Reaction mechanism may even change during the course of substrate titration, a so called 'mechanism switching'. Only membranes of low PS content displayed Michaelis-Menten behavior. As the PS content increased, several steps could be rate-limiting step(s), and Michaelis-Menten mechanism did not apply.;The interference of annexin with the protein-membrane interaction and the prothrombinase reaction were also studied. Depending on membrane composition and vesicle type, annexin showed differential impacts on the dynamics of prothrombin-membrane interaction and the prothrombinase reaction and variant efficiency in displacement of prothrombin from the membrane surface.;Annexin-membrane association induced many changes in the membrane, including the clustering of acidic phospholipids. The kinetic study showed that the majority of clustering and declustering occurred in parallel with protein-membrane binding and EGTA-induced dissociation, respectively. A small portion of the clustering continued after protein binding was complete, suggesting that annexin binding might happen through the attachment to a small number of acidic phospholipids followed by clustering of acidic phospholipids to form the thermodynamically most stable structure.