Binding of a C-Terminal Fragment (Residues 369 to 435) of Vitamin D-Binding Protein to Actin
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S. Buch
Abstract
The vitamin Dbinding protein (DBP) binds to monomeric actin with high affinity. The variation in DBP isoforms is due to genetic polymorphism and varying glycosylation. To obtain a homogeneous preparation, the cDNA for human DBP and truncations thereof were cloned and various systems were applied for heterologous bacterial and yeast expression. The fulllength protein and the N and Cterminal halves of DBP remained insoluble probably because the protein did not fold to its native threedimensional structure due to formation of accidental intra and intermolecular disulfide bonds during expression in bacteria or yeast. This problem was overcome by cloning of a Cterminal fragment comprising residues 369 to 435 that did not contain disulfide bonds and was completely soluble. Binding of the Cterminal fragment to monomeric actin was demonstrated by comigration with actin during native polyacrylamide gel electrophoresis and surface plasmon resonance, however, at considerably lower affinity than fulllength DBP. This suggests that in addition to the Cterminal amino acid sequence other parts (amino acid residues or sugar moieties) of DBP participate in actin binding. The Cterminal fragment was found to inhibit denaturation of actin and to decrease the rate of actin polymerisation both at the barbed and at the pointed end in a concentrationdependent manner. According to a quantitative analysis of the polymerisation kinetics, association of actin monomers to nucleate filaments was not prevented by binding of the Cterminal fragment to actin. These data suggest that the sites on the surface of actin that are involved in actin nucleation and elongation are different.
Copyright © 2002 by Walter de Gruyter GmbH & Co. KG
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- Immunopharmacology of CpG DNA
- Induction and Regulation of Endogenous Granulocyte Colony-Stimulating Factor Formation
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