Molecular Aspects of Biological Hydroxyapatite Formation

Coleman, Joseph

Abstract

The specific aims of the project are: 1) to determine the structure and mechanism of the matrix vesicle alkaline phospatase (AP) from mammalian bone and its role in Ca++ and HPO4 transport and hydroxyapatite formation; and 2) to determine the structural features of the bone Gamma-carboxyglutamate (Gla)-containing protein (BGP or osteocalcin) and the particular topology of this molecule responsible for its recognition of the hydroxyapatite lattice and to explore the function of this vitamin K dependently-carboxylated protein. Both alkaline phosphatase and the bone Gla-containing protein are molecules participating in the formation and dynamic equilibrium of the mineral phase of bone. The bone alkaline phosphatase will be isolated and characterized as regards MW, Zn(II), Mg(II) and Ca(II) binding properties, formation of covalent and noncovalent phosphoenzyme intermediates. It will be determined if alkaline phosphatase is one of several or the major peptide associated with the matrix vesicle and its membrane-binding domain determined as well as the relationship of the phosphate ester hydrolysis to Ca transport. Formation of artificial vesicles containing AP will be attempted. The solution structure of BGP and its Ca complex will be determined and attempts will be made to determine the topology of the protein when bound to the surface of hydroxyapatite. Methods to be used are multinuclear NMR (1H for the solution structure of BGP; 31P for the phosphoenzyme intermediates of AP, 113Cd NMR for the metal complexes of BGP and AP, 13C for the conformation of BGP in both solution and the solid state. The membrane binding domain of AP and the hydroxyapatite binding domain of BGP will be explored by limited proteolysis, the latter also by chemical modification in the solution vs. the solid state. The 45Ca++, H32PO4 will be used to measure the effect of BGP on the CaHPO42H20-hydroxyapatite equilibrium and EM, X-ray, and binding studies will be applied to the interaction of BGP with hydroxyapatite and the bone matrix.