title: Direct identification of the calcium-binding amino acid, gamma-carboxyglutamate, in mineralized tissue.
abstract: A direct approach has been developed for quantitative identification of the calcium-binding amino acid, gamma-carboxyglutamate, in proteins. This should be advantageous for the study of numerous systems where specific roles for the binding of calcium or other divalent cations are suspected. Investigation of mineralized tissue, where calcium-binding proteins are implicated in the mineralization process, revealed that gamma-carboxyglutamate was present in proteins solubilized from chicken bone with neutral aqueous ethylenediamine tetraacetic acid. This was established by direct isolation of the amino acid from alkaline hydrolysates and its quantitative conversion to glutamic acid by decarboxylation in 0.05 M HCl at 100 degrees. The kinetics of decarboxylation and chromatographic behavior are identical to those of gamma-carboxyglutamate from human prothrombin. After resolution of the soluble bone proteins by phosphate gradient elution from hydroxyapatite, gamma-carboxyglutamate was found to be concentrated primarily in one BaSO4-adsorbable anionic protein species; bone collagen was devoid of the amino acid. In view of the recently discovered requirement of vitamin K for generation of calcium binding sites (gamma-carboxyglutamate) by gamma-carboxylation of specific glutamic acid residues in prothrombin, our findings may implicate vitamin K metabolism in normal bone development and suggest a role for the gamma-carboxyglutamate-rich protein in regulation of calcium salt deposition in mineralized tissues.
mesh_terms: D000818:Animals; D001665:Binding Sites; D001842:Bone and Bones / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002645:Chickens; D005971:Glutamates / Q000032:analysis*; D006801:Humans; D011485:Protein Binding; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D011516:Prothrombin / Q000378:metabolism
chemical_list: D005971:Glutamates; D011506:Proteins; D011516:Prothrombin; D002118:Calcium