Abstract
The gamma-carboxyglutamate-containing protein from human bone has been isolated from proteins released upon demineralization of bone by using a combination of gel filtration and immunoadsorption onto a column of immobilized antibody. The sequence of the protein has been determined by automatic sequence analysis of the whole protein and of peptides isolated from tryptic and Staphylococcus aureus protease digests. The protein consists of 49 amino acids in a single polypeptide chain, contains 2 residues of gamma-carboxyglutamic acid, and a single disulfide bond. There are 4 translational substitutions between the human and calf bone proteins and 23 translational substitutions between the human and swordfish bone proteins. Glu-17 was found to be only 9% gamma-carboxylated, while sequence positions 21 and 24 are fully gamma-carboxylated. The identification of glutamic acid at position 17 represents the first instance where a partially gamma-carboxylated glutamate has been found in a sequence position which is to the NH2-terminal side of a gamma-carboxyglutamate residue. The possible significance of this observation is discussed.
Highlights
From the $Department of Biology (8-022), University of California, Sun Diego, La Jolla, California 92093 and nthe Salk Institute for Biological Studies, La Jotla, Californi9a2138
We have described previously the isolation and sequence of the 49-residue calf bone
Isolation a n d Purification of Human Bone Gla ProteinDemineralization of human bone in 20%formic acid followed by gel filtration of the soluble extract on Sephacryl S-200 in 6 M GuHCl (Fig. 2) proved to be a useful initial step in the isolation of the Gla protein
Summary
Portions of this paper (including “Materials and Methods,” Figs. 1s through 4S, and Tables 11through IX) are presented in miniprint at the end of this paper. 1s through 4S, and Tables 11through IX) are presented in miniprint at the end of this paper. The miniprint is read with the aid of a standard magnifyingglass. Full sized photocopies are available from the Journal of Biological Chemistry, 9650 RockviIle Pike, Bethesda, MD 20014. Request Document 80M-683, cite author(s), and include a check or money order for $2.70 per set of photocopies.
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