ISOLATION AND COMPLETE AMINO-ACID-SEQUENCE OF OSTEOCALCIN FROM CANINEBONE

Osteocalcin was purified in high yield and to homogeneity from the dia physis of dog femora by the following steps: (1) acid demineralization of bone powder, (2) solid-phase extraction of acid-soluble proteins o n Sep-Pak C18 cartridges, (3) gel filtration on Sephadex G-50, and (4) fast protein liquid chromatography on an Accell-QMA anion-exchange co lumn. Starting from 30 g washed bone powder, approximately 7-10 mg pur e protein was obtained in 2 days. The key step is the initial solid-ph ase extraction of osteocalcin from a large volume of a demineralized b one solution. The primary structure was established by automated seque nce analyses of two tryptic peptides, of two endoproteinase Glu-C carb oxy-terminal peptides, and of the first 30 amino acid residues of the intact protein. Dog osteocalcin contains 49 amino acids, has a molecul ar mass of 5654 daltons, contains no Thr, Met, Hyp, or Trp, has a disu lfide bond between Cys 23 and 29, and is fully gamma-carboxylated at r esidues 17, 21, and 24. Dog osteocalcin does not contain a pair of bas ic amino acids found at positions 43-44 in most other osteocalcins fro m mammals and birds. A computer search for homology indicated 88, 90, 84, 88, 66, and 57% sequence identity of dog osteocalcin with human, b ovine, cat, monkey, chicken, and swordfish osteocalcin, respectively, and weaker homologies with the gamma-carboxylated domains of blood-clo tting proteins and the Pro-rich N-terminal extensions of myosin light- chain A1 and beta-crystalline B1. The possible relevance of these homo logies to the structure and potential functions of osteocalcin is disc ussed.