Isolation and complete amino acid sequence of osteocalcin from canine bone.
Osteocalcin was purified in high yield and to homogeneity from the diaphysis of dog femora by the following steps: (1) acid demineralization of bone powder, (2) solid-phase extraction of acid-soluble proteins on Sep-Pak C18 cartridges, (3) gel filtration on Sephadex G-50, and (4) fast protein liquid chromatography on an Accell-QMA anion-exchange column. Starting from 30 g washed bone powder, approximately 7-10 mg pure protein was obtained in 2 days. The key step is the initial solid-phase extraction of osteocalcin from a large volume of a demineralized bone solution. The primary structure was established by automated sequence analyses of two tryptic peptides, of two endoproteinase Glu-C carboxy-terminal peptides, and of the first 30 amino acid residues of the intact protein. Dog osteocalcin contains 49 amino acids, has a molecular mass of 5654 daltons, contains no Thr, Met, Hyp, or Trp, has a disulfide bond between Cys 23 and 29, and is fully gamma-carboxylated at residues 17, 21, and 24. Dog osteocalcin does not contain a pair of basic amino acids found at positions 43-44 in most other osteocalcins from mammals and birds. A computer search for homology indicated 88, 90, 84, 88, 66, and 57% sequence identity of dog osteocalcin with human, bovine, cat, monkey, chicken, and swordfish osteocalcin, respectively, and weaker homologies with the gamma-carboxylated domains of blood-clotting proteins and the Pro-rich N-terminal extensions of myosin light-chain A1 and beta-crystalline B1. The possible relevance of these homologies to the structure and potential functions of osteocalcin is discussed.
Colombo
G
G
Department of Biochemistry, Albert B. Chandler Medical Center, University of Kentucky, Lexington.
Fanti
P
P
Yao
C
C
Malluche
H H
HH
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001842:Bone and Bones / Q000737:chemistry; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D005971:Glutamates / Q000302:isolation & purification; D018698:Glutamic Acid; D006801:Humans; D008969:Molecular Sequence Data; D015675:Osteocalcin / Q000737:chemistry / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000302:isolation & purification; D013057:Spectrum Analysis; D014357:Trypsin
D000596:Amino Acids; D005971:Glutamates; D010446:Peptide Fragments; D015675:Osteocalcin; D018698:Glutamic Acid; D010450:Endopeptidases; D014357:Trypsin