Isolation and complete amino acid sequence of osteocalcin from canine bone

Giovanna Colombo, Paolo Fanti, Chinghua Yao, Hartmut H. Malluche

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

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 γ-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 γ-carboxylated domains of blood-clotting proteins and the Pro-rich N-terminal extensions of myosin lightchain Al and β-crystalline B1. The possible relevance of these homologies to the structure and potential functions of osteocalcin is discussed.

Original languageEnglish (US)
Pages (from-to)733-743
Number of pages11
JournalJournal of Bone and Mineral Research
Volume8
Issue number6
StatePublished - Jun 1993
Externally publishedYes

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Osteocalcin
Canidae
Amino Acid Sequence
Bone and Bones
Dogs
Solid Phase Extraction
Powders
Proteins
Amino Acids
Diaphyses
Basic Amino Acids
Peptides
Acids
Blood Coagulation
Myosins
Liquid Chromatography
Disulfides
Femur
Birds
Haplorhini

ASJC Scopus subject areas

  • Surgery

Cite this

Colombo, G., Fanti, P., Yao, C., & Malluche, H. H. (1993). Isolation and complete amino acid sequence of osteocalcin from canine bone. Journal of Bone and Mineral Research, 8(6), 733-743.

Isolation and complete amino acid sequence of osteocalcin from canine bone. / Colombo, Giovanna; Fanti, Paolo; Yao, Chinghua; Malluche, Hartmut H.

In: Journal of Bone and Mineral Research, Vol. 8, No. 6, 06.1993, p. 733-743.

Research output: Contribution to journalArticle

Colombo, G, Fanti, P, Yao, C & Malluche, HH 1993, 'Isolation and complete amino acid sequence of osteocalcin from canine bone', Journal of Bone and Mineral Research, vol. 8, no. 6, pp. 733-743.
Colombo, Giovanna ; Fanti, Paolo ; Yao, Chinghua ; Malluche, Hartmut H. / Isolation and complete amino acid sequence of osteocalcin from canine bone. In: Journal of Bone and Mineral Research. 1993 ; Vol. 8, No. 6. pp. 733-743.
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