title: Plasmin-mediated proteolysis of osteocalcin.
abstract: Plasmin cleaves osteocalcin at a site within its carboxyl end, thus creating an N-midterminal 1-43 and a short C-terminal 44-49 peptides. The products of the cleavage were identified by matrix assisted laser desorption ionization time of flight mass spectrophotometry and by reversed phase high performance liquid chromatography followed by N-terminal sequence determination. When separated by sodium dodecyl sulfide-polyacrylamide gel electrophoresis in the presence of reducing agents, large (LF; N-midterminal) and a small molecular weight (SF; C-terminal) fragments can be identified. The major cleavage site involves arg43-arg44 amino acid residues, and the resulting 44-49 C-terminal fragment appears as a slow migrating band on native gels (SFnat). Elevated levels of calcium ion inhibit the plasmin-mediated lysis of osteocalcin. Plasmin-mediated cleavage of osteocalcin occurs both in solution and when bound to hydroxyapatite. Both osteocalcin cleavage products detach from the hydroxyapatite substrate. Diisopropyl fluorophosphate-inhibited plasmin does not displace osteocalcin from the hydroxyapatite surface. Previously, the C-terminal pentapeptide has been shown to be chemotactic for bone cells while bone particles lacking osteocalcin were resistant to bone resorption. We therefore hypothesize that the plasmin-mediated digestion of free and hydroxyapatite-bound osteocalcin could play a role in the regulation of bone remodeling.
authors.affiliation: Biology Department, Bucknell University, Lewisburg, Pennsylvania, USA.
authors.forename: J F
authors.initials: JF
authors.lastname: Novak
authors.forename: J D
authors.initials: JD
authors.lastname: Hayes
authors.forename: S K
authors.initials: SK
authors.lastname: Nishimoto
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D016723:Bone Remodeling / Q000502:physiology; D002851:Chromatography, High Pressure Liquid; D005341:Fibrinolysin / Q000378:metabolism*; D006801:Humans; D006882:Hydroxyapatites; D066298:In Vitro Techniques; D008970:Molecular Weight; D015675:Osteocalcin / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012996:Solutions; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D006882:Hydroxyapatites; D010446:Peptide Fragments; D012996:Solutions; D015675:Osteocalcin; D005341:Fibrinolysin