Abstract
The mineralized scale of the freshwater sunfish Lepomis macrochirus (bluegill) contains a Gla protein. The protein was identified in extracts of scale by a new colorimetric assay for Gla-containing proteins. The protein was purified by gel filtration chromatography followed by reversed phase high performance liquid chromatography (HPLC). Several tests establish the identity of scale Gla protein and bone Gla protein (BGP). First, the proteins exhibit identical mobilities on electrophoresis and by reversed phase HPLC. Second, they have identical amino-terminal amino acid sequences. Finally, identical peptides are generated by proteolytic digestion. The 45-residue amino acid sequence of the bone Gla protein from L. macrochirus has a high sequence homology with swordfish, as well as homology to mammalian bone Gla protein. The BGP of bluegill shares with swordfish BGP a truncated NH2 terminus and an extended COOH terminus. These features may be unique to fish, as they have not been observed in terrestrial vertebrates. The bluegill BGP is the first vitamin K-dependent protein to contain a non-gamma-carboxylated residue to the NH2-terminal side of all of its Gla residues. In all other vitamin K-dependent proteins, Gla always appears to the NH2-terminal side of the first Glu. The implications of this result are discussed. The bluegill rib bone is curiously enriched in BGP, as are other mineralized tissues of this species. One hypothesis is that this may be due to the acellular nature of the bone in this species. The abundance of BGP in the bones of this fish may provide clues to the unknown function of this bone protein.
Highlights
From the $Biochemistry Department, University of Tennessee, Memphis, Tennessee 38163 and the Wollegeof Marine Studies, University of Delaware, Lewes, Delaware 19958
The amino acids between swordfish and calfbone Gla proteins protein was purified by gelfiltration chromatography followed by reversed phase high performance liquid chromatography (HPLC)
Several tests establish the identity of scale Gla proteinand bone Gla protein (BGP)
Summary
The pattern and amount of protein extractable analyzed by Sephacryl S-200 or acrylamide gel electrophoresis was identical in rib bones boiled for 2 min or unboiled. Proteolytic Peptides and Protein Sequence-Samples of scale and bone proteins were digestedby cyanogen bromide (CNBr)or S.aureus V8 protease by established methods [12], andthe peptides were separated by a modified gradient (7% acetonitrile initially to 56% over 35 min) reversed phase HPLC described above. The gels were either Coomassie Blue-stained or -blotted and DBS-stained (reacted with 4-diazobenzenesulfonicacid) to visualize the y-carboxyglutamic acid or Gla-containing proteins as described previously [17]. Using this blotting and DBS staining technique, even impure preparations could be seen to contain Gla-containing proteins and peptides. Sodium dodecyl sulfate gel electrophoresis was performed as described by Laemmli, and stained with Coomassie Blue [18]
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