168 S. M. Krane, M. J. Glimcher 



Protein Phosphorus and Phosphokinases In Connective Tissue 



S. M. Krane, M. J. Glimcher 



Departments of Medicine and Orthopedic Surgery, Harvard Medical School, 

 Massachusetts General Hospital, Boston, Mass., U. S. A. 



We have previously discussed the evidence which suggested that interactions 

 betweeen phosphorus and groups on the side chains of the structural proteins of 

 mineralized tissues played a major role in the nucleation of apatite crystals. (Glim- 

 cher and Krane, 1962). It was shown that purified, reconstituted collagen fibrils, 

 incubated in vitro with inorganic orthophosphate, bound as many as 150 — 170 moles 

 of phosphorus per mole collagen (Glimcher and Krane, 1964 a). Furthermore the 

 interaction between collagen and phosphate was found to encompass a wide spectrum 

 of bond types from readily dissociable, electrostatic bonds to covalent bonds. 

 Covalently bound phosphorus would be ideally suited for a role in the nucleation 

 process, since such groups would be fixed at specific sites on the molecule as well as 

 have relatively fixed distance, direction and orientation, and still be reactive. Sub- 

 sequently we were able to demonstrate that ^-P-orthophosphate administered to 

 rabbits and guinea pigs in vivo, was found associated with both the neutral and acid 

 soluble collagens of skin and healing wounds (Glimcher et al., 1964). Furthermore 

 organic phosphorus was detected chemically, in varying amounts in different species, 

 in soft-tissue collagens and gelatins derived from collagens. In addition the single- 

 stranded a chains of several of these gelatins were purified by chromatography on 

 carboxymethyl-cellulose resin columns and shown still to contain the organic phos- 

 phorus, with the higher concentration in the a, compared to the a^ chains. C. J. Fran- 

 cois (personal communication) has recently found organic phosphorus in purified 

 a gelatin chains from chicken bone. Veis and Schlueter (1964) and Schlueter and 

 Veis (1964) have also reported the presence of organic phosphorus in fragments 

 produced by periodate treatment of bovine dentin. We observed that after digestion 

 of the collagens and gelatins with bacterial collagenase and partial acid hydrolysis 

 followed by various chromatographic and electrophoretic procedures, that the organic 

 phosphorus was present in several peptide fractions rich in glycine and serine and 

 containing relatively large amounts of sugar. However, in all but one of these frac- 

 tions more phosphorus was present than could be accounted for by the content of 

 serine and threonine alone. In addition, the phosphorus linkage was more stable to 

 alkali than is usually found for peptide-bound phosphoserine. It was, therefore, 

 suggested that at least part of the organic phosphorus in collagens was bound to a 

 carbohydrate moiety. 



Since tooth enamel is the most highly mineralized of all the calcified tissues in 

 vertebrates, phosphorus analyses were performed on the decalcified, soluble organic 

 matrix proteins of both fetal and adult bovine enamel (Glimcher and Krane, 

 1964 b). These proteins contained large amounts of organic phosphorus, some frac- 

 tions as many as 90 residues per 1000 amino acid residues. After partial acid 

 hydrolysis it was possible to identify O-phosphoserine and O-phosphoserine- 

 containing peptides. More recently we have demonstrated the presence of organic 

 phosphorus in the proteins of newly formed enamel from the incisor teeth of adult 

 rabbits and rats. 



