Identification of organic phosphorus covalently bound to collagen and non-collagenous proteins of chicken-bone matrix. The presence of O-phosphoserine and O-phosphothreonine in non-collagenous proteins, and their absence from phosphorylated collagen

Abstract

Non-collagenous phosphoproteins, almost all of which can be extracted in EDTA at neutral pH in the presence of proteinase inhibitors, are identified in the matrix of chicken bone, and are therefore not covalently bound to collagen. Similarly, all the peptides containing y-carboxyglutamic acid are present in the EDTA extract and none in the insoluble residue, confirming that none is covalently linked to chicken bone collagen. However, organic phosphorus is also found to be present in chicken bone collagen, principally in the a2-chains. Of the total protein-bound organic phosphorus present in chicken bone matrix, approx. 80 % is associated with the non-collagenous proteins and 20 % with collagen. The soluble non-collagenous proteins contain both O-phosphoserine and 0-phosphothreonine and these account for essentially all of their organic phosphorus content. In contrast, collagen contains neither 0-phosphoserine nor 0-phosphothreonine. Indeed, no phosphorylated hydroxy amino acid, phosphoamidated amino acid or phosphorylated sugar could be identified in purified components of collagen, which contain approximately four to five atoms of organic phosphorus per molecule of collagen. Peptides containing organic phosphorus were isolated from partial acid hydrolysates and enzymic digests of purified collagen components, which contain an as-yet-unidentified cationic amino acid. These data, the very high concentrations of glutamic acid in the phosphorylated peptides, and the pH-stability of the organic phosphorus moiety in intact collagen chains strongly suggest that at least part of the organic phosphorus in collagen is present as phosphorylated glutamic acid. This would indicate that the two major chemically different protein fractions in chicken bone matrix that contain organic phosphorus may represent two distinct metabolic pools of organic phosphorus under separate biological control.Phosphoproteins have been identified as part of the non-collagenous components of the organic matrices of mineralized vertebrate tissues such as enamel (Seyer & Glimcher, 1971;Papas et al., 1977;Seyer & Glimcher, 1977a), dentine Veis & Perry, 1967;Butler, 1972;Butler et al., 1972;Veis et al., 1972;Carmichael & Dodd, 1973;Pieri et al., 1975;Menanteau et al., 1977) and bone (Spector & Glimcher, 1972;Shuttleworth & Veis, 1972) [see Leaver et al. (1975b) for a review]. There still exists, however, at least two major points of controversy with regard to bone and dentine: (1) how much, if any, of the phosphorylated protein(s) is covalently bound to the collagen, and (2), if present, Abbreviation used: P,, organic phosphate. * Present address: I.N.S.E.R.M. U12 and U18, Hc6pital des Enfants malades, Paris, France. t To whom requests for reprints should be addressed. Vol. 177 the nature of the organic phosphate moiety covalently bound to the collagen.Although evidence has been presented that none of the phosphoprotein is covalently bound to the collagen of rat dentine (Butler, 1972;Butler et al., 1972Butler et al., , 1976, equally substantia...

Radioautographic visualization and biochemical identification of O-phosphoserine- and O-phosphothreonine-containing phosphoproteins in mineralizing embryonic chick bone.

“…We believe it may be concluded that the "P-labeled components visualized in this study by radioautography represent principally the phosphoproteins isolated and characterized previously (14,(21)(22)(23). The localization of silver grains over endoplasmic reticulum and Golgi apparatus of osteoblasts provides further strong evidence that the phosphoproteins are synthesized principally by these cells, a conclusion consistent with the results recently obtained in cell culture by Gotoh et al (15).…”

“…The "P-labeled phosphoprotein(s) identified in the present experiments are, like those isolated and characterized from whole postnatal chick bone (21) and those synthesized in organ and cell culture (14,15) in EDTA, are nondiffusible and contain both Ser(P) and Thr(P) . We believe it may be concluded that the "P-labeled components visualized in this study by radioautography represent principally the phosphoproteins isolated and characterized previously (14,(21)(22)(23).…”

“…Aliquot portions of these samples were hydrolyzed in 4 N HCI, 106°C, for 6 h and subjected to preparative ion exchange amino acid chromatography for the isolation of Ser("P) and Thr("P) (21,22).…”

An unusual bovine pancreatic protein exhibiting pH-dependent globule-fibril transformation and unique amino acid sequence.

“…The phosphate content was analyzed chromatographically on a Beckman 121M amino acid analyzer as phosphoserine and phosphothreonine after hydrolysis in 4 M HCl for 6 hr at 105°C as described by Cohen-Solal et al (18) in the laboratory of Melvin Glimcher (Childrens Hospital Medical Center).…”

Prothymosin α in Vivo Contains Phosphorylated Glutamic Acid Residues

“…Very few bona fide examples of glutamyl phosphates in eukaryotes have been described. The best documented case is that of the ␣2 chains of type I chicken bone collagen (46), where 4 -5 atoms of organic phosphorus/mol of collagen were found in the absence of phosphorylated hydroxyamino acids, phosphoamidated amino acids, or phosphorylated sugars (47). These glutamyl phosphate groups, which survived in part during the several hours needed for purification of the protein, were later localized to a specific peptide (48).…”