The Mr 24,000 phosphoprotein from developing bone is the NH2-terminal propeptide of the alpha 1 chain of type I collagen.
Using nondegradative isolation procedures, we have purified and characterized the Mr 24,000 phosphoprotein from developing bovine and human bone where it constitutes 5% of the noncollagenous protein in the mineral compartment. This hydroxyproline-containing protein could not be cleaved by cyanogen bromide. The purified, intact product spontaneously formed a complex consistent with a collagen-like trimer that remained a trimer even in sodium dodecyl sulfate-polyacrylamide gels. The ability to form the complex was lost upon treatment with bacterial collagenase, a treatment that resulted in an NH2-terminally blocked fragment of Mr 17,000. After deblocking, the NH2-terminus of the intact, Mr 24,000 bovine product was shown to have virtually the same amino acid sequence (residues 1-24 with asparagine rather than aspartic acid at position 20 as reported earlier by Horlein et al. (Horlein, D., Fietzek, P. P., Wachter, E., Lapiere, C. M., and Kuhn, K. (1979) Eur. J. Biochem. 90, 31-38) as the amino-terminal segment of dermatosparatic calf skin alpha 1 type I procollagen. Furthermore, pulse-chase studies showed a precursor-product relationship between procollagen and the Mr 24,000 protein. Anti-serum made against the bovine bone protein bound to bands on electrotransfers that were consistent with the positions of both alpha 1(I) procollagen and the procollagen chain missing its COOH-terminal extension peptide (pN-alpha 1(I), as well as the original Mr 24,000 product in extracts of bone, skin, tendon, cornea, and other type I collagen-containing tissues. Fetal calf serum contained an average of 106 micrograms/ml of the Mr 24,000 protein as determined by quantitative enzyme-linked immunosorbent assay. The only serine residue in the bovine bone protein was phosphorylated. It is unknown whether the corresponding collagen NH2-terminal pro-peptides in other tissues and serum are similarly phosphorylated.
Fisher
L W
LW
Bone Research Branch, National Institute of Dental Research, Bethesda, Maryland 20892.
Robey
P G
PG
Tuross
N
N
Otsuka
A S
AS
Tepen
D A
DA
Esch
F S
FS
Shimasaki
S
S
Termine
J D
JD
D000818:Animals; D001846:Bone Development; D001842:Bone and Bones / Q000032:analysis* / Q000378:metabolism; D002417:Cattle; D005333:Fetus; D006801:Humans; D007231:Infant, Newborn; D046911:Macromolecular Substances; D008970:Molecular Weight; D010012:Osteogenesis; D010750:Phosphoproteins / Q000302:isolation & purification*; D011347:Procollagen / Q000096:biosynthesis* / Q000302:isolation & purification; D013045:Species Specificity
D046911:Macromolecular Substances; D010750:Phosphoproteins; D011347:Procollagen