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.

Inactivation of the bovine mitochondrial F1-ATPase with dicyclohexyl[14C]carbodiimide leads to the modification of a specific glutamic acid residue in the beta subunit.

When the bovine mitochondrial F1-ATPase is inactivated with dicyclohexyl[14C]carbodiimide and then gel-filtered, from 2 to 3 g atoms of 14C are incorporated/mol of enzyme. Prior inactivation of the enzyme by the modification of an essential tyrosine residue with 4-chloro-7-nitrobenzofurazan, a reaction that can be reversed by thiols, does not affect the irreversible inactivation of the ATPase by dicyclohexyl[14C]carbodiimide. During the large scale modification of the F1-ATPase by dicyclohexyl[14C]carbodiimide which led to 70% inactivation, 1.9 g atoms of 14C were incorporated/mol of enzyme. Isolation of the alpha, beta, and gamma subunits from this large scale inactivation revealed that the gram atoms of 14C bound per mol of each of the subunits was: alpha, 0.04; beta, 0.56; and gamma, 0.04. The majority of the radioactivity in a cyanogen bromide digest of the 14C-labeled beta subunit was isolated in a fragment that has the following amino acid sequence: Glu-Leu-Ile-Asn-Asn-Val-Ala-Lys-Ala-His-Gly-Gly-Tyr-Ser-Val-Phe-Ala-Gly-Val-Gly -Glu-Arg-Thr-Arg-Glu-Gly-Asn-Asp-Leu-Tyr-Glu*-His-Met; where Glu* represents the N gamma-glutamyl derivative of dicyclohexyl[14C]urea.