Synthetic bovine prothrombin precursor 13-29 for studies of vitamin K dependent carboxylase.
The synthesis of the amino acid sequence found in bovine prothrombin precursor 13-29 (PTP 13-29) has been achieved by solid-phase synthesis of the bis(acetamidomethyl)-protected linear peptide followed by cyclization to the monomeric disulfide. Synthesis of the disulfide bond was achieved by deprotection with mercuric acetate in acetic acid followed by oxidation with potassium ferricyanide. Experimental conditions for closure of the disulfide bond were identified by obtaining the circular dichroism spectra of the linear precursor in a variety of solvent systems. Cyclization in organic solvent systems was not successful but led to the formation of insoluble polymers. Synthetic PTP 13-29 was tested as a substrate for the vitamin K dependent carboxylase. Neither the linear nor cyclic synthetic 17 amino acid peptides were carboxylated as well as the standard, Boc-Glu-Glu-Leu-OMe, at mM concentrations. The estimated Km of synthetic PTP 13-29 is greater than 1 mM. Thus, bovine prothrombin precursor 13-29 is not an unusually effective substrate for the carboxylase as reported by Soute et al.
D000818:Animals; D019735:Carbon-Carbon Ligases; D002417:Cattle; D007202:Indicators and Reagents; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000378:metabolism; D011498:Protein Precursors / Q000138:chemical synthesis* / Q000378:metabolism; D011516:Prothrombin / Q000138:chemical synthesis* / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
D007202:Indicators and Reagents; D010446:Peptide Fragments; D011498:Protein Precursors; C050961:prothrombin precursor (13-29); D011516:Prothrombin; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase