title: Evidence for the vitamin K-dependent gamma-carboxylation of the first glutamic acid residue in peptide substrates containing a diglutamyl sequence.
abstract: The peptide substrate commonly used in vitamin K-dependent carboxylation, Phe-Leu-Glu-Glu-Val, has been shown, by the use of high-voltage paper electrophoresis, to be degraded from the N-terminus by a microsomal leucine amino-peptidase. The replacement of phenylalanine with a N-t-butoxycarbonyl group resulted in a tetrapeptide substrate with a blocked N-terminus resistant to enzymic degradation. Vitamin K-dependent carboxylation of this non-degradable substrate gave a unique carboxylated product, which was separated from microsomal protein and unchanged substrate by using DEAE-Sephadex A25 as a final step. The carboxylated product was subsequently decarboxylated in 2HCl and analysed by using g.l.c. coupled to a mass spectrometer. This showed that only the first glutamic acid residue in the peptide substrate was carboxylated.
mesh_terms: D000818:Animals; D002262:Carboxy-Lyases / Q000378:metabolism; D002863:Chromogenic Compounds; D004590:Electrophoresis, Paper; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D066298:In Vitro Techniques; D008297:Male; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000201:enzymology; D009842:Oligopeptides / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D014812:Vitamin K / Q000494:pharmacology*
chemical_list: D002863:Chromogenic Compounds; D005971:Glutamates; C039432:N-tert-butyloxycarbonyl-leucyl-glutamyl-glutamyl-valine; D009842:Oligopeptides; D014812:Vitamin K; D018698:Glutamic Acid; C029471:phenylalanyl-leucyl-glutamyl-glutamyl-valine; D002262:Carboxy-Lyases