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.
Evidence for the vitamin K-dependent γ-carboxylation of the first glutamic acid residue in peptide substrates containing a diglutamyl sequence
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A I Burgess, M P Esnouf, K Rose, R E Offord; Evidence for the vitamin K-dependent γ-carboxylation of the first glutamic acid residue in peptide substrates containing a diglutamyl sequence. Biochem J 1 October 1983; 215 (1): 75–81. doi: https://doi.org/10.1042/bj2150075
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