Detection of D-amino acids in purified proteins synthesized in Escherichia coli.
Amino Acids
; 38(5): 1377-85, 2010 May.
Article
en En
| MEDLINE
| ID: mdl-19768521
ABSTRACT
It has long been believed that amino acids comprising proteins of all living organisms are only of the L-configuration, except for Gly. However, peptidyl D-amino acids were observed in hydrolysates of soluble high molecular weight fractions extracted from cells or tissues of various organisms. This strongly suggests that significant amounts of D-amino acids are naturally present in usual proteins. Thus we analyzed the D-amino acid contents of His-tag-purified beta-galactosidase and human urocortin, which were synthesized by Escherichia coli grown in controlled synthetic media. After acidic hydrolysis for various times at 110 degrees C, samples were derivatized with 4-fluoro-7-nitro-2, 1, 3-benzoxadiazole (NBD-F) and separated on a reverse-phase column followed by a chiral column into D- and L-enantiomers. The contents of D-enantiomers of Ala, Leu, Phe, Val, Asp, and Glu were determined by plotting index D/(D + L) against the incubation time for hydrolysis and extrapolating the linear regression line to 0 h to eliminate the effect of racemization of amino acids during the incubation. Significant contents of D-amino acids were reproducibly detected, the D-amino acid profile being specific to an individual protein. This finding indicated the likelihood that D-amino acids are in fact present in the purified proteins. On the other hand, the D-amino acid contents of proteins were hardly influenced by the addition of D- or L-amino acids to the cultivation medium, whereas intracellular free D-amino acids sensitively varied according to the extracellular conditions. The origin of these D-amino acids detected in proteins was discussed.
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Beta-Galactosidasa
/
Escherichia coli
/
Urocortinas
/
Aminoácidos
Tipo de estudio:
Diagnostic_studies
Límite:
Humans
Idioma:
En
Revista:
Amino Acids
Asunto de la revista:
BIOQUIMICA
Año:
2010
Tipo del documento:
Article
País de afiliación:
Japón