Purification and characterization of a novel imidazole dipeptide synthase from the muscle of the Japanese eel Anguilla japonica.

We have purified a novel enzyme from eel white muscle which catalyzes the syntheses of imidazole dipeptides, such as carnosine (beta-alanyl-L-histidine), anserine (beta-alanyl-pi-methyl-L-histidine), and balenine (ophidine; beta-alanyl-tau-methyl-L-histidine), directly from their precursors. The enzyme was purified 1130-fold from eel muscle by a series of column chromatographies. Although eel muscle contains a large amount of carnosine and only trace amounts of anserine and balenine, the anserine synthesizing activity was by far the highest. From gel permeation chromatography, the molecular mass of the enzyme was calculated to be 275kDa. SDS-PAGE of the purified enzyme represented a band around 43kDa, suggesting that the native enzyme is a hexamer or heptamer. The optimal pH and temperature were around 9.5 and 60 degrees C, respectively. K(m) values for beta-alanine and pi-methyl-L-histidine were 44 and 89mM, respectively. The enzyme was greatly activated by Zn(2+) and inhibited by EDTA. The N-terminal amino acid sequence of 25 residues of the purified enzyme showed 52% amino acid identity to 38-62 residues of zebrafish haptoglobin precursor. The purified enzyme also exhibited hydrolytic activity against these imidazole dipeptides.

Protection of neuronal cells against reactive oxygen species by carnosine and related compounds.

Carnosine and related compounds were compared in terms of their abilities to decrease the levels of reactive oxygen species (ROS) in suspensions of isolated neurons activated by N-methyl-D-aspartic acid (NMDA) using both stationary fluorescence measurements and flow cytometry. Carnosine was found to suppress the fluorescent signal induced by ROS production and decreased the proportion of highly fluorescent neurons, while histidine showed opposite effects. N-Acetylated derivatives of both carnosine and histidine demonstrated weak (statistically indistinguishable) suppressive effects on the ROS signal. N-Methylated derivatives of carnosine suppressed intracellular ROS generation to the same extent as carnosine. This rank of effectiveness is distinct from that previously obtained for the anti-radical ability of CRCs (anserine>carnosine>ophidine). These differences suggest that the similar ability of carnosine and its N-methylated derivatives to protect neuronal cells against the excitotoxic effect of NMDA is not solely related to the antioxidant properties of these compounds.