Metabolism of the alpha-amino nitrogen of glutamine in rat lens.

Rat lenses were cultured 4-24 h at 37 degrees C in balance salt medium containing 5 mM [15N]-(amino)-glutamine or 5 mM [15N]-glutamate. Free amino acids were extracted with 6% trichloroacetic acid containing alpha-aminoisobutyrate as an internal standard, and trifluoroacetyl-n-butyl (TAB) derivatives were prepared. Amino acids were quantified by gas chromatography, and 15N enrichment in amino groups of several lenticular free amino acids was determined by mass spectrometry. Culture of lenses with [15N]-(amino)-glutamine resulted in more rapid [15N]-labeling of the lenticular glutamate-glutamine pool than with [15N]-glutamate. The [15N]-(amino)-glutamine entered the lenses, and within 4 hr the lenticular glutamine-glutamate pool contained 70% amino-[15N] and had more than doubled in concentration. In contrast, [15N]-glutamate entered the lenses more slowly and lenticular glutamate-glutamine reached 30% 15N in 4 hr and 60% in 24 hr, with little change in concentration. The more rapid entry of [15N]-(amino)-glutamine resulted in more labeling of other amino acids than with [15N]-glutamate. For example, after 8 hr of culture, the lenses in [15N]-(amino)-glutamine contained a % 15N enrichment of 31 in alanine, 8 in glycine, 44 in proline, 24 in serine, 62 in aspartate-asparagine, and 78 in glutamate-glutamine, compared to values of 15, 5, 20, 13, 38, and 37 respectively for the lenses in [15N]-glutamate. The results indicate that in rat lenses the two-step process of glutamine transport and deamidation is more rapid than direct transport of glutamate. Lenses cultured with glutamine released sufficient ammonia into the culture medium to account for most of the added glutamine amido nitrogen. The data indicate that the amido nitrogen of glutamine is not utilized by the lens, but is lost to the surrounding medium. In contrast, the alpha-amino nitrogen can serve as a source of nitrogen for many important pathways of lens amino acid metabolism.