Jernigan H M, Laranang A S
Exp Eye Res. 1984 Jul;39(1):113-22. doi: 10.1016/0014-4835(84)90120-9.
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.
将大鼠晶状体在含有5 mM [¹⁵N]-(氨基)-谷氨酰胺或5 mM [¹⁵N]-谷氨酸的平衡盐培养基中于37℃培养4 - 24小时。用含有α-氨基异丁酸作为内标的6%三氯乙酸提取游离氨基酸，并制备三氟乙酰正丁酯（TAB）衍生物。通过气相色谱对氨基酸进行定量，并通过质谱法测定几种晶状体游离氨基酸氨基中的¹⁵N富集情况。与用[¹⁵N]-谷氨酸培养相比，用[¹⁵N]-(氨基)-谷氨酰胺培养晶状体导致晶状体谷氨酸 - 谷氨酰胺池的[¹⁵N]标记更快。[¹⁵N]-(氨基)-谷氨酰胺进入晶状体，4小时内晶状体谷氨酰胺 - 谷氨酸池中含有70%的氨基-[¹⁵N]，浓度增加了一倍多。相比之下，[¹⁵N]-谷氨酸进入晶状体的速度较慢，晶状体谷氨酸 - 谷氨酰胺在4小时内达到30%的¹⁵N，24小时内达到60%，浓度变化不大。[¹⁵N]-(氨基)-谷氨酰胺更快的进入导致比[¹⁵N]-谷氨酸更多的其他氨基酸被标记。例如，培养8小时后，[¹⁵N]-(氨基)-谷氨酰胺中的晶状体丙氨酸的¹⁵N富集百分比为31，甘氨酸为8，脯氨酸为44，丝氨酸为24，天冬氨酸 - 天冬酰胺为62，谷氨酸 - 谷氨酰胺为⁷⁸，而[¹⁵N]-谷氨酸中的晶状体相应值分别为15、5、20、13、38和37。结果表明，在大鼠晶状体中，谷氨酰胺转运和脱酰胺的两步过程比谷氨酸的直接转运更快。用谷氨酰胺培养的晶状体向培养基中释放了足够的氨，以解释大部分添加的谷氨酰胺酰胺氮。数据表明，谷氨酰胺的酰胺氮未被晶状体利用，而是流失到周围培养基中。相比之下，α-氨基氮可作为晶状体氨基酸代谢许多重要途径的氮源。
