Creek K E, Shankar S, De Luca L M
Arch Biochem Biophys. 1987 May 1;254(2):482-90. doi: 10.1016/0003-9861(87)90127-5.
In studies designed to reexamine the in vivo occurrence of retinyl phosphate mannose we injected hamsters intraperitoneally with either [2-3H]mannose or [15-3H]retinol and sacrificed the animals 15 min later. The small intestine was removed, the epithelial cells were scraped, and a methanolic extract of the labeled cells was prepared and chromatographed on a Mono Q anion-exchange column. Intraperitoneal administration of either [2-3H]mannose or [15-3H]retinol lead to the formation of a tritium-labeled anionic compound with a retention time on the Mono Q column similar to that of standard retinyl phosphate mannose. However, the biochemical properties of this labeled anionic compound were those expected of an organic acid and not retinyl phosphate mannose. The compound was resistant to both strong acid hydrolysis and mild base hydrolysis, as well as digestion with alpha- or beta-mannosidase, phosphodiesterase I, nucleotide pyrophosphatase, or beta-glucuronidase. When chromatographed on an Aminex HPX-87H organic acid analysis column or a silicic acid column the labeled anionic compound derived from either [2-3H]mannose or [15-3H]retinol comigrated with standard lactic acid. Treatment of the anionic compound derived from [2-3H]mannose with lactate oxidase or L-lactate 2-monooxygenase resulted in the formation of a tritium-labeled product that cochromatographed, respectively, with pyruvate or acetate on the Aminex HPX-87H column. However, treatment of the anionic compound derived from [15-3H]retinol with these same two enzymes resulted in a labeled product that migrated on the Aminex column at the same position as tritiated water. This result demonstrated that the labeled hydrogen was removed during enzymatic digestion and suggested that it was present on the second carbon of lactic acid. During the course of these studies no evidence for the in vivo labeling of a compound with the properties of retinyl phosphate mannose was found. Since [2-3H]mannose leads to labeled lactic acid in vivo the tritium label must not always be lost, as expected, during the entry step into glycolysis in which mannose 6-phosphate is converted to fructose 6-phosphate. The results suggest that an intramolecular hydrogen transfer from the C-2 position of mannose 6-phosphate to the C-1 position of fructose 6-phosphate can occur during the phosphomannose isomerase reaction. The finding that the position of the tritium label on lactic acid derived from [15-3H]retinol is on the second carbon is consistent with it coming from NADH labeled with tritium in the transferable hydrogen which was formed intracellularly during the NAD+-linked oxidation of retinol to retinaldehyde.
在旨在重新审视磷酸甘露糖视黄酯在体内生成情况的研究中，我们给仓鼠腹腔注射了[2-³H]甘露糖或[15-³H]视黄醇，并在15分钟后处死动物。取出小肠，刮下上皮细胞，制备标记细胞的甲醇提取物，并在Mono Q阴离子交换柱上进行色谱分析。腹腔注射[2-³H]甘露糖或[15-³H]视黄醇均导致形成一种³H标记的阴离子化合物，其在Mono Q柱上的保留时间与标准磷酸甘露糖视黄酯相似。然而，这种标记阴离子化合物的生化特性是有机酸的特性，而非磷酸甘露糖视黄酯的特性。该化合物对强酸水解、温和碱水解以及用α-或β-甘露糖苷酶、磷酸二酯酶I、核苷酸焦磷酸酶或β-葡萄糖醛酸酶消化均具有抗性。当在Aminex HPX-87H有机酸分析柱或硅酸柱上进行色谱分析时，源自[2-³H]甘露糖或[15-³H]视黄醇的标记阴离子化合物与标准乳酸同时洗脱。用乳酸氧化酶或L-乳酸2-单加氧酶处理源自[2-³H]甘露糖的阴离子化合物，分别导致形成一种³H标记产物，该产物在Aminex HPX-87H柱上与丙酮酸或乙酸共色谱。然而，用这两种相同的酶处理源自[15-³H]视黄醇的阴离子化合物，导致一种标记产物在Aminex柱上迁移到与³H2O相同的位置。这一结果表明，标记氢在酶促消化过程中被去除，提示其存在于乳酸的第二个碳原子上。在这些研究过程中，未发现体内标记具有磷酸甘露糖视黄酯特性的化合物的证据。由于[2-³H]甘露糖在体内导致标记乳酸，因此³H标记并不总是如预期那样在进入糖酵解的步骤中丢失，在该步骤中6-磷酸甘露糖转化为6-磷酸果糖。结果表明，在磷酸甘露糖异构酶反应过程中，可能发生从6-磷酸甘露糖的C-2位置到6-磷酸果糖的C-1位置的分子内氢转移。源自[15-³H]视黄醇的乳酸上³H标记位于第二个碳原子上这一发现，与它来自在视黄醇向视黄醛的NAD⁺连接氧化过程中细胞内形成的可转移氢中被³H标记的NADH一致。
