Nakanishi N, Ozawa S, Iwanaga M, Akatsuka I, Sawada E, Asaumi R, Hasegawa H, Yamada S
Department of Oral Biochemistry, Meikai University School of Dentistry, Saitama, Japan.
Meikai Daigaku Shigaku Zasshi. 1990;19(2):197-203.
A method to measure the GTP cyclohydrolase and biopterin (BP) content at the same time in the sonicate of pheochromocytoma PC12h cells was described. The cells cultured in a 5-cm dish were sonicated with 0.4 to 0.5 ml of 50 mM Tris-HCl, pH 7.8, and a 50 microliters aliquot was used for one assay. After the GTP cyclohydrolase reaction, dihydroneopterin triphosphate, the reaction product, was oxidized and dephosphorylated to form neopterin (NP); and the pterins in the reaction mixture were then analyzed by high-performance liquid chromatography (HPLC). Mainly three peaks of pterins were detected in the eluate from the HPLC column: they were NP, BP, and 2-amino-4-hydroxypteridine (AHP). The amount of NP was a measure of the enzyme activity. On the other hand, a significant part of the biopterin contained in the enzyme sample was converted to AHP during the GTP cyclohydrolase reaction. Therefore, the sum amount of BP plus AHP was a measure of total BP content in the enzyme sample (sonicate of PC12h cells). The method is convenient and useful to study the actions and action mechanisms of various biologically active substances on the BP level of pheochromocytoma cells in culture.
本文描述了一种同时测定嗜铬细胞瘤PC12h细胞超声裂解液中GTP环化水解酶和生物蝶呤（BP）含量的方法。在5厘米培养皿中培养的细胞用0.4至0.5毫升50 mM Tris-HCl（pH 7.8）进行超声处理，每次测定取50微升等分试样。GTP环化水解酶反应后，反应产物三磷酸二氢新蝶呤被氧化并去磷酸化形成新蝶呤（NP）；然后通过高效液相色谱（HPLC）分析反应混合物中的蝶呤。在HPLC柱洗脱液中主要检测到三个蝶呤峰：它们是NP、BP和2-氨基-4-羟基蝶啶（AHP）。NP的量是酶活性的指标。另一方面，酶样品中所含的生物蝶呤在GTP环化水解酶反应过程中有很大一部分转化为AHP。因此，BP与AHP的总量是酶样品（PC12h细胞超声裂解液）中总BP含量的指标。该方法对于研究各种生物活性物质对培养的嗜铬细胞瘤细胞BP水平的作用及作用机制方便且有用。
