Simultaneous determination of GTP cyclohydrolase activity and biopterin content in pheochromocytoma PC12h cells.

Abstract

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.