Direct visualization of IMP--GMP:pyrophosphate phosphoribosyltransferase in polyacrylamide gels

Direct visual detection of IMP-GMP: pyrophosphate phosphoribosyl-transferase (HGPRT) activity after separation by polyacrylamide gel electrophoresis has not been easy to achieve. Radiochemical assays have been used but they have the disadvantage of requiring considerable amounts of time before the results are available (1.2). A fluorescence assay that couples inosine 5′-monophosphate (IMP) to IMP dehydrogenase with concomitant formation of NADH has been described (3) but is inconvenient because the coupling enzyme is not commercially available. Bieber (4) has developed a fluorescence assay that can be used for HGPRT detection but it requires that the gels be sliced before they are incubated with the substrate mixture. A rapid, convenient method for visual observation of HGPRT activity would facilitate the study of this enzyme and its variants. In this paper we report the development of such a method based on precipitation of inorganic pyrophosphate, one of the reaction products, with Mn²⁺.