[30] Glutaminase from mammalian tissues

This chapter provides an overview of glutaminase from mammalian tissues. The mitochondrial phosphate activated glutaminase (PAG), which is the most important glutaminase in mammalian tissues, is described. Measurement of the reaction products ammonia or glutamate may be used for assay of PAG. Measurement of ammonia suffers from two disadvantages—the great water solubility of this compound makes it difficult to keep the background values sufficiently low and some undissociated ammonia may escape, particularly at prolonged incubations at 37° when the pH exceeds 8.0. Ammonia can be monitored, for example, with Nessler's reagent after microdistillation, by the ammonia electrode, or by coupling to the glutamate dehydrogenase (GDH) reaction, whereby NADH oxidation is measured. The two latter methods permit measurement of enzyme rates. The assay of PAG in isolated mitochondria, synaptosomes, cultured neurons, granulocytes, and astrocytes is reviewed in the chapter. Glutamate formed in a prefixed time is assayed following incubation of the cellular material with L-glutamine (preferably in the physiological concentration range), two concentrations of phosphate (to ensure that PAG is assayed), and inhibitors to prevent the metabolism of glutamate.