Kinetics and localization of brain phosphate activated glutaminase

Abstract

The cellular concentration of phosphate, the main activator of phosphate activated glutaminase (PAG) is rather constant in brain and kidney. The enzyme activity, however, is modulated by a variety of compounds affecting the binding of phosphate, such as glutamate, calcium, certain long chain fatty acids, fatty acyl CoA derivatives, members of the tricarboxylic acid cycle and protons (Kvamme et al. [2000] Neurochem. Res. 25:1407-1419). Therefore, the kinetic and allosteric properties of the enzyme are essential for regulating the enzyme activity in situ, especially because the enzymically active pool of PAG is assumed to have an external localization in the inner mitochondrial membrane, being exposed to cytosolic variation in the content of effectors. This has largely been overlooked. A hypothetical model for the allosteric interactions based on the sequential induced fit allosteric model by Koshland et al. ([1966] Biochemistry 5:365-385) is presented. Furthermore, it has been generally accepted that there exist only two isoforms of PAG, the kidney PAG that is similar to brain PAG, and the liver PAG. Therefore, the immunoreactivity of brain cells against kidney PAG antibodies has been considered a measure of PAG protein. Gomez-Fabre et al. ([2000] Biochem. J. 345:365-375) recently found, however, that a PAG mRNA from human breast cancer ZR75 cells is present in human brain and liver, but not in the kidney. We observed only traces of PAG immunoreactivity in cultured astrocytes and cultured neuroblastoma cells, regardless whether antibodies against the C- and N-termini of kidney PAG or antibodies against liver PAG were used, but considerable enzyme activity, demonstrating hitherto unknown isoforms of PAG (Torgner et al. [2001] FEBS Lett. 268(Suppl 1):PS2-031).

A Tale of Two Glutaminases: Homologous Enzymes with Distinct Roles in Tumorigenesis

“…The N-termini of the GLS variants begin with a 16-residue sequence generally predicted to localize the proteins to the mitochondria, and indeed, there is near predictive and experimental consensus that GLS is localized to this organelle (Table 1 & Table 2) [34][35][36][37][38][39][40][41][42][43][44][45]. To predict GLS localization, we utilized five freely available predictive algorithms, one of which predicted localization based upon the N-terminal sequence only (TargetP) [42], one of which used the whole protein sequence (SCLPred) [44] and the remaining three of which made predictions based on whole-protein genetic ontology (GO) calculations [41,43,45].…”

“…The mechanisms underlying these activating and inhibitory events are not known. As shown in Figure 3, the amino GLS2 † Liver N Mitochondrial OI [34] GLS † Ascites Y Mitochondrial and lysosomal OI [35] GLS † Kidney N Mitochondrial OI [36] GAC Breast Y Mitochondrial IF, OI [37] KGA Breast Y Cytosolic IF, OI [37] GAC Prostate Y Mitochondrial OI [37] KGA Prostate Y Cytosolic OI [37] GAC Lung Y Mitochondrial OI [37] KGA Lung Y Cytosolic OI [37] GLS † Brain N Synaptosomal OI [38] GLS † Kidney N Mitochondrial OI [38] GLS † Kidney N Mitochondrial OI [39] LGA Brain N Nuclear IF, OI [40] KGA Brain N Mitochondrial IF, OI [40] LGA(r) Insect cells N Mitochondrial and nuclear OI [51] GLS2 † Liver N Mitochondrial OI [56] GLS2 † Liver N Mitochondrial OI [57] GAC…”

The identification of vesicular glutamate transporter 3 suggests novel modes of signaling by glutamate

“…However, it has been difficult to identify unambiguously the cells that release glutamate as a transmitter because this ubiquitous amino acid is a key intermediate in cellular metabolism and is required for protein synthesis. The enzyme glutaminase that produces glutamate from glutamine may be enriched in glutamatergic neurons, but is also expressed by many other cells and tissues (3)(4)(5)(6). Plasma membrane glutamate transporters are also expressed primarily on astrocytes (7,8).…”

13C Magnetic Resonance Spectroscopy in Neurobiology - Its Use in Monitoring Brain Energy Metabolism and in Identifying Novel Metabolic Substrates and Metabolic Pathways

“…Glutamine is exported from astrocytes to the extracellular fluid, from where it is taken up by neurons, both glutamatergic and GABAergic. Inside neurons glutamine becomes deamidated to glutamate by phosphate-activated glutaminase (Kvamme et al, 2001), a mitochondrial enzyme, which in the brain appears to be expressed only by neurons. In GABAergic neurons the glutamine-derived glutamate becomes decarboxylated to GABA by glutamic acid decarboxylase (GAD), an enzyme that in the brain is expressed in inhibitory GABAergic neurons (Fonnum et al, 1970).…”