Novel Form of Phosphate Activated Glutaminase in Cultured Astrocytes and Human Neuroblastoma Cells, PAG in Brain Pathology and Localization in the Mitochondria

Abstract

A novel form of phosphate activated glutaminase (PAG), catalyzing the synthesis of glutamate from glutamine, has been detected in cultured astrocytes and SH-SY5Y neuroblastoma cells. This enzyme form is different from that of the kidney and liver isozymes. In these cells we found high enzyme activity, but no or very weak immunoreactivity against the kidney type of PAG, and no immunoreactivity against the liver type. PAG was also investigated in brain under pathological conditions. In patients with Down's syndrome the immunoreactivity in the frontoparietal cortex was significantly reduced. The findings leading to our conclusion of a functionally active PAG on the outer face of the inner mitochondrial membrane are discussed, and a model is presented.

Kinetics of a Novel Isoform of Phosphate Activated Glutaminase (PAG) in SH-SY5Y Neuroblastoma Cells

“…We have previously found that SH-SY5Y neuroblastoma cells express a novel form of PAG [9] We here report that this enzyme shows a high enzyme activity, which agrees with the general view that that tumor cells have the capacity to metabolize Gln at high rates. During growth of the cells, we have observed that the enzyme activity varies with the proliferation state of the cells similar to Ehrlich ascites tumor cells [19] and ZR-75 human breast cancer cells [2].…”

“…We have recently found that the neuroblastoma cell line SH-SY5Y, which show neuron-like properties [8] expresses a novel form of PAG [9]. In spite of high enzyme activity, Western blots of cellular homogenates from these cells show very weak immunoreactivity against our PAG antibodies [3].…”

Phosphate-activated glutaminase (GLS2), a p53-inducible regulator of glutamine metabolism and reactive oxygen species

“…Glutamate participates in regulation of mitochondrial bioenergetics in many normal and cancer cells via the tricarboxylic acid cycle for ATP production as well as antioxidant defense through GSH synthesis. The two different phosphate activated GLS isoforms GLS1 (kidney-type) and GLS2 (liver-type) in mammals are encoded by separate genes on different chromosomes (17)(18)(19). Previous data have suggested that GLS1 up-regulation is associated with increased rates of proliferation, whereas GLS2 prevalence seems to correspond with resting or quiescent cell states (17).…”

Role of Glutamate Dehydrogenase in Cancer Growth and Homeostasis

“…Glutaminolysis as illustrated in Figure 2 is associated with the increased expression of both the extrinsic cell membrane phosphate independent glutaminase/gamma glutamyltransferase/gamma glutamyltranspeptidase (PIG, GGT, GGTP) which generates extracellular glutamate [2,12] and intracellular phosphate dependent glutaminases, Phosphate dependent glutaminases (PDG,GLS1 and GAC, [13,14]) which generates glutamate cytosolically [2,13]; extracellular glutamate can be transported(GLAST, Figure2) into the cytosol functioning as an inhibitor of the intracellular glutaminases [2]. Noteworthy, c-myc signaling up-regulates both the cell membrane glutamine transporter (ASC, Figure 2) and the intracellular glutaminases in cancer cells [15].…”

“…NHE(sodium hydrogen ion exchanger 1) mediated acid extrusion is up-regulated coupled to anaerobic glycolysis acidifying extracellular milieu while cell membrane glutamate transporter(GLAST1) relocates to mitochondria with PIG produced GLU -accumulating extracellularly and contributing with reduced pHe to host defense barrier. Glutamine(GLN) transported into cell by ASC and hydrolyzed to glutamate on outer surface of inner mitochondrial membrane by GAC [13] coupled with GLAST1. GLC removal (dashed line) accentuates GDH flux by "pull" mechanism while NHE mediated acid extrusion supported by GDH and accelerated αKG input with cytosolic malate(MAL) conversion to PYR supplying anaplerosis.…”