Phosphate-dependent glutaminase in enterocyte mitochondria and its regulation by ammonium and other ions

Summary.  The effects of ammonium and other ions on phosphate dependent glutaminase (PDG) activity in intact rat enterocyte mitochondria were investigated. Sulphate and bicarbonate activated the enzyme in absence and presence of added phosphate. In presence of 10 mM phosphate, ammonium at concentrations <1 mM inhibited the enzyme. This inhibition was reversed by increased concentration of phosphate or sulphate. The inhibition of PDG by ammonium in presence of 10 mM phosphate was biphasic with respect to glutamine concentration, its effect being through a lowering of V_max at glutamine concentration of ≤5 mM, and increased K_m for substrate concentration above 5 mM. The activation of the enzyme by bicarbonate was through an increase in V_max. Ammonium and bicarbonate ions may therefore be important physiological regulators of PDG. It is suggested that phosphate and other polyvalent ions may function by preventing product inhibition of the enzyme through promotion of PDG dimer formation. The dimerized enzyme may have a high affinity for glutamine and reduced sensitivity to inhibition by ammonium ions. Received August 10, 2001 Accepted April 1, 2002 Published online August 30, 2002 Acknowledgement This work was supported by University of Zimbabwe research grant to Dr. B. Masola. Authors' address: Dr. Bubuya Masola, Department of Biochemistry, University of Zimbabwe, P O Box MP167, Mount Pleasant, Harare, Zimbabwe, E-mail: masolab@yahoo.co.uk     

Hepatic glutaminase flux regulation of glutamine homeostasis. Studies in vivo

The role of hepatic glutaminase flux in regulating plasma glutamine homeostasis was studied in the intact rat. Interorgan glutamine flow during chronic metabolic acidosis was away from the splanchnic bed and to the kidneys. Hindquarter and hepatic glutamine release were the major sources of glutamine removed by the kidneys. Interorgan glutamate flow was from the liver to the hindquarters and kidneys. Chronic metabolic acidosis reduced arterial glutamine concentration 30%. Acute respiratory acidosis (pH 7.12 +/- 0.02) returned arterial glutamine concentration to normal values, increasing and decreasing hepatic glutamine and glutamate release respectively; renal and gut glutamine removal rates were not decreased. Hepatic unidirectional glutamine utilization measured isotopically was decreased 51% by acute acidosis; unidirectional glutamine production was unchanged. The results are consistent with the proposed role of ammonia-activated hepatic glutaminase in the regulation of glutamine homeostasis during acute acidosis.     

Amphetamine stimulation of glutaminase is blocked by neuroleptics

The ability of several classes of neuroleptics to inhibit the activity of phosphate-activated glutaminase was studied in several brain regions. These agents decreased glutaminase activity only in the amygdala. Amphetamine elevated glutaminase activity in this region. This stimulation was not blocked by (-) butaclamol, but was blocked by (+) butaclamol, haloperidol, chlorpromazine or clozapine.     

The regulation of phosphate-activated glutaminase activity and glutamine metabolism in the streptozotocin-diabetic rat.

The activity of phosphate-activated glutaminase was increased in the kidney, liver and small intestine of rats made diabetic for 6 days with injection of streptozotocin (75 mg/kg body wt.). Insulin prevented this increase in all three tissues. Treatment with NaHCO3, to correct the acidosis that accompanies diabetes, prevented the increase in renal glutaminase activity, but not that in liver or small intestine. Chemically induced acidosis (NH4Cl solution as drinking water) or alkalosis (NaHCO3 solution as drinking water) increased and decreased, respectively, glutaminase activity in the kidney, but were without significant effect on the activity in liver and small intestine. The increase in glutaminase activity in the small intestine during diabetes was due to an overall increase in the size of this organ, and was only detectable when activity was expressed in terms of whole organ, not mucosal scrapings or isolated enterocytes. Prolonged diabetes (40 days) resulted in an even greater increase in the size and glutaminase activity of the small intestine. Despite this marked increase in capacity for glutamine catabolism, arteriovenous-difference measurements showed a complete suppression of plasma glutamine utilization by the small intestine during diabetes, confirming the report by Brosnan, Man, Hall, Colbourne & Brosnan [(1983) Am. J. Physiol. 235, E261-E265].     

In vitro glutaminase regulation and mechanisms of glutamate generation in HIV-1-infected macrophage

Mononuclear phagocyte (MP, macrophages and microglia) dysfunction plays a significant role in the pathogenesis of HIV-1-associated dementia (HAD) through the production and release of soluble neurotoxic factors including glutamate. Glutamate production is greatly increased following HIV-1 infection of cultured MP, a process dependent upon the glutamate-generating enzyme glutaminase. Glutaminase inhibition was previously found to significantly decrease macrophage-mediated neurotoxicity. Potential mechanisms of glutaminase-mediated excitotoxicity including enzyme up-regulation, increased enzyme activity and glutaminase localization were investigated in this report. RNA and protein analysis of HIV-infected human primary macrophage revealed up-regulation of the glutaminase isoform GAC, yet identified no changes in the kidney-type glutaminase isoform over the course of infection. Glutaminase is a mitochondrial protein, but was found to be released into the cytosol and extracellular space following infection. This released enzyme is capable of rapidly converting the abundant extracellular amino acid glutamine into excitotoxic levels of glutamate in an energetically favorable process. These findings support glutaminase as a potential component of the HAD pathogenic process and identify a possible therapeutic avenue for the treatment of neuroinflammatory states such as HAD.     

Regulation of the kidney xanthine dehydrogenase by glutaminase.

The steady-state concentrations of glutamine, glutamate and ammonia in the kidney cells might regulate the rate of renal xanthine dehydrogenase activity. Both glutamate and glutamine were found to be effective inhibitors of the renal xanthine dehydrogenase activity in vivo. The inhibition by glutamate depends essentially on the glutaminase inhibition.     

Ammonia regulation of phosphate-activated glutaminase displays regional variation and impairment in the brain of aged rats

The regulation of PAG by ammonia in whole brain (Sprague-Dawley) and regional (Fischer-344) synaptosomal preparations from adult and aged animals was assessed. Whole brain synaptosomal preparations from both age groups displayed a significant decrease in PAG activity with increasing ammonium chloride concentrations, however, the aged rats exhibited a significant attenuation in ammonia-induced PAG inhibition. PAG activity measured in synaptosomes prepared from the striatum (STR), temporal cortex (TCX) and hippocampus (HIPP) was also inhibited by ammonium chloride. The STR showed the greatest degree of ammonia-induced PAG inhibition (55%) followed by the HIPP (30–35%) and the TCX (25–30%). This reduction in PAG activity was significantly attenuated in STR from aged rats at ammonium chloride concentrations greater than 50 M and in the TCX, PAG activity was significantly attenuated in the aged rats at ammonia concentrations of 0.5 and 1.0 mM. Ammonia regulation of PAG activity in the HIPP appeared to be unaffected by age. Ammonium chloride concentrations up to 5 mM had no effect on GLU release from cortical slices, although GLN efflux was significantly enhanced. These findings suggest that isozymes of PAG may exist in different brain regions based on their differential sensitivity to ammonia. The attenuation of ammonia-induced PAG inhibition seen in aged rats may have deleterious effects in the aged brain.Abbreviations PAG phosphate-activated glutaminase: L-glutamine amidohydrolase; EC 3.5.1.2 - STR striatum - TCX temporal cortex - HIPP hippocampus     

Inhibition by glutamate of phosphate-dependent glutaminase of rat kidney.

A membrane-associated form of phosphate-dependent glutaminase was derived from sonicated mitochondria and purified essentially free of gamma-glutamyl transpeptidase activity. Increasing concentrations of phosphate cause a sigmoidal activation of the membrane-bound glutaminase. Phosphate also causes a similar effect on the rate of glutaminase inactivation by the two affinity labels, L-2-amino-4-oxo-5-chloropentanoic acid and 6-diazo-5-oxo-L-norleucine, as observed previously for the solubilized and purified enzyme. Therefore the two forms of glutaminase undergo similar phosphate-induced changes in conformation. A sensitive radioactive assay was developed and used to determine the kinetics of glutamate inhibition of the membrane-associated glutaminase. The Km for glutamine decreases from 36 to 4 mM when the phosphate concentration is increased from 5 to 100 mM. Glutamate is a competitive inhibitor with respect to glutamine at both high and low concentrations of phosphate. However, the Ki for glutamate is increased from 5 to 52 mM with increasing phosphate concentration. Therefore glutamine and glutamate interact with the same site on the glutaminase, but the specificity of the site is determined by the available phosphate concentration.     

Regulation of phosphate-activated glutaminase (PAG) by glutamate analogues

The ability of structural analogues of glutamate (GLU) to modulate phosphate activated glutaminase (PAG) was assessed in the present series of studies. A number of GLU receptor agonists and antagonists were tested for their ability to inhibit synaptosomal PAG activity. PAG activity was determined by measuring GLU formation from 0.5mM glutamine (GLN) in the presence of 10 mM phosphate. GLU analogues at 5–10 mM were found to significantly inhibit PAG activity. It was determined that PAG inhibition occurred regardless of whether the GLU analogues were receptor agonists or antagonists, however, PAG inhibition was influenced by analogue chain length, isomeric form and substituent substitution. The glutamate uptake blockers, dihydrokainic acid and DL-threo--hydroxyaspartic acid were relatively weak inhibitors of PAG (<25% inhibition) as were the receptor agonists, ibotenic acid and (±)cis-2,3-piperidine-dicarboxylic acid. Other GLU analogues produced inhibition of PAG in the range of 40–70%. PAG inhibition by GLU analogues did not appear to differ substantially among the brain regions evaluated (cortex, striatum and hippocampus). The endogenous amino acids, glycine, taurine and N-acetylaspartic acid, also significantly inhibited PAG activity in the 5–10 mM range. The noncompetitive NMDA antagonists, (+)MK801 and ketamine, at a concentration of 5 mM, significantly stimulated PAG activity 1.5–2 fold over control values. The activation of PAG by (+)MK801 was dose-related, stereoselective and appeared to result from a synergistic interaction with phosphate to enhance substrate (GLN) binding to PAG. The results of these studies suggest that GLU analogues could potentially alter neurotransmitter GLU synthesis if sufficient concentrations of these drugs are used in in vitro or in vivo studies. Furthermore, preliminary evidence suggests that other endogenous amino acids (glycine, taurine, N-acetylaspartic acid) may modulate PAG activity. These studies have further characterized the structural requirements for the allosteric regulation of PAG by glutamate and its analogues.     

Characteristics of Triticale glutaminase]

The glutaminase (EC 3.5.1.2) isolated from seedlings of triticale (Triticale sp.) had a pH optimum of about 8, was inhibited with excess substrate (glutamine), and reaction products (glutamate and NH4+). A monovalent anion (Cl-) and a multivalent anion (phosphate) were shown to activate the glutaminase. Some features of the glutaminase from triticale were similar to those of animal glutaminase activated by phosphate and were different from features of the enzyme from Escherichia coli.     

Nitrogen anabolism underlies the importance of glutaminolysis in proliferating cells

Glutaminolysis and the Warburg effect are the two most noticeable metabolic features of tumor cells, whereas their biological significance in cell proliferation remains elusive. A widely accepted current hypothesis is that tumor cells use glutamine as a preferred carbon source for energy and reducing power, which has been used to explain both glutaminolysis and the Warburg effect. Here we provide evidence to show that supplying nitrogen, not the carbon skeleton, underlies the major biological importance of glutaminolysis for proliferating cells. We show that alternative nitrogen supplying mechanisms rescue cell proliferation in glutamine-free media. Particularly, we show that ammonia is sufficient to maintain a long-term survival and proliferation of Hep3B in glutamine-free media. We also observed that nitrogen source restriction repressed carbon metabolic pathways, including glucose utilization. Based on these new observations and metabolic pathways well-established in published literature, we propose an alternative model that cellular demand for glutamate is a key molecule in nitrogen anabolism, which is the driving force of glutaminolysis in proliferating cells. Our model suggests that the Warburg effect may be a metabolic consequence secondary to the nitrogen anabolism.Key words: glutaminolysis, cancer, Warburg effect, transamination, glycolysis, hypoxia     

Effect of succinylation on the oligomeric structure of glycinin

By varying the ratio of succinic anhydride to the protein, glycinin, one of the major fractions of soybean proteins, is succinylated to various levels. Sedimentation velocity experiments indicate the dissociation of the protein due to succinylation. Viscosity increases and a blue shift occurs in the absorption spectrum. The rate of proteolysis increases. Both dissociation and denaturation of the protein appear to occur. The effect of syccinylation on glycinin and arachin, the major protein of groundnuts, appears to be different.