Regulation of glutaminase by exogenous glutamate,ammonia and 2-oxoglutarate in synaptosomal enriched preparation from rat brain

Phosphate activated glutaminase in synaptosomal enriched preparation from rat brain is very sensitive to inhibition by low concentration of glutamate, ammonia and 2-oxoglutarate when added to the incubation medium at pH 7.6. By increasing the concentration of either of these compounds up to 0.5 mM a pronounced initial inhibition is followed by little or no further effect when the concentration is increased beyond this level. By lowering the pH of the reaction mixture to 7.0, the inhibition by glutamate is almost abolished and that of ammonia reduced. Glutamate inhibits mainly the N-ethylmaleimide-sensitive fraction of glutaminase which previously is suggested to be localized to the outer phase of the mitochondrial inner membrane, whereas ammonia inhibits both the N-ethylmaleimidesensitive and-insensitive fraction. Evidence has been produced to show that the inhibition by 2-oxoglutarate is caused by glutamate formation by aminotransferase reactions. Since 2-oxoglutarate is produced by the tricarboxylic acid cycle, the operation of this cycle may regulate the glutaminase reaction by controlling glutamate formation via the aminotransferase reactions.Abbreviations used NEM N-ethylmaleimide - PAG phosphate activated glutaminase - AOA aminooxyacetic acid     

Evidence indicating that pig renal phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane

Phosphate-activated glutaminase in intact pig renal mitochondria was inhibited 50-70% by the sulfhydryl reagents mersalyl and N-ethylmaleimide (0.3-1.0 mM), when assayed at pH 7.4 in the presence of no or low phosphate (10 mM) and glutamine (2 mM). However, sulfhydryl reagents added to intact mitochondria did not inhibit the SH-enzyme beta-hydroxybutyrate dehydrogenase (a marker of the inner face of the inner mitochondrial membrane), but did so upon addition to sonicated mitochondria. This indicates that the sulfhydryl reagents are impermeable to the inner membrane and that regulatory sulfhydryl groups for glutaminase have an external localization here. The inhibition observed when sulfhydryl reagents were added to intact mitochondria could not be attributed to an effect on a phosphate carrier, but evidence was obtained that pig renal mitochondria have also a glutamine transporter, which is inhibited only by mersalyl and not by N-ethylmaleimide. Mersalyl and N-ethylmaleimide showed nondistinguishable effects on the kinetics of glutamine hydrolysis, affecting only the apparent Vmax for glutamine and not the apparent Km calculated from linear Hanes-Woolf plots. Furthermore, both calcium (which activates glutamine hydrolysis), as well as alanine (which has no effect on the hydrolytic rate), inhibited glutamine transport into the mitochondria, indicating that transport of glutamine is not rate-limiting for the glutaminase reaction. Desenzitation to inhibition by mersalyl and N-ethylmaleimide occurred when the assay was performed under optimal conditions for phosphate activated glutaminase (i.e. in the presence of 150 mM phosphate, 20 mM glutamine and at pH 8.6). Desenzitation also occurred when the enzyme was incubated with low concentrations of Triton X-100 which did not affect the rate of glutamine hydrolysis. Following incubation with [14C]glutamine and correction for glutamate in contaminating subcellular particles, the specific activity of [14C]glutamate in the mitochondria was much lower than that of the surrounding incubation medium. This indicates that glutamine-derived glutamate is released from the mitochondria without being mixed with the endogenous pool of glutamate. The results suggest that phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane.     

Phosphate-Activated Glutaminase in the Crude Mitochondrial Fraction (P2 Fraction) from Human Brain Cortex

The kinetics and other properties of phosphate-activated glutaminase have for the first time been studied in the crude mitochondrial fraction (P2 fraction) from human brain. The enzyme is for unexplained reasons inactivated postmortem. The enzyme activity decreases by storing the tissue or homogenate at 37 degrees C. The inactivation is not caused by formation of a dialysable inhibiting compound. No large proteolytic degradation has occurred, since the phosphate-activated glutaminase-like immunoreactive band did not disappear during the storage. The molecular weight of the subunit of the enzyme as determined by immunoblots of sodium dodecyl sulfate-treated homogenates from human brain is estimated to be approximately 64 K. The enzyme has been shown to have a pH optimum of 8.6; it is activated by phosphate, inhibited by glutamate, and partially inhibited by ammonia. Double-inverse plots of enzyme activity against phosphate are concave-upward, and more so in the presence of an inhibitor. The inhibition by glutamate appears to be noncompetitive with the substrate glutamine, and competitive with the activator phosphate. These kinetic properties are not significantly different from our earlier observations concerning phosphate-activated glutaminase from pig brain and pig kidney.     

Regulatory effects of fatty acyl-coenzyme A derivatives on phosphate-activated pig brain and kidney glutaminase in vitro.

1. Fatty n-acyl-CoA derivatives in the concentration range 5muM-0.1mM and with 5-18 fatty acyl carbons have dual effects on phosphate-activated glutaminase from pig brain and kidney. Generally, fatty acyl-CoA derivatives in low concentrations activate the enzyme, but inhibit at higher concentrations; phosphate and citrate potentiate the activation, displaying positive co-operatively, and protect against inactivation. The fatty acyl-CoA derivatives affect glutaminase similarly to Bromothymol Blue, but differently from acetyl-CoA, which activates the enzyme only at very low phosphate or citrate concentrations. 2. Saturated fatty acyl-CoA derivatives, with 5-10 fatty acyl carbons, only activate the enzyme in the concentration range 0-0.1 mM. When the fatty acyl chain is elongated, the fatty acyl-CoA derivatives gradually become more powerful inhibitors of glutaminase at the expense of their activating capacity. In particular, palmitoyl-CoA and stearoyl-CoA are strong inhibitors at concentrations (10 muM) at which the corresponding free fatty acids and fatty acyl-carnitine derivatives have no effect. 3. The unsaturated fatty acyl-CoA derivatives, oleoyl-CoA and linoleoyl-CoA, behave as potent activators in the lower part of the concentration range tested (0-0.05mM), and as inhibitors in the upper part of this range (0.02-0.10mM). Oleic acid and linoleic acid have similar properties, but their activating capacity is less pronounced. 4. Phosphate both prevented and reversed the inhibition, but no restoration of activity was possible once the enzyme became inactivated. 5. By changing the pH from 7.0 to 8.0 the activating capacity of the fatty acyl-CoA derivatives is increased, as is their concentration range for activation. 6. The fatty acyl-CoA derivatives are somewhat more potent activator for brain glutaminase, but otherwise they affect the two enzymes similarly.     

Properties and submitochondrial localization of pig and rat renal phosphate-activated glutaminase

Two pools ofphosphate-activated glutaminase (PAG) were separated from pig and ratrenal mitochondria. The partition of enzyme activity corresponded withthat of the immunoreactivity and also with the postembedding immunogoldlabeling of PAG, which was associated partly with the inner membraneand partly with the matrix. The outer membrane was not labeled. PAG inintact mitochondria showed enzymatic characteristics that were similarto that of the membrane fraction and also mimicked that of thepolymerized form of purified pig renal PAG. PAG in the soluble fractionshowed properties similar to that of the monomeric form of purifiedenzyme. It is indicated that the pool of PAG localized inside the innermitochondrial membrane is dormant due to the presence of highconcentrations of the inhibitor glutamate. Thus the enzymaticallyactive PAG is assumed to be localized on the outer face of the innermitochondrial membrane. The activity of this pool of PAG appears to beregulated by compounds in the cytosol, of which glutamate may be most important.

     

Intestinal barrier function of Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.) post smolts is reduced by common sea cage environments and suggested as a possible physiological welfare indicator

Background  

Fish farmed under high intensity aquaculture conditions are subjected to unnatural environments that may cause stress. Therefore awareness of how to maintain good health and welfare of farmed fish is important. For Atlantic salmon held in sea cages, water flow, dissolved oxygen (DO) levels and temperature will fluctuate over time and the fish can at times be exposed to detrimentally low DO levels and high temperatures. This experimental study investigates primary and secondary stress responses of Atlantic salmon post smolts to long-term exposure to reduced and fluctuating DO levels and high water temperatures, mimicking situations in the sea cages. Plasma cortisol levels and cortisol release to the water were assessed as indicators of the primary stress response and intestinal barrier integrity and physiological functions as indicators of secondary responses to changes in environmental conditions.     

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

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.     

Ultrastructure of pig renal glutaminase. Evidence for conformational changes during polymer formation

Highly purified pig renal glutaminase has been examined by electron microscopy. It is demonstrated that the Tris·HCl form of the enzyme contains cylindrical particles with a diameter of about 60 Å and a length of about 82 Å. Treatment of the Tris·HCl form of the enzyme with sodium dodecyl sulphate and mercapto-ethanol, followed by polyacrylamide gel electrophoresis in sodium dodecyl sulphate, shows that the enzyme contains two types of sub units with molecular weights of 53,000 and 61,000. In agreement with earlier data, it is further demonstrated that the phosphate form is a simple dimer of the Tris·HCl form. Evidence that major conformational changes are involved in the formation of large, helical polymers of the enzyme described earlier, is also presented.