Glutamate grabbers, such as glutamate oxaloacetate transaminase (GOT), have been proposed to prevent excitotoxicity secondary to high glutamate levels in stroke patients. However, the efficacy of blood glutamate grabbing by GOT could be dependent on the extent and severity of the disruption of the blood-brain barrier (BBB). Our purpose was to analyze the relationship between GOT and glutamate concentration with the patient\'s functional status differentially according to BBB serum markers (soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK) and leukoaraiosis based on neuroimaging). This retrospective observational study includes 906 ischemic stroke patients. We studied the presence of leukoaraiosis and the serum levels of glutamate, GOT, and sTWEAK in blood samples. Functional outcome was assessed using the modified Rankin Scale (mRS) at 3 months. A significant negative correlation between GOT and glutamate levels at admission was shown in those patients with sTWEAK levels > 2900 pg/mL (Pearson\'s correlation coefficient: -0.249; p < 0.0001). This correlation was also observed in patients with and without leukoaraiosis (Pearson\'s correlation coefficients: -0.299; p < 0.001 vs. -0.116; p = 0.024). The logistic regression model confirmed the association of higher levels of GOT with lower odds of poor outcome at 3 months when sTWEAK levels were >2900 pg/mL (OR: 0.41; CI 95%: 0.28-0.68; p < 0.0001) or with leukoaraiosis (OR: 0.75; CI 95%: 0.69-0.82; p < 0.0001). GOT levels are associated with glutamate levels and functional outcomes at 3 months, but only in those patients with leukoaraiosis and elevated sTWEAK levels. Consequently, therapies targeting glutamate grabbing might be more effective in patients with BBB dysfunction.

Nitrogen metabolism can regulate mycelial growth and secondary metabolism in Ganoderma lucidum. As an important enzyme in intracellular amino acid metabolism, glutamate oxaloacetate transaminase (GOT) has many physiological functions in animals and plants, but its function in fungi has been less studied. In the present study, two GOT isoenzymes were found in G. lucidum; one is located in the mitochondria (GOT1), and the other is located in the cytoplasm (GOT2). The reactive oxygen species (ROS) level was increased in got1 silenced strains and was approximately 1.5-fold higher than that in the wild-type (WT) strain, while silencing got2 did not affect the ROS level. To explore how GOT affects ROS in G. lucidum, experiments related to the generation and elimination of intracellular ROS were conducted. First, compared with that in the WT strain, the glutamate content, one of the substrates of GOT, decreased when got1 or got2 was knocked down, and the glutathione (l-γ-glutamyl-l-cysteinylglycine) (GSH) content decreased by approximately 38.6%, 19.3%, and 40.1% in got1 silenced strains, got2 silenced strains, and got1/2 co-silenced strains respectively. Second, GOT also affects glucose metabolism. The pyruvate (PA), acetyl-CoA and α-ketoglutarate (α-KG) contents decreased in got1 and got2 silenced strains, and the transcription levels of most genes involved in the glycolytic pathway and the tricarboxylic acid cycle increased. The NADH content was increased in got1 silenced strains and got2 silenced strains, and the NAD+/NADH ratio was decreased, which might result in mitochondrial ROS production. Compared with the WT strain, the mitochondrial ROS level was approximately 1.5-fold higher in the got1 silenced strains. In addition, silencing of got1 or got2 resulted in a decrease in antioxidant enzymes, including superoxide dismutase, catalase, glutathione reductase, and ascorbate peroxidase. Finally, ganoderic acid (GA) was increased by approximately 40% in got1 silenced strains compared with the WT strain, while silencing of got2 resulted in a 10% increase in GA biosynthesis. These findings provide new insights into the effect of GOT on ROS and secondary metabolism in fungi. KEY POINTS: • GOT plays important roles in ROS level in Ganoderma lucidum. • Silencing of got1 resulted in decrease in GSH content and antioxidant enzymes activities, but an increase in mitochondrial ROS level in G. lucidum. • Silencing of got1 and got2 resulted in an increase in ganoderic acid biosynthesis in G. lucidum.

Molybdenum cofactor deficiency and isolated sulfite oxidase deficiency are two rare genetic disorders that are caused by impairment of the mitochondrial enzyme sulfite oxidase. Sulfite oxidase is catalyzing the terminal reaction of cellular cysteine catabolism, the oxidation of sulfite to sulfate. Absence of sulfite oxidase leads to the accumulation of sulfite, which has been identified as a cellular toxin. However, the molecular pathways leading to the production of sulfite are still not completely understood. In order to identify novel treatment options for both disorders, the understanding of cellular cysteine catabolism - and its alterations upon loss of sulfite oxidase - is of utmost importance. Here we applied a new detection method of sulfite in cellular extracts to dissect the contribution of cytosolic and mitochondrial glutamate oxaloacetate transaminase (GOT) in the transformation of cysteine sulfinic acid to sulfite and pyruvate. We found that the cytosolic isoform GOT1 is primarily responsible for the production of sulfite. Moreover, loss of sulfite oxidase activity results in the accumulation of sulfite, H2S and persulfidated cysteine and glutathione, which is consistent with an increase of SQR protein levels. Surprisingly, none of the known H2S-producing pathways were found to be upregulated under conditions of sulfite toxicity suggesting an alternative route of sulfite-induced shift from oxidative to H2S dependent cysteine catabolism.

For the measurement of malate by an enzyme sensor, we did a comparative study using malate dehydrogenase (MDH) alone, MDH and glutamate oxaloacetate transaminase (GOT) together, a malic enzyme (ME) that requires NADP as a cofactor, and MDH and NADH oxidase together. With respect to the response of each reactor to 0.5 mM L-malate, the systems using ME alone and MDH plus NADH oxidase gave high values. The ranges of measurements were 0.05-1.00 mM (MDH alone), 0.01-0.05 mM (MDH plus GOT), 0.01-0.50 mM (ME alone) and 0.02-1.00 mM (MDH plus NADH oxidase). In the system with MDH alone, however, reducing sugars in the sample interfered with measurements and it was impossible to use this system for practical analysis of fruit samples. By contrast, the systems using ME alone or MDH plus NADH oxidase were unaffected by the presence of reducing sugars and were suitable for analysis of samples. Thus, the MDH-NADH oxidase system is recommended for practical analyses of samples.

Glutamate oxaloacetate transaminase (GOT) catalyzes the reversible reaction of l-aspartate and α-ketoglutarate into oxaloacetate and L-glutamate and plays a key role in carbon and nitrogen metabolism in all organisms. In human tissues, GOTs are pyridoxal 5\'-phosphate-dependent (PLP) enzymes which exist in cytoplasm and mitochondrial forms, GOT1 and GOT2, respectively. GOT1 expression correlates with the growth of several tumors because cancer cells can utilize the amino acid glutamine to fuel anabolic processes, and therefore, GOT1 represents a new therapeutic target in cancer. In this work, human GOT1 was expressed in Escherichia coli periplasmic space, and purified by a combination of His-tag immobilized metal-ion affinity chromatography and anion exchange chromatography. Optimal activity of the enzyme occurred at a temperature of 37 °C and a pH of 7.5. Cations such as Na(+), K(+) and Mg(2+) slightly inhibited the activity of recombinant human GOT1, while Zn(2+), Mn(2+), Cu(2+), Ni(2+), Co(2+) and Ca(2+) had stronger inhibitory effects. Crystals of human GOT1 were grown using the hanging-drop vapor diffusion method at 4°C with 0.1M Bis-Tris pH 6.0% and 21% (w/v) PEG 3350. The crystals diffracted to 2.99Å resolution and belonged to space group P43212 with the unit cell parameters a = b = 93.4, c = 107.4Å, α = β = γ = 90°.

Diabetes has been reported to affect salivary glands adversely in humans and experimental models. Glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and lactate dehydrogenase (LDH) are salivary enzymes that also are widely distributed in animal tissues. We determined GOT and GPT levels in saliva samples of 100 type 1 and 30 type 2 diabetic patients using reflectance spectrophotometry and compared them to 30 age and sex matched healthy controls. Statistically significant differences were observed in the mean values of GOT and GPT in type 1 diabetics compared to type 2 and control groups. Significantly higher GOT levels were found in the 1-20 year age group of type 1 diabetics. Our findings suggest that salivary gland damage is due to the same immunological attack that affects pancreatic β cells and results in type 1 diabetes.

OBJECTIVE: Based on the capacity of the blood-resident enzyme glutamate oxaloacetate transaminase (GOT) to metabolize blood glutamate, our aim was to study the association of GOT activity with serum glutamate levels and clinical parameters in patients with migraine.
METHODS: This case-control study included 45 episodic migraine patients (IHS 2004 criteria) and 16 control subjects. We analyzed glutamate and GOT activity in peripheral blood samples obtained during interictal periods and migraine attacks ( N  = 15). Frequency, severity, and duration of attacks and time of evolution were also recorded.
RESULTS: Migraine patients showed lower GOT activity than controls (15.2 ± 2.9 vs. 18.7 ± 3.8 U/l) and higher levels of glutamate (153.7 ± 68.6 vs. 121.5 ± 59.2 μM) (all P  < 0.05). A negative correlation was found between GOT activity and glutamate levels ( R  = -0.493; P  < 0.0001) in interictal periods; however, this negative correlation was lost during attacks ( R  = -0.026; P  = 0.925). During attacks, we found a positive correlation between the time elapsed from attack onset and glutamate levels ( R  = 0.738; P  < 0.0001), but not for GOT activity ( R  = -0.075; P  = 0.809).
CONCLUSIONS: Migraine patients showed reduced GOT activity and increased levels of blood glutamate levels as compared to control subjects. Furthermore, a negative correlation was found between GOT activity and glutamate levels in interictal periods.