Anti-GLUD1 monoclonal antibody, clone FQS22481

Glutamate dehydrogenase (GDH), an enzyme that is central to the metabolism of glutamate, is present at high levels in the mammalian brain. Studies on human leukocytes and rat brain suggested the presence of two GDH activities differing in thermal stability and allosteric regulation, but molecular biological investigations led to the cloning of two human GDH-specific genes encoding highly homologous polypeptides. The first gene, designated GLUD1, is expressed in all tissues (housekeeping GDH), whereas the second gene, designated GLUD2, is expressed specifically in neural and testicular tissues. In this study, we obtained both GDH isoenzymes in pure form by expressing a GLUD1 cDNA and a GLUD2 cDNA in Sf9 cells and studied their properties. The enzymes generated showed comparable catalytic properties when fully activated by 1 mM ADP, However, in the absence of ADP, the nerve tissue-specific GDH showed only 5% of its maximal activity, compared with similar to 40% showed by the housekeeping enzyme. Low physiological levels of ADP (0.05-0.25 mM) induced a concentration-dependent enhancement of enzyme activity that was proportionally greater for the nerve tissue GDH (by 550-1,300%) than of the housekeeping enzyme (by 120-150%). Magnesium chloride (1-2 mM) inhibited the nonactivated housekeeping GDH (by 45-64%); this inhibition was reversed almost completely by ADP. In contrast, Mg2+ did not affect the nonstimulated nerve tissue-specific GDH, although the cation prevented much of the allosteric activation of the enzyme at low ADP levels (0.05-0.25 mM). Heat-inactivation experiments revealed that the half-life of the housekeeping and nerve tissue-specific GDH was 3.5 and 0.5 h, respectively, Hence, the nerve tissue-specific GDH is relatively thermolabile and has evolved into a highly regulated enzyme, These allosteric properties may be of importance for regulating brain glutamate fluxes in vivo under changing energy demands.

Congenital hyperinsulinism/hyperammonemia (HI/HA) syndrome gives rise to unregulated protein-induced insulin secretion from pancreatic beta-cells, fasting hypoglycemia and elevated plasma ammonia levels. Mutations associated with HI/HA were identified in the Glud1 gene, encoding for glutamate dehydrogenase (GDH). We aimed at identifying the molecular causes of dysregulation in insulin secretion and ammonia production conferred by the most frequent HI/HA mutation Ser445Leu. Following transduction with adenoviruses carrying the human GDH-wild type or GDH-S445L-mutant gene, immunoblotting showed efficient expression of the transgenes in all the investigated cell types. Enzymatic activity tested in INS-1E beta-cells revealed that the mutant was much more sensitive to the allosteric activator ADP, rendering it highly responsive to substrates. INS-1E cells expressing either the wild type or mutant GDH responded similarly to glucose stimulation regarding mitochondrial activation and insulin secretion. However, at basal glucose glutamine stimulation increased mitochondrial activity and insulin release only in the mutant cells. In mouse and human islets, expression of mutant GDH resulted in robust elevation of insulin secretion upon glutamine stimulation, not observed in control islets. Hepatocytes expressing either the wild type or mutant GDH produced similar levels of ammonia when exposed to glutamine, although alanine response was strongly elevated with the mutant form. In conclusion, the GDH-S445L mutation confers hyperactivity to this enzyme due to higher sensitivity to ADP allosteric activation. This renders beta-cells responsive to amino acid stimulation, explaining protein-induced hypoglycemia secondary to non-physiological insulin release. Hepatocytes carrying mutant GDH produced more ammonia upon alanine exposure, which underscores hyperammonemia developed by the patients.