NERVE TISSUE-SPECIFIC HUMAN GLUTAMATE-DEHYDROGENASE THAT IS THERMOLABILE AND HIGHLY REGULATED BY ADP

Glutamate dehydrogenase (GDH), an enzyme that is central to the metabo lism of glutamate, is present at high levels in the mammalian brain. S tudies on human leukocytes and rat brain suggested the presence of two GDH activities differing in thermal stability and allosteric regulati on, 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 (housekeepin g GDH), whereas the second gene, designated GLUD2, is expressed specif ically in neural and testicular tissues. In this study, we obtained bo th 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 AD P, However, in the absence of ADP, the nerve tissue-specific GDH showe d only 5% of its maximal activity, compared with similar to 40% showed by the housekeeping enzyme. Low physiological levels of ADP (0.05-0.2 5 mM) induced a concentration-dependent enhancement of enzyme activity that was proportionally greater for the nerve tissue GDH (by 550-1,30 0%) 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+ d id not affect the nonstimulated nerve tissue-specific GDH, although th e 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 w as 3.5 and 0.5 h, respectively, Hence, the nerve tissue-specific GDH i s relatively thermolabile and has evolved into a highly regulated enzy me, These allosteric properties may be of importance for regulating br ain glutamate fluxes in vivo under changing energy demands.