Nerve Tissue‐Specific Human Glutamate Dehydrogenase that Is Thermolabile and Highly Regulated by ADP

Abstract

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 -~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, Mg 2~did not affect the nonstimulated nerve tissuespecific 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 tissuespecific 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. Key Words: Glutamate dehydrogenase-Human brain -Glutamate metabolism-ADP enzyme regulation-Enzyme thermolability.

Different Antigenic Reactivities of Bovine Brain Glutamate Dehydrogenase Isoproteins

“…Taken together with the differences in their amino acid sequences and in their regulatory properties Shashidharan et al, 1997), the present findings support the possibility that different types of GDHs may function differently in a biological system, as many proteins have functions distinct from those for which they were originally identified. Actually, other roles of GDHs have been reported.…”

“…Our work also led to the finding that GDH is present in bovine brain in "heatlabile" (GDH I) and "heat-stable'' (GDH 11) forms . It has been reported that the activities of the GDH isotypes differ in their relative resistance to thermal inactivation, detergent extractability, and allosteric regulation characteristics (Colon et al, 1986;Shashidharan et al, 1997). Similar results were reported by other investigators showing that reduction in GDH activity in patients with neurodegenerative disorders was largely limited to the heat-labile form (Plaitakis et al, 1984).…”

“…Therefore, further studies are required to elucidate the physiological roles of various types of GDH isoproteins. An issue not addressed in this work is whether our GDH isoproteins are the bovine counterparts of the human brain pair (Shashidharan et al, 1994(Shashidharan et al, , 1997. Further sequence characterization is in progress.…”

The Role of Glutamate Dehydrogenase Activity in Development of Neurodegenerative Disorders

“…Its function is to catalyse reversible deamination of glutamate into α-ketoglutarate and ammonium ion [2] [3] [4]. There are two forms: thermostable and thermolabile [1] [5] [6]. With thermal inactivation of serum, GLDH was found that almost one third of GLDH serum originated from the rough endoplasmic reticulum and the rest from mitochondria [4].…”

Human glutamate dehydrogenase is immunologically distinct from other mammalian orthologues

“…However, the activity of GDH is also enriched in the nuclear fraction as well as in the mitochondria fraction (Lai et al, 1986). Recent biochemical works led to the finding that multiple forms of GDH are present in mammalian system and that the activities of the GDH isotypes differ in their relative resistance to thermal inactivation, detergent extractability, and allosteric regulation characteristics (Plaitakis et al, 1984;2000;Abe et al, 1992;Shashidharan et al, 1997). These forms have been designated soluble and particulate GDH (Plaitakis et al, 1984;Colon et al, 1986;Hussain et al, 1989).…”