Two Soluble forms of Glutamate Dehydrogenase Isoproteins from Bovine Brain

Abstract

Two soluble forms of novel glutamate dehydrogenase isoproteins, designated GDH I and GDH 11, have been purified from bovine brain. GDH I and GDH I1 were separated on a hydroxyapatite column and eluted by a step gradient at different phosphate concentrations (30 mM and 50 rnM for GDH I and GDH 11, respectively). The preparations were homogeneous on SDS/PAGE. GDH I and GDH II showed similarity in their molecular sizes and are composed of six identical subunits having a molecular size of 57500 Da. Differences between the biochemical properties of GDH I and GDH 11, such as N-terminal amino acid sequences of intact and tryptic-digested enzymes, kinetic parameters, optimum pH and heat stability, were extensively examined in both reductive amination of a-oxoglutarate and oxidative deamination of glutamate. The different effects of ADP on GDH isoproteins were also studied under various conditions. These results indicate that GDH I and GDH 11, isolated from bovine brain, are novel and distinct polypeptides.

Inhibition of glutamate dehydrogenase and insulin secretion by KHG26377 does not involve ADP-ribosylation by SIRT4 or deacetylation by SIRT3

“…Insulin-free bovine serum albumin fraction, guinea pig anti-porcine insulin serum, and rabbit anti-guinea pig globulin serum were from the Cell Signalling Technology (Beverly, MA, USA) Fetal calf serum and Dulbecco's Modified Eagle's Medium were obtained from Gibco-BRL Life Technologies (Rockville, MD, USA) All other chemicals and solvents were of reagent grade or better. Bovine brain GDH and anti-GDH monoclonal antibodies were produced in our laboratory as previously described (23,24). Recombinant human SIRT3 was purchased from Cayman Chemical (Ann Arbor, MI, USA), and recombinant human SIRT4 was purchased from Sigma-Aldrich.…”

“…A number of recent studies have used inhibitors to decrease GDH activity in pancreatic islets (7,12,(22)(23)(24)(25)(26). Upon glucose stimulation, GDH inhibition results in both diminished insulin release and decreased cellular glutamate levels (26).…”

“…Only islets that were undamaged and completely free of acinar and connective tissue were used. In experiments, isolated islets were cultured for 1 h in RPMI 1640 medium containing 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin in an atmosphere of humidified 95% air and 5% CO2 at 37 o C. Insulin secretion was stimulated by incubating islets in Krebs-Ringer bicarbonate solution (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.18 mM potassium phosphate, 1.16 mM magnesium sulfate, and 25 mM NaHCO3, pH 7.4) containing different concentrations of glucose in the absence and presence of KHG26377 (1 mg) at 37 o C for 1-2 h. Total islet insulin was measured in islets treated with ethanol overnight at −20 o C. GDH activity was measured spectrophotometrically in both the direction of reductive amination of 2-oxoglutarate and in the direction of oxidative deamination of glutamate (23). For kinetic studies, Km and Vmax were calculated by linear regression of double-reciprocal plots and are expressed with standard errors.…”

Molecular Gene Cloning, Expression, and Characterization of Bovine Brain Glutamate Dehydrogenase

“…The allosteric control of mammalian GDH activity by positive (ADP) and negative effectors (GTP) has been studied (Colman, 1991;Hudson and Daniel, 1993). The mammalian GDH is strictly regulated by allosteric activators and inhibitors (McPherson and Wootton, 1983;Cho et al, 1995). GTP inhibits enzyme turnover over a wide range of conditions by increasing the affinity of the enzyme for the product, making product release rate-limiting under all conditions in the presence of ADP (Peterson and Smith, 1999;Smith et al, 2001).…”

“…The GDH enzyme is expressed at high levels in the brain, liver, pancreas, and kidney, but not in muscles. There are three types of GDH that vary according to the co-enzymes; NAD(H)-specific GDH, NADP(H)-specific GDH, and GDH with mixed specificity, as well as the structure of GDHs, according to the sources (Veronese et al, 1974;Smith et al, 1975;McPherson and Wootton, 1983;Rice et al, 1985;Cho et al, 1995). The largest difference between the mammalian and bacterial GDH is the long antenna domain in mammalian GDH that is formed by the 48-amino acid insertion, beginning *To whom correspondence should be addressed.…”