Camp. Biochem. Physiol. Vol. 97C, No. 2, pp. 265-267, 1990 0306~4492/90 $3.00 + 0.00 Printed in Great Britain 0 1990 Pergamon Press plc zyxwvutsrq GLUTAMATE DEHYDROGENASE: SOME PROPERTIES OF THE RAT BRAIN ENZYME FROM DIFFERENT CELLULAR COMPARTMENTS C. ARCE, S. CA~~ADAS, M. J. OSET-GASQUE, E. CASTRO and M. P. GONZALEZ Instituto de Bioquimica (Centro Mixto C.S.I.C.-U.C.M.), Facultad de Farmacia, Ciudad Universitaria, 28040-Madrid, Spain (Receioed 30 April 1990) Abstract-l. Differences in the GDH activity of neuronal, glial cells and synaptosomes were detected. 2. The enzyme was measured in both directions: synthesis and degradation of glutamate. 3. Synaptosomes were the region with the highest GDH activity. 4. ADP plays an imporatant role in the regulation of the reaction sense. 5. This effector produced higher activation on the enzyme measured in the direction of glutamate synthesis than in the sense of its degradation. 6. The enhancement produced by ADP was dependent on the enzyme localization. The ADP effect is discussed. INTRODUCTION In the brain, as in many other tissues, glutamate dehydrogenase (GDH) forms part of a complex enzymatic machine for transferring amino groups between cr-keto acids and the NH: ion. GDH is not the only enzyme involved in the cellular ammonia metabolism, since glutamate can be amidated by glutamine synthetase (GS) in most tissues, including the brain where this enzyme is specially important since this tissue does not have a functional urea cycle for clearing ammonia. Although the role of GDH in this process is not known, the fact that GDH is a reversible enzyme and it operates in two ways (syn- thetizing glutamate and degradating it), suggests that the regulation of the sense reaction by enzyme could be important in order to understand its possible role in the regulation of NH: levels in the brain. Although there are several studies on brain GDH (GonzBlez et al., 1976; Che et al., 1979; McCarty et al., 1980, 1981; Nicklas, 1984; McCarty and Tipton, 1984), the properties of the brain enzyme have not been studied in detail. Its activity in this organ is about one tenth of the activity of the enzyme in liver (Schmit, 1963) and immunological studies have suggested that the enzyme from the two sources may be similar (Talal and Tomkins, 1964). With respect to its compartmental localization, there has been some suggestion that GDH is a glial enzyme (Berl et al., 1978), although other studies indicate that it may be more active in neurons (Quastel, 1978; Nicklas et al., 1979). The controversy concerning this point is due to the high compartmen- tation of glutamate and related enzymes in brain. In the present paper we give data which demon- strate that GDH is found in both glial and neural cells, the synaptic region of neural cells being the compartment with the highest GDH activity. We also studied the regulatory role of ADP in these compartments. MATERIAL AND METHODS Brains Brains from two-week-old albino rats of the Wistar strain were obtained by decapitation. Cell separation Brains were processed and glial and neural cells and synaptosomes were separated according to Bedoya et al. (1988). Cellular populations were identified by morpho- logic and biochemistry parameters and their integrities were checked by dry exclusion method using Trypan blue and by measuring the LDH in the absence and pres- ence of TritonX-100 according to Clark and Nicklas (1970). Enqmatic extract preparations Glial and neural cells and synaptosomal fractions pre- pared according to L6pez-P6rez et al. (1981) were extracted, separately, with 0.02 M potassium phosphate buffer pH 7.4 containing 0.5% Triton X-100. This Triton concentration was chosen because it was the best one to solubilize the enzyme without GDH activity loss. The homogenates were centrifuged at 30,OOOg for 20 min at 4°C and supematants were used to measure the enzymatic activity. GDH activity in the direction of oxidative deamination was determined spectrophotometrically at 37°C by follow- ing the rate of reduction of NAD+ in a reaction mixture containing 43 mM potassium phosphate buffer pH 7.4, 5.6 mM NAD+. 17 mM glutamate and enzymatic extracts with about 1 mg protein. Enzymatic activity in the direction of glutamate for- mation was determined following the rate of oxidation of NADH+ in a reaction medium containing 90mM potass- ium phosphate buffer pH 7.4, 136 mM ammonium acetate, 0.23 mM NADH+, 45 mM 2-oxoglutarate and enzymatic extracts as before. The reaction rate was monitored at 340 nm with a Pye Unicam SP-1800 recording spectropho- tometer. Proteins were determined by the method of Lowry et al. (1951). 265