Glutamic acid decarboxylase (GAD) activity was measured in homogenates of conidia and both submerged and aerial mycelia of Trichoderma viride. The GAD activity in conidia had a temperature optimum at 30 degrees C and a pH optimum at pH 4. GAD was stimulated by EDTA (2 mM) and was insensitive to treatment with calmodulin antagonists calmidazolium (10 microM) or phenothiazine neuroleptics (60 microM). Cyclosporin A (up to 300 microM) partially inhibited GAD in the homogenate, but not in the supernatant obtained after centrifuging the homogenate. Attempts to release GAD activity from the homogenate using high ionic strength, detergents, or urea failed. Freezing-thawing led to the partial increase of activity in the conidial homogenate. These results indicate that GAD is a membrane-bound enzyme. The highest specific activity of GAD was present in the mitochondrial/vacuolar organellar fraction. Germination of conidia in the submerged culture led to a temporary decrease in GAD activity. After prolonged cultivation, the activity displayed quasi-oscillatory changes. The stationary state was characterized by a high GAD activity. The presence of gamma-aminobutyric acid in the submerged mycelia was demonstrated. In surface culture in the dark, GAD activity increased in a monophasic manner until conidia formation. The illumination of dark-cultivated mycelia by a white-light pulse caused a dramatic increase in GAD activity. Light-induced changes were not observed in mutants with delayed onset of conidiation. In the dark or upon illumination by light pulse, the increase of GAD activity preceded the appearance of conidia. Thus, GAD activity in T. viride is closely associated with its developmental status and may represent a link between differentiation events and energy metabolism.
ATP acts as an inhibitor of brain glutamate decarboxylase in a water brain tissue extract with low ATPase activity. This inhibition is not caused by competition of ATP with glutamate or pyridoxal‐5‐phosphate. The concentration of ATP, which causes a 50% inhibition of glutamate decarboxylase, [I]50%, depends on the pyridoxal‐5‐phosphate concentration. At 15 μM pyridoxal‐5‐phosphate the [I]50% is 0.3 mM ATP. At 75 μM pyridoxal‐5‐phosphate the [I]50% is 1.3 mM ATP. AT 375 μM pyridoxal‐5‐phosphate glutamate decarboxylase is not inhibited to 50% even with 10 mM ATP. ADP, GTP and UTP are strong inhibitors of glutamate decarboxylase at high concentration. At lower concentrations ATP is the most potent inhibitor. AMP and adenosine do not effect glutamate decarboxylase, nor do they affect the inhibitory action of ATP. Inorganic phosphate in concentrations of 5—40 mM does not influence the activity of glutamate decarboxylase. At the same concentrations Pi diminishes the inhibitory effect of ATP. The [I]50% increases from 0.3 mM ATP (without Pi) to 1.7 mM ATP in the presence of 20 mM Pi.
KCN poisoning causes a pronounced decrease in the y-aminobutyric acid (GABA) concentration in the brain of rats (TURSK+, 1960). It is known that KCN inhibits many metabolic processes: it inhibits cytochrome oxidase and thus a whole series of oxido-reductive processes in the cell. KCN also inhibits the group of enzymesrequiring pyridoxal phosphate as coenzyme; and several other enzymes. In view of the relatively unspecific action of cyanides we considered it to be of interest to study in greater detail the question of where this compound interferes with the metabolic pathways of glutamic acid and GABA in the brain. From the known actions of KCN, two mechanisms leading to a decrease of GABA in the brain as a consequence of cyanide poisoning can be expected :(a) As a result of the defect in cell respiration the integrity of nerve cells is destroyed and GABA is released from the damaged cells.(b) Cyanide inhibits glutamate decarboxylase to a greater extent than GABA transaminase (TURSK~, in press). (Both of these enzymes contain pyridoxal-phosphate.) GABA is therefore broken down more rapidly than it is synthesized, MATERIALS A N D M E T H O D S Materials used. Adult white rats weighing 200 g, and 30-day-old rats were used. The guinea-pigs used weighed from 300 to 400 g, and the white mice, which were of a controlled breed, weighed about 20 g. [U14Clglutamic acid was obtained from the Radiochemical Centre Amersham, Great Britain ; potassium cyanide from Lachema; GABA was synthesized by Dr. BERANEK from the Chemical Institute of the Czechoslovak Academy of Sciences in Prague; glutamic acid p.a. was obtained from Lachema.Administration of KCN and preparation of the brain homogenate. KCN was administered intraperitoneally to rats at a dose of 8 mg in 0.5 ml of water. The amount of KCN given to mice and guinea pigs was calculated on the basis of the body weight of the animal so that each animal received 40 mg of KCN/kg in 0.5 ml water. The animals were decapitated after the onset of convulsions, which was usually 1-3 min after the administration of cyanide.After weighing, the brains were homogenized in ice-cold twice distilled water in a glass homogenizer according to POTTER and ELVEHJEM. The homogenate was deproteinized by boiling. When trichloroacetic acid was used for deproteinization the results of the GABA assays were 5-7 per cent higher. However, this increase was paralleled in the control and KCN-intoxicated animals.GABA and glutamic acid assay. The deproteinized homogenate was centrifuged and the supernatant was applied to Whatman No. 1 chromatographic paper for electrophoretic separation according to MIKES (1957), using pyridine acetate buffer, pH 3.8. Detection was with 0.5% ninhydrine in a mixture of acetone and pyridine (9: 1, v/v). The quantitative evaluation of the coloured complex with Cu(NO& was done according to FISCHER and DORFEL (1953).Administration of labelled glutamic acid and measurement of the activity of glutamic acid and of GABA. About 1 PC of Ul4C-1abelled glutamic acid in 0.01 ml was administe...
The renal glutamic acid decarboxylase (GAD) differs from the brain and pancreatic enzyme by its strong binding to membranes that is not influenced by detergents. After centrifugation of freshly prepared homogenate of the rat renal cortex, only 10±15% of GAD activity was found in supernatants and 15±30% in pellets. The majority of the GAD activity was lost. The bound GAD was found in the pellet. A thermolabile activator was present in the supernatant, which was not lost on dialysis. Approximately 55% of the total GAD activity was solubilized in homogenates stored for 24 h at 4 8C without detergent, whereas in homogenates stored with Triton X-100, the solubilized GAD increased to 80%. This solubilization was decreased by inhibitors of thioproteases such as leupeptin, antipain and trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane (E-64). Solubilized GAD was applied to DEAE Toyopearl resin and the GAD activator was eluted with 35 mm P i . GAD was eluted with 250 mm P i . The effect of ATP on the activity of renal GAD was also different to its effect on brain GAD. ATP is a strong inhibitor of the brain enzyme at physiological concentrations. ATP (and P i ), together with chlorides (another brain GAD inhibitor), stabilize the renal GAD. However, renal GAD was inhibited by ATP in the presence of leupeptin in freshly prepared homogenates. Similarly, ATP inhibits solubilized GAD from homogenates stored without Triton X-100 for 24 h at 4 8C, but P i retains its stabilizing effect in this preparation.A significant finding of the work presented here is the obligatory requirement of an endogenous activator for renal GAD activity. Whether this activator is an enzyme converting the inactive GAD to active enzyme (as hypothesized for brain GAD), or whether it is a protein affecting the activity of renal GAD by binding (as observed for GAD in some plants) remains to be established.
Changes in morphology and in transformations of [U-'4C]glucose and [l-14C]acetate into amino acids of the brain cortex were followed on the Sth, 10th and 21st days after production of mechanical lesions and compared with control tissue. In the experimental tissue, proliferation of astroglia and reduction of the number of neurons had taken place. On the 10th day, accumulation of mitochondria and of some gliofilaments in the cytoplasm of astroglia was observed. On the 21st day, the gliofilaments occupied a substantial portion of the astroglial cytoplasm and the mitochondria were reduced in number and compressed to the cell membrane. Incorporation of 14C from acetate into amino acids was substantially increased on the 10th day (up to 240% with respect to controls) and normalized again on the 21st day. Incorporation of ['4Clglucose into amino acids decreased somewhat during the experimental period. It has been proposed that the proliferation of astrocytes and their ultrastructural changes may account for the increased transformation of ["Clacetate into amino acids, in particular into glutamine which is formed from the small glutamate pool.
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