Pharmacology & Toxicology 1991. 69. 238-241.
On the Inhibition of Glutamic Acid Decarboxylase and y-Aminobutyric Acid Transaminase by Sodium Cyanide Gudrun Cassel, Lena Karlsson and Ake Sellstrom Division of Experimental Medicine, Department of NBC Defence. National Defense Research Establishment. S-901 82 Umea. Sweden (Received November 26. 1990: Accepted April 3. 1991)
Abstract: The effects of sodium cyanide (NaCN) on the y-aminobutyric acid metabolizing enzymes glutamic acid decarboxylase (GAD) and y-aminobutyric acid transaminase (GABA-T) were studied in ritro. With no pyridoxal-5phosphate added , GAD was non-competitively inhibited by NaCN . with an IC50 of 280 !IM . GAD was also inhibited when exposed to an equimolar amount of NaCN and pyridoxal-5-phosphate. NaCN inhibited GABA-T. The inhibition kinetics suggests that NaCN may react with more than one of the substrates and products present during the reaction. i.e. pyridoxal-5-phosphate. a-ketoglutarate and/or succinic semialdehyde. The presence of pyridoxal-5-phosphate in the reaction mixture completely protected GABA-T from inhibition by NaCN. The y-aminobutyric acid synthesizing enzyme. GAD may thus be inhibitied in rho by NaCN or by a reaction product of NaCN and pyridoxal-5-phosphatc. The yaminobutyric acid catabolizing enzyme. GABA-T is not as vulnerable to inhibition by NaCN . since the cyanide-pyridoxal5-phosphate complex is ineffective as inhibitor. Abbreriations: GAD. glutamic acid decarboxylase; GABA- T. y-aminobutyric acid transaminase: GABA. y-aminobutyric acid; NaCN , sodium cyanide: OPT. ortho-phthaldialdehyd: AET, 2-aminoethylisothionium bromide hydrobromidc: PLP. pyridoxal-5-phosphate: CN - . cyanide ion.
Acute cyanide poisoning will, in severe cases, cause symptoms such as fatigue, dizziness, unconsciousness. convulsions, cessation of breath. heart failure and ultimately death (Egekeze & Oehme 1980; Ballantyne 1983; Way 1984). The cause of death is most likely an inhibition of the cellular respiration at the cytochrome oxidase level. Studies of the inhibition of cytochrome oxidase activity in various organs following cyanide intoxication show some correlation between the severity of the intoxication and the degree of inhibition of the cytochrome oxidase (Aibaum et a/. 1946; Isom & Way 1976; lsom et a/. 1982). However. animals given antidotes. sodium nitrate or thiosulfate. appeared to have recovered cytochrome oxidase activity of the liver but not that of the brain (Isom & Way 1976; Isom eta/. 1982). This may in part be an effect of the poor distribution of the antidotes to the brain. but it also reflects the absence of the cyanide metabolizing enzyme rhodanese in the brain. The brain thus appears to be vulnerable to cyanide during intoxication for an extended period of time. Inhibition of the brain cytochrome oxidase by cyanide results in a so-called histotoxic anoxia, which secondarily influences the metabolism of a number of neurotransmitters (Shimada eta/. 1974; Gibson eta/. 1978. 1981 a & b). During insufficient energy metabolism of the brain. convulsions frequently occur. This is also true during cyanide intoxication (Ballentyne 1983). In addition to the known effect of cyanide on the cytochrome oxidase activity. cyanide also interacts with a number of other enzymes (Dixon & Webb 1958; Solomonson 1982). In fact, many of these enzymes are as sensitive or more to cyanide as the cytochrome oxidase (Solomonson 1982). In a previous study we have examined the effects of cyanide on the GABA metabolism in vi1•o
(Persson et a/. 1985). We found that y-aminobutyric acid (GABA) levels decrease after cyanide intoxication. a finding which recently has been confirmed by Yamamoto ( 1990). A decreased GABA activity during cyanide intoxication would in part explain the increased susceptibility to convulsions (Tapia 1975). Inhibition of the pyridoxal-5-phosphate (PLP) requiring enzymes glutamic acid decarboxylase (GAD. EC 4.1.1.15) and y-aminobutyric acid transaminase (GABA-T. EC 2.6.1.19) by potassium cyanide. was reported by Tursky & Sajter ( 1962). The decreased GABA levels observed by us in l'iro may suggest that GAD is more susceptible to sodium cyanide (NaCN) than GABA-T. Accordingly. in the present study we decided to reexamine the inhibition of GAD and GA BAT by NaCN .
Materials and Methods Animals. Male Sprague Dawley rats weighing approximately 200 g (ALA B. Sweden) were used. The animals were housed 4-5 per cage. The room temperature was 21 - 24 and humidity 50± 5% . They had free access to Ewos commercial pelleted diet (RJ) and tap water. En=yme assays. The rats were sacriliced by decapitation and the brains were homogenized (1 /20. wfv) in 50 mM potassium phosphate buffer (pH 7.2) containing I mM 2-amino-cthylisothionium bromide hydrobromidc (AET) and 0.1 mM EDTA.
GABA-transaminase. The radiochemical method or Hall & Kravitz (1967) was used with some modifications. The GABA-T activity was assayed using 100 !II of homogenate to a reaction medium containing 0.1 M Tris-HCI (pH 8.Q). I mM AET. I mM succinic
EFFECTS OF CYAN ID E ON GABA METABOLISM CAD y - aminobulyriCclnoc temialdohyd
aucclnlc acid
NAO
• - kotogtutmt
)I CN '
glutomlc eel
On the Inhibition of Glutamic Acid Decarboxylase and y-Aminobutyric Acid Transaminase by Sodium Cyanide Gudrun Cassel, Lena Karlsson and Ake Sellstrom Division of Experimental Medicine, Department of NBC Defence. National Defense Research Establishment. S-901 82 Umea. Sweden (Received November 26. 1990: Accepted April 3. 1991)
Abstract: The effects of sodium cyanide (NaCN) on the y-aminobutyric acid metabolizing enzymes glutamic acid decarboxylase (GAD) and y-aminobutyric acid transaminase (GABA-T) were studied in ritro. With no pyridoxal-5phosphate added , GAD was non-competitively inhibited by NaCN . with an IC50 of 280 !IM . GAD was also inhibited when exposed to an equimolar amount of NaCN and pyridoxal-5-phosphate. NaCN inhibited GABA-T. The inhibition kinetics suggests that NaCN may react with more than one of the substrates and products present during the reaction. i.e. pyridoxal-5-phosphate. a-ketoglutarate and/or succinic semialdehyde. The presence of pyridoxal-5-phosphate in the reaction mixture completely protected GABA-T from inhibition by NaCN. The y-aminobutyric acid synthesizing enzyme. GAD may thus be inhibitied in rho by NaCN or by a reaction product of NaCN and pyridoxal-5-phosphatc. The yaminobutyric acid catabolizing enzyme. GABA-T is not as vulnerable to inhibition by NaCN . since the cyanide-pyridoxal5-phosphate complex is ineffective as inhibitor. Abbreriations: GAD. glutamic acid decarboxylase; GABA- T. y-aminobutyric acid transaminase: GABA. y-aminobutyric acid; NaCN , sodium cyanide: OPT. ortho-phthaldialdehyd: AET, 2-aminoethylisothionium bromide hydrobromidc: PLP. pyridoxal-5-phosphate: CN - . cyanide ion.
Acute cyanide poisoning will, in severe cases, cause symptoms such as fatigue, dizziness, unconsciousness. convulsions, cessation of breath. heart failure and ultimately death (Egekeze & Oehme 1980; Ballantyne 1983; Way 1984). The cause of death is most likely an inhibition of the cellular respiration at the cytochrome oxidase level. Studies of the inhibition of cytochrome oxidase activity in various organs following cyanide intoxication show some correlation between the severity of the intoxication and the degree of inhibition of the cytochrome oxidase (Aibaum et a/. 1946; Isom & Way 1976; lsom et a/. 1982). However. animals given antidotes. sodium nitrate or thiosulfate. appeared to have recovered cytochrome oxidase activity of the liver but not that of the brain (Isom & Way 1976; Isom eta/. 1982). This may in part be an effect of the poor distribution of the antidotes to the brain. but it also reflects the absence of the cyanide metabolizing enzyme rhodanese in the brain. The brain thus appears to be vulnerable to cyanide during intoxication for an extended period of time. Inhibition of the brain cytochrome oxidase by cyanide results in a so-called histotoxic anoxia, which secondarily influences the metabolism of a number of neurotransmitters (Shimada eta/. 1974; Gibson eta/. 1978. 1981 a & b). During insufficient energy metabolism of the brain. convulsions frequently occur. This is also true during cyanide intoxication (Ballentyne 1983). In addition to the known effect of cyanide on the cytochrome oxidase activity. cyanide also interacts with a number of other enzymes (Dixon & Webb 1958; Solomonson 1982). In fact, many of these enzymes are as sensitive or more to cyanide as the cytochrome oxidase (Solomonson 1982). In a previous study we have examined the effects of cyanide on the GABA metabolism in vi1•o
(Persson et a/. 1985). We found that y-aminobutyric acid (GABA) levels decrease after cyanide intoxication. a finding which recently has been confirmed by Yamamoto ( 1990). A decreased GABA activity during cyanide intoxication would in part explain the increased susceptibility to convulsions (Tapia 1975). Inhibition of the pyridoxal-5-phosphate (PLP) requiring enzymes glutamic acid decarboxylase (GAD. EC 4.1.1.15) and y-aminobutyric acid transaminase (GABA-T. EC 2.6.1.19) by potassium cyanide. was reported by Tursky & Sajter ( 1962). The decreased GABA levels observed by us in l'iro may suggest that GAD is more susceptible to sodium cyanide (NaCN) than GABA-T. Accordingly. in the present study we decided to reexamine the inhibition of GAD and GA BAT by NaCN .
Materials and Methods Animals. Male Sprague Dawley rats weighing approximately 200 g (ALA B. Sweden) were used. The animals were housed 4-5 per cage. The room temperature was 21 - 24 and humidity 50± 5% . They had free access to Ewos commercial pelleted diet (RJ) and tap water. En=yme assays. The rats were sacriliced by decapitation and the brains were homogenized (1 /20. wfv) in 50 mM potassium phosphate buffer (pH 7.2) containing I mM 2-amino-cthylisothionium bromide hydrobromidc (AET) and 0.1 mM EDTA.
GABA-transaminase. The radiochemical method or Hall & Kravitz (1967) was used with some modifications. The GABA-T activity was assayed using 100 !II of homogenate to a reaction medium containing 0.1 M Tris-HCI (pH 8.Q). I mM AET. I mM succinic
EFFECTS OF CYAN ID E ON GABA METABOLISM CAD y - aminobulyriCclnoc temialdohyd
aucclnlc acid
NAO
• - kotogtutmt
)I CN '
glutomlc eel
