Determination of glutamic acid decarboxylase activity and inhibition by an H2O2-sensing glutamic acid oxidase biosensor.
The catalytic activity of the enzyme L-glutamic acid decarboxylase (GAD) is determined by an amperometric method based on a recently developed glutamate-selective biosensor. The biosensor is composed of an amperometric H2O2 electrode and a biocatalytic membrane containing the enzyme glutamic acid oxidase (GAO). The biosensor allows the direct and continuous measurement of GA levels by monitoring the H2O2 produced at the electrode interface as a coproduct of the GAO-catalyzed GA oxidation to alpha-ketoglutaric acid. Since GA is transformed to gamma-aminobutyric acid and CO2 under the catalytic activity of GAD, the rate of GA consumption in solution, monitored by the GAO biosensor, represents a reliable measure of GAD catalytic activity. Additional experiments performed in the presence of different concentrations of the GAD inhibitor valproic acid have shown the suitability of the proposed approach for the study of GAD inhibitors also. Discussion of the main experimental characteristics of this new analytical method is given in terms of sensitivity, reproducibility, and reliability of the experimental results and ease, time, and cost of operation.
Botrè
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Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Università La Sapienza, Rome, Italy.
Botrè
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F
Galli
M
M
Lorenti
G
G
Mazzei
F
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Porcelli
F
F
D000594:Amino Acid Oxidoreductases / Q000737:chemistry*; D015374:Biosensing Techniques; D005968:Glutamate Decarboxylase / Q000032:analysis* / Q000037:antagonists & inhibitors; D005971:Glutamates / Q000737:chemistry; D018698:Glutamic Acid; D006861:Hydrogen Peroxide / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics
D005971:Glutamates; D018698:Glutamic Acid; D006861:Hydrogen Peroxide; D000594:Amino Acid Oxidoreductases; C023061:L-glutamate oxidase; D005968:Glutamate Decarboxylase