Enzyme inhibition XI: glutamate decarboxylase activity relationship with the reaction products as determined by the colorimetric and radioisotopic methods.
The relationship of glutamate acid decarboxylase (GAD) activity with the reaction products was developed. It was based on incubating sodium glutamate substrate (S) with GAD enzyme (E) when the enzyme-substrate-complex (ES) product was obtained along with gamma aminobutyric acid (GABA) and unreacted sodium glutamate. The reaction products were separated by paper chromatography. The ES, GABA and S products were sprayed with ninhydrin reagent when ninhydrin-colored-complex (NCC) was formed on the paper chromatogram. The products were extracted with 75% ethanol containing 0.5% cupric sulfate. The NCC absorption readings of ES and S products were measured by a spectrophotometer. A standard curve was prepared by plotting absorption readings against different concentrations of sodium glutamate. This curve was the basis of determining GAD activity of E. coli and C. welchii. It was observed that NCC absorption of ES and S products was directly related with the enzyme activity. The qualities of ES and S products in the reaction mixture increased as the enzyme activity increased with the incubation time. On the other hand, some products in the reaction mixture decreased in the presence of an inhibitor of GAD activity. The relationship of reaction products with GAD activity was also established by the radioisotopic method. The results obtained by the chromatographic separation of products followed by the spectrophotometric method of determining GAD activity is a simple, safe and less expensive compared to the other methods.
Sethi
M L
ML
Department of Pharmaceutical Sciences, College of Pharmacy, Nursing and Allied Health Sciences, Howard University, Washington, DC 20059, USA.
D001149:Arsenates / Q000494:pharmacology; D002250:Carbon Radioisotopes / Q000032:analysis / Q000737:chemistry; D003124:Colorimetry / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology; D005968:Glutamate Decarboxylase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D018698:Glutamic Acid / Q000032:analysis / Q000378:metabolism; D013379:Substrate Specificity; D014635:Valproic Acid / Q000494:pharmacology
D001149:Arsenates; D002250:Carbon Radioisotopes; D004791:Enzyme Inhibitors; D018698:Glutamic Acid; D014635:Valproic Acid; C009277:sodium arsenate; D005968:Glutamate Decarboxylase