Related Experiment Videos
Inhibition of NAD(H)/NADP(H)--requiring enzymes by aurintricarboxylic acid
1Department of Medical Pharmacology & Toxicology, College of Medicine, Texas A & M University Health Science Center, College Station 77843-1114, USA.
Abstract:
Aurintricarboxylic acid (ATA), an inhibitor of Ca(2+)-dependent endonuclease activity, is often used to implicate a role for increased intracellular calcium in mechanistic toxicology studies. We report here on the ability of ATA to inhibit the activity of several NAD(H)/NADP(H)-requiring enzymes (purified or cellular homogenates), including lactic dehydrogenase, alcohol dehydrogenase, cytochrome c reductase, ethoxycoumarin o-dealkylase, isocitric dehydrogenase, glutathione reductase and glucose-6-phosphate dehydrogenase. These results were compared with the ability of ATA to inhibit micrococcal nuclease and rat liver Ca(2+)-dependent endonuclease activity in similar incubations. With the exception of alcohol dehydrogenase, ATA was a potent inhibitor of each of the purified enzymes, with IC50s ranging from 0.5 to 82 microM. In cell homogenates, however, ATA was from 10 to 100-fold less potent at inhibiting these enzymes. When exogenous protein was added to purified enzyme incubations, the effect of ATA was similarly diminished. Our results demonstrate that ATA inhibits a wide range of NAD(H)/NADP(H)-requiring enzymes in in vitro incubations using purified enzymes, but that the inhibitory effects are markedly reduced in incubations which more closely resemble a cellular milieu.
Insights
Aurintricarboxylic acid (ATA) inhibits many NAD(H)/NADP(H)-requiring enzymes in vitro. However, ATA’s inhibitory effects are significantly reduced in cellular environments, challenging its use in toxicology studies.
Area of Science:
- Biochemistry
- Toxicology
- Enzymology
Background:
- Aurintricarboxylic acid (ATA) is commonly used to investigate the role of intracellular calcium in toxicology.
- ATA is known to inhibit Ca(2+)-dependent endonuclease activity.
Purpose of the Study:
- To investigate the inhibitory effects of ATA on various NAD(H)/NADP(H)-requiring enzymes.
- To compare ATA's inhibitory potency in purified enzyme systems versus cellular homogenates.
Main Methods:
- Enzyme inhibition assays were performed using purified NAD(H)/NADP(H)-requiring enzymes and rat liver Ca(2+)-dependent endonuclease.
- Enzyme activity was also assessed in cellular homogenates.
- The impact of exogenous protein on ATA's inhibitory activity was evaluated.
Main Results:
- ATA potently inhibited most purified NAD(H)/NADP(H)-requiring enzymes (IC50s 0.5–82 μM), except for alcohol dehydrogenase.
- In cellular homogenates, ATA was 10 to 100-fold less potent in inhibiting these enzymes.
- The presence of exogenous protein diminished ATA's inhibitory effects on purified enzymes.
Conclusions:
- ATA inhibits a broad spectrum of NAD(H)/NADP(H)-requiring enzymes in vitro.
- ATA's inhibitory efficacy is substantially reduced in conditions mimicking a cellular environment, suggesting limitations for its use in mechanistic toxicology.