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Analytical Techniques for Assaying Nitric Oxide Bioactivity
Published on: June 18, 2012
Caveat: mycoplasma arginine deiminase masquerading as nitric oxide synthase in cell cultures
1Department of Surgery, Chungbuk National University Hospital, Chungbuk, South Korea.
Insights
Researchers mistook arginine deiminase for inducible nitric oxide synthase (NOS) in dog cells due to a Mycoplasma contaminant. This highlights the risk of confusing these enzymes when using arginine assays without checking cofactors.
Area of Science:
- Cell Biology
- Enzymology
- Microbiology
Background:
- Inducible nitric oxide synthase (iNOS) plays a crucial role in various physiological processes.
- Accurate identification of enzymes in cell cultures is essential for reliable research.
- Mycoplasma contamination is a common issue in cell culture systems.
Purpose of the Study:
- To investigate the presence of inducible nitric oxide synthase (NOS) in dog gallbladder epithelial cells.
- To identify a potential contaminant in fibroblast-conditioned medium affecting enzyme assays.
- To differentiate between NOS and arginine deiminase activity in cell culture models.
Main Methods:
- Utilized confluent cultures of dog gallbladder epithelial cells stimulated by conditioned medium from human neonatal foreskin fibroblasts.
- Assayed enzyme activity via the conversion of radiolabeled arginine to citrulline.
- Performed enzyme purification and analyzed reaction products (ammonia liberation) to confirm enzyme identity.
Main Results:
- Observed high enzyme activity after 4 days of conditioned medium exposure.
- Determined the enzyme did not require standard NOS cofactors (NADPH, FAD, FMN, tetrahydrobiopterin) for maximal activity.
- Confirmed the enzyme was arginine deiminase, not NOS, and identified Mycoplasma contamination in the fibroblast culture.
Conclusions:
- Dog gallbladder epithelial cells stimulated with conditioned medium exhibited arginine deiminase activity, not iNOS.
- Mycoplasma contamination in the fibroblast culture was the source of arginine deiminase.
- There is a significant risk of confusing arginine deiminase with NOS when using the [3H]arginine assay, necessitating cofactor checks.
Abstract:
We used confluent cultures of dog gallbladder epithelial cells, stimulated by conditioned medium from a culture of human neonatal foreskin fibroblasts, to establish the presence of inducible nitric oxide synthase (NOS, EC 1.14.13.39). Assay was by conversion of radiolabeled arginine to citrulline. By 4 days after addition of the conditioned medium, a relatively high level of activity was observed. However, further study showed that the enzyme did not require addition of the usual cofactors for maximal activity (NADPH, FAD, FMN and tetrahydrobiopterin) and was stable in the absence of anti-proteolytic agents. Our suspicion that this enzyme might not be NOS but arginine deiminase (EC 3.5.3.6) was confirmed by enzyme purification and by the liberation of ammonia during enzyme reaction. This enzyme, which is absent from primates and virtually confined to single-cell organisms, suggested the presence of Mycoplasma, a common contaminant of cell cultures, and it was subsequently confirmed that the fibroblast culture was a source of Mycoplasma. With the widespread interest in nitric oxide and NOS, and common use of the convenient [3H]arginine assay, there is a considerable danger of the two enzymes being confused. At the very least, it is necessary to check for activity in the absence of added cofactors.
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