CAVEAT - MYCOPLASMA ARGININE DEIMINASE MASQUERADING AS NITRIC-OXIDE SYNTHASE IN CELL-CULTURES

We used confluent cultures of dog gallbladder epithelial cells, stimul ated by conditioned medium from a culture of human neonatal foreskin f ibroblasts, to establish the presence of inducible nitric oxide syntha se (NOS, EC 1.14.13.39), Assay was by conversion of radiolabeled argin ine to citrulline. By 4 days after addition of the conditioned medium, a relatively high level of activity was observed. However: further st udy showed that the enzyme did not require addition of the usual cofac tors for maximal activity (NADPH, FAD, FMN and tetrahydrobiopterin) an d 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 o f Mycoplasma, a common contaminant of cell cultures, and it was subseq uently confirmed that the fibroblast culture was a source of Mycoplasm a. With the widespread interest in nitric oxide and NOS, and common us e of the convenient [H-3]arginine assay, there is a considerable dange r of the two enzymes being confused. At the very least, it is necessar y to check for activity in the absence of added cofactors. 0167-4889/9 8/$ - see front matter (C) 1998 Elsevier Science B.V. All rights reser ved.