Identification of a nicotinamide adenine dinucleotide glycohydrolase and an associated inhibitor in isoniazid-susceptible and -resistant Mycobacterium phlei.
Nicotinamide adenine dinucleotide glycohydrolase (NADase) activity was demonstrated in the catalases fraction of Sephadex G-200-chromatographed sonic extracts of isoniazid (INH)-susceptible (Inhs) and -resistant (Inhr) Mycobacterium phlei. Since crude extracts had no demonstrable activity even after heating, active fractions of the NADase were purified chromatographically by removing the inhibitor with Sephadex G-200. Assays for oxidized nicotinamide adenine dinucleotide (NAD+) hydrolytic activity were done by following the disappearance of NAD+ by the methods of alcohol dehydrogenase or cyanide addition. The NADase activity was linear with respect to time as well as concentration of enzyme and was inhibited in the presence of 0.04 M NADP, benzoic acid hydrazide, or nicotinamide. Crude extracts or pooled concentrated Sephadex G-200 fractions eluting after the catalase inhibited NADase activity by at least 70%. Inhibitor activity was present in both the Inhs and Inhr strains of M. phlei. The activity of the partially purified inhibitors was reversible by INH or nicotinic acid hydrazide at levels between 10 and 100 mM. These findings indicate that an NADase inhibitor system which is sensitive to reversal by INH functions in both the Inhs and Inhr strains; however, unlike previous studies with other mycobacterial species, the enzyme is sensitive to inhibition by nicotinamide. Furthermore, the inhibitors are heat stable and sensitive to reversal by nicotinic acid hydrazide as well as INH.
D002850:Chromatography, Gel; D004352:Drug Resistance, Microbial; D006358:Hot Temperature; D007538:Isoniazid / Q000494:pharmacology*; D009161:Mycobacterium / Q000201:enzymology*; D009168:Mycobacterium phlei / Q000187:drug effects / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D009244:NAD+ Nucleosidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D009536:Niacinamide / Q000494:pharmacology
D009243:NAD; D009536:Niacinamide; D009244:NAD+ Nucleosidase; D007538:Isoniazid