Abstract
Lactobacillus fructosus requires nicotinamide for growth and maintenance. The biosynthetic pathway of nicotinamide adenine dinucleotide (NAD) in this microorganism shows that the microorganism contains a phosphoribosyltransferase and an NAD pyrophosphorylase that are specific for nicotinamide and nicotinamide mononucleotide (NMN), respectively. This chapter discusses the assay method, purification procedure, and properties of nicotinamide phosphoribosyltransferase and NAD pyrophosphorylase from Lactobacillus fructosus . The activity of nicotinamide phosphoribosyltransferase is assayed by measuring the formation of NMN- 14 C from nicotinamide- 14 C in the presence of 5-phosphoribosyl-l-pyrophosphate (PRPP) and adenosine triphosphate (ATP). The ribonucleotide produced is isolated by paper chromatography and the radioactivity is determined. Enzyme activity may be assayed by measuring the formation of radioactive NAD from NMN- 14 C and ATP. The activity may be assayed in larger-scale incubation with higher concentrations of nonradioactive NMN, ATP, and the enzyme preparation. The NAD produced is determined by measuring the increase in absorbance at 340 m μ upon the addition of alcohol and alcohol dehydrogenase. The enzyme is specific for NMN and does not react with nicotinic acid ribonucleotide. The final preparation is practically free of nicotinamide phosphoribosyltransferase.
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