Assay method using NAD synthetase

A method for assaying a component in a specimen, said component being selected from the group consisting of ATP, desamido-NAD+, and an amide donor, said donor being an ammonia compound, L-glutamine or L-asparagine;

• said method comprising:
• performing a main reaction comprising incubating said specimen with NAD+ synthetase from Bacillus licheniformis B-0844 FERM P-6809 in the presence of ATP, desamido-NAD+, an amide donor, and Mg++ to generate NAD+, wherein the components not being measured are in excess of the component being measured and wherein said NAD synthetase is capable of retaining at least 95% of its activity after treatment at 40.degree. for 10 minutes at pH 6.8 in the absence of potassium chloride and bovine serum albumin;
• subjecting the thus-generated NAD+ to a coenzyme cycling reaction comprising reducing said generated NAD+ to reduced NAD in a reaction system comprising a dehydrogenase while dehydrogenating a substrate of said dehydrogenase, and then oxidizing the thus-generated reduced NAD to NAD+ in said coenzyme cycling reaction system further comprising diaphorase while reducing a tetrazolium salt to a formazan;
• and
• measuring the progress of said cycling reaction by measuring a compound participating in said cycling reaction, said compound being selected from the group consisting of said substrate of said dehydrogenase, said dehydrogenated substrate, said tetrazolium salt and said formazan.