Determination of NAD pyrophosphorylase activity in biological samples.
A sensitive and simple method was developed for the accurate measurement of NAD pyrophosphorylase (NMN adenylyltransferase; EC 2.7.7.1) activity in biological samples. The reaction product of [4-3H]NAD was separated from the substrates [4-3H]NMN and ATP by HPLC. Under the standardized conditions of the assay, the enzyme activity in human chronic myelogenous leukemia K562 cells was found mainly in the nucleus (97%) with a sp act of 183.5 +/- 3.5 nmol/h/mg protein. The Km's for substrates NMN and ATP were 0.11 +/- 0.01 mM and 0.55 +/- 0.04 mM, respectively. This technique is highly reproducible with a 5% variation (SD) in five separate determinations. The lowest number of cells used for this enzyme assay was 41,000 with a protein content of 4 micrograms. The range of NAD produced during the assay was 2 to 200 microM. NAD pyrophosphorylase activities in the mononuclear cells of leukemic patients, human ovarian carcinoma cells, and rat liver were assayed.
Paulik
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Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis 46202-5200.
Jayaram
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HN
Weber
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G
D000255:Adenosine Triphosphate; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008297:Male; D009243:NAD; D009537:Nicotinamide Mononucleotide; D009612:Nicotinamide-Nucleotide Adenylyltransferase / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D009243:NAD; D009537:Nicotinamide Mononucleotide; D000255:Adenosine Triphosphate; D009612:Nicotinamide-Nucleotide Adenylyltransferase