Enzyme activities leading to NAD synthesis in human lymphocytes

Pyridine nucleotide levels and the activities of enzymes involved in NAD sy nthesis (nicotinic acid phosphoribosyltransferase, nicotinic acid- and nico tinamide mononucleotide-adenylyltransferase) have been assayed in human nor mal lymphocytes by an HPLC method using radioactive or nonradioactive subst rates, NAD concentration was 46.4 +/- 17.2 pmol 10(-6) cells, and that of N ADP was 14.5 +/- 3.9 pmol 10(-6) cells (mean +/- standard deviation). The a denylyltransferase activity using nicotinic acid mononucleotide as substrat e was 1.530 +/- 0.216 nmol h(-1) 10(-6) cells, using nicotinamide mononucle otide was 1.466 +/- 0.354 nmol h(-1) 10(-6) cells. The apparent K-M values were 0.015 mM for the former substrate and 0.167 mM for the latter. The mea n activity of nicotinic acid phosphoribosyl-transferase was 0.038 +/- 0.014 nmol h(-1) 10(-6) cells, and the apparent K-M for nicotinic acid was 0.165 mM. The proposed methods, easy and rapid to perform, are reliable and sens itive, avoiding the use of radiolabels except for NAPRT and displaying a ve ry low activity. The reported findings, together with the previous ones in human erythrocytes, can provide an useful base to investigate NAD metabolis m in humans through the study of blood cells. (C) 2000 Academic Press.