Enzyme activities leading to NAD synthesis in human lymphocytes.
Pyridine nucleotide levels and the activities of enzymes involved in NAD synthesis (nicotinic acid phosphoribosyltransferase, nicotinic acid- and nicotinamide mononucleotide-adenylyltransferase) have been assayed in human normal lymphocytes by an HPLC method using radioactive or nonradioactive substrates. NAD concentration was 46.4 +/- 17.2 pmol 10(-6) cells, and that of NADP was 14.5 +/- 3.9 pmol 10(-6) cells (mean +/- standard deviation). The adenylyltransferase activity using nicotinic acid mononucleotide as substrate was 1.530 +/- 0.216 nmol h(-1) 10(-6) cells, using nicotinamide mononucleotide 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 mean activity of nicotinic acid phosphoribosyltransferase 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 sensitive, avoiding the use of radiolabels except for NAPRT and displaying a very low activity. The reported findings, together with the previous ones in human erythrocytes, can provide an useful base to investigate NAD metabolism in humans through the study of blood cells.
Sestini
S
S
Department of Molecular Biology, University of Siena, Siena, Italy. sestinis@unisi.it
Jacomelli
G
G
Pescaglini
M
M
Micheli
V
V
Pompucci
G
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D000328:Adult; D002457:Cell Extracts; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D008875:Middle Aged; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D009612:Nicotinamide-Nucleotide Adenylyltransferase / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000378:metabolism; D010430:Pentosyltransferases / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D002457:Cell Extracts; D009243:NAD; D009249:NADP; C018348:nicotinic acid adenine dinucleotide; D000255:Adenosine Triphosphate; D010430:Pentosyltransferases; D009713:Nucleotidyltransferases; C484009:NMNAT3 protein, human; D009612:Nicotinamide-Nucleotide Adenylyltransferase; C038480:nicotinic acid mononucleotide adenylyltransferase; C021864:nicotinate phosphoribosyltransferase