Specific binding and uptake of extracellular nicotinamide in human leukemic k-562 cells

Abstract

Extracellular nicotinamide is well recognized as the primary precursor to the cellular synthesis of NAD. NAD is a pivotal molecule in regulating the energy and redox potentials of cells via synthesis of ATP and NAD(P)/NAD(P)H ratios. NAD turnover in cells is very rapid due to NAD catabolism via ADP-ribosylation reactions. These facts suggest that the cellular uptake and transport of nicotinamide may not be a passive process but a highly regulated cellular event. We have utilized radiometric procedures to characterize the uptake of [ 14]nicotinamide in human leukemic K-562 cells. At physiologically relevant doses of nicotinamide (<100 μM), the uptake was saturable with a K m of 2.3 ± 1.0 μM and a V max of about l.5 ± 0.5 pmol/10 6 cells/min. Kinetic studies revealed that nicotinamide was first taken up intracellularly and then immediately converted to NAD and 1-methyl nicotinamide. All of the nicotinamide taken up into the cell was bound tightly to plasma membranes (25,000 g pellet) with K d values between 3.2 and 12.7 μM and a B max of 1.56 pmol/10 6 cells. The specificity of nicotinamide binding was demonstrated by competitive inhibition experiments using NAD analogs, nicotinamide derivatives, and agonists or antagonists of plasma membrane receptors. We conclude that there is specific binding of nicotinamide, followed by intracellular uptake and immediate synthesis to NAD.