Partial purification and properties of nicotinamide adenine dinucleotide synthetase from human erythrocytes: evidence that enzyme activity is a sensitive indicator of lead exposure

Abstract

We have examined properties of nicotinamide adenine dinucleotide (NAD) synthetase from human erythrocytes. The enzyme was found to be cold labile and extremely unstable in crude hemolysate, with complete loss of activity occurring after 24 hours at 4 degrees C. However, maintenance of crude hemolysate at 20 to 25 degrees C in the presence of EDTA and KCl increased NAD synthetase stability substantially (half- life = 10 days). Using these conditions, NAD synthetase was purified 3,100-fold with a 29% yield using DEAE-cellulose column chromatography, ammonium sulfate fractionation, and dialysis. The apparent Michaelis- Menten constants for nicotinic acid adenine dinucleotide (NAAD), adenosine triphosphate, Mg2+, glutamine, and K+ were 0.108, 0.154, 1.36, 2.17, and 8.32 mmol/L, respectively. The pH optimum ranged between 6.8 and 7.4, and the molecular weight was estimated to be 483 +/- 5 Kd. The enzyme was markedly inhibited by Pb2+ and Zn2+, with concentrations necessary for 50% inhibition of activity of 1.3 and 2.0 mumol/L, respectively. The incubation of intact red blood cells with lead followed by rigorous washing to remove lead abolished nearly all NAD synthetase activity. In contrast, glucose-6-phosphate dehydrogenase activity, which is not sensitive to lead, was unaffected, whereas pyrimidine 5′-nucleotidase activity, which is sensitive to lead, was decreased 30% to 50% under these conditions. More importantly, patients with lead overburden (34 to 72 micrograms Pb2+/dL blood) all had markedly decreased NAD synthetase activity. These data together with other results suggest that erythrocyte NAD synthetase activity is a sensitive indicator of lead exposure in humans.

Metabolic Profiling of Alternative NAD Biosynthetic Routes in Mouse Tissues

“…In contrast, the NADS-catalyzed reaction is considered physiologically irreversible [14] , [53] . The equilibrium constant and substrate affinities of mouse NADS are not available, but the first parameter can be approximated to exceed 10 6 based on reported standard free energy change [54] , whereas affinities can be assumed to be similar to those of human NADS [43] .…”

“… a Sum of the three NMNAT isozyme activities individually calculated as in Table 2 but versus the NaMN substrate ( K m values used are reported in ref [19] ). b Values are calculated from the steady-state kinetic equation (3 ) shown in Methods, using the NADS activities measured at saturating substrates ( Figure 3A ) as V max values, the NaAD, ATP, and glutamine levels (nmol/g, Figure 4 ) as micromolar concentrations, and the K m values from [43] . …”

“… b Values are calculated from the steady-state kinetic equation (3 ) shown in Methods, using the NADS activities measured at saturating substrates ( Figure 3A ) as V max values, the NaAD, ATP, and glutamine levels (nmol/g, Figure 4 ) as micromolar concentrations, and the K m values from [43] . …”

“…Equation (3 ) was used for NADS. where [A], [B], [C], [D], and [E] are the concentrations of ATP, NMN, NaMN, NaAD, and Gln, respectively, assuming the tissue values “nmol/g” as equal to micromolar; V max values are approximated as the “maximal rates” above; K ma , K mb , K mc are the Michaelis constants of NMNAT isozymes, respectively, for ATP (33.5 µM NMNAT1, 82 µM NMNAT2, 39 µM NMNAT3), for NMN (25.2 µM NMNAT1, 38.5 µM NMNAT2, 117.6 µM NMNAT3), and for NaMN (67.7 µM NMNAT1, 14.5 µM NMNAT2, 111 µM NMNAT3) [18] , [19] ; K ma' K md , K me are the Michaelis constants of NADS, respectively, for ATP (154 µM), NaAD (108 µM), and Gln (2170 µM) [43] . Note that [B] = NMN and [C] = NaMN, and their Michaelis constants, were inverted when using equation (2 ) for the evaluation of the deamidated reaction catalyzed by NMNAT.…”

Oral L-glutamine therapy for sickle cell anemia: I. subjective clinical improvement and favorable change in red cell NAD redox potential

“…The intracellular level of glutamine on the other hand was not elevated; instead, the level of glutamate, a byproduct of glutamine in NAD synthesis, was increased in sickle RBC in comparison to high reticulocyte controls [10]. As the Km of NAD synthetase for glutamine has been known to be higher than the mean concentration of intracellular glutamine [11], we hypothesized, in conjunction with these data, that supplemental glutamine may increase the activity of NAD synthesis, thereby countering the oxidant-dependent pathophysiology of sickle RBC. Therefore, we conducted a pilot trial to examine the clinical and biochemical effects of oral L-glutamine supplementation in sickle cell anemia patients.…”