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.

Enzymology of mammalian NAD metabolism in health and disease

“…More recently, the human enzyme gene NADsyn1 (yeast qns1), encoding a 706 amino acids protein, has been identified, and the protein has been characterized after overexpression in COS-7 cells (144). The recombinant enzyme in solution behaves as a 500-kDa homohexamer (144), consistent with previous observation from human erythrocytes and yeast enzyme preparations (137,138). Kinetic characterization of human NADS, performed at pH 7.5, showed K m values for NaAD, glutamine, and ATP of 0.49 mM, 1.44 mM, and 0.089 mM, respectively.…”

“…The first one is strictly ammonia-dependent, while the other uses glutamine as the nitrogen donor. Enzymes belonging to the first class are typically found only in prokaryotes, e.g., E. coli (133), B. subtilis (134), S. typhimurium (135), and B. anthracis (136), while glutamine-dependent NADSs have been reported in eukaryotes (137,138) and selected prokaryotes like M. tuberculosis (139). Structural alignments evidence that the two classes of NADSs share highly conserved C-terminal Figure 6.…”

“…In eukaryotes, glutamine-dependent NADS activity has long been known in human erythrocytes and yeast (137,138). More recently, the human enzyme gene NADsyn1 (yeast qns1), encoding a 706 amino acids protein, has been identified, and the protein has been characterized after overexpression in COS-7 cells (144).…”

A novel deamido-NAD+-binding site revealed by the trapped NAD-adenylate intermediate in the NAD+ synthetase structure

“…On the other hand, the enzyme from Mycobacterium tuberculosis, a 738-residue polypeptide chain, has been isolated recently and shown to be able to use both ammonia and glutamine as nitrogen sources [15]. Furthermore, both the human and tobacco NAD + synthetases have been reported to use glutamine as a nitrogen source [16,17].…”

Comparative genomics of NAD(P) biosynthesis and novel antibiotic drug targets

“…Ammonia‐dependent NADS has been typically found in most bacteria, such as E. coli (Allibert et al, 1987), B. subtilis (Nessi et al, 1995), S. typhimurium (Hughes et al, 1988), Helicobacter pylori (Kang et al, 2005a,b) and B. anthracis (McDonald et al, 2007), while glutamine‐dependent NADS activity appears in eukaryotes (Zerez et al, 1990) and several prokaryotes, such as human (Sakai et al, 1997) and M. tuberculosis (Bellinzoni et al, 2005). Both types contain a highly conserved C‐terminal synthetase domain and a P‐loop motif of N‐type ATP pyrophosphatases family.…”