title: 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.
authors.affiliation: Department of Medicine, Harbor-UCLA Medical Center, Torrance 90502.
authors.forename: C R
authors.initials: CR
authors.lastname: Zerez
authors.forename: M D
authors.initials: MD
authors.lastname: Wong
authors.forename: K R
authors.initials: KR
authors.lastname: Tanaka
mesh_terms: D019732:Amide Synthases; D015415:Biomarkers / Q000097:blood*; D002848:Chromatography, DEAE-Cellulose; D004492:Edetic Acid / Q000494:pharmacology; D004795:Enzyme Stability; D004912:Erythrocytes / Q000201:enzymology*; D005260:Female; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D007854:Lead / Q000494:pharmacology*; D007855:Lead Poisoning / Q000097:blood / Q000175:diagnosis* / Q000201:enzymology; D008025:Ligases / Q000037:antagonists & inhibitors / Q000097:blood* / Q000302:isolation & purification; D008297:Male; D008970:Molecular Weight; D009942:Organometallic Compounds / Q000494:pharmacology; D012016:Reference Values; D015032:Zinc / Q000494:pharmacology
chemical_list: D015415:Biomarkers; D009942:Organometallic Compounds; D007854:Lead; D004492:Edetic Acid; D008025:Ligases; D019732:Amide Synthases; C038479:NAD+ synthase; D005978:Glutathione; D015032:Zinc; C008261:lead acetate