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1990 75: 1576-1582

Partial purification and properties of nicotinamide adenine dinucleotide synthetase from human erythrocytes: evidence that enzyme activity is a sensitive indicator of lead exposure
CR Zerez, MD Wong and KR Tanaka

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Partial Purification and Properties of Nicotinamide Adenine Dinucleotide Synthetase From Human Erythrocytes: Evidence That Enzyme Activity Is a Sensitive Indicator of Lead Exposure
By Charles R. Zerez, Mitchell D. Wong, and Kouichi R. Tanaka
We have examined properties of nicotinamide adenine dinucleotide (NAD) synthetase from human erythrocytes. The enzyme was found t o be cold labile and extremeky unstable in crude hemolysate, with completdoss of activity occurring after 24 hours at 4C. However, maintenance of crude hemolysate at 20 t o 25C in the presence of EDTA and KCI 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 MichaelisMenten constants for nicotinic acid adenine dinucleotide (NAAD), adenosine triphosphate, Mg", 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 tebe 483 f 5 Kd. The
enzyme was markedly inhibited by Pb2+ and Zn", with concentrations necessary for 50% inhibition of activity of 1.3 and 2.0 pmol/L, respectively. The incubation of intact red blood cells with lead followed by rigorous washing t o 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 t o lead, was decreased 30% to 50% under these conditions. More importantly, patients with lead overburden (34to 72 pg Pb2+/dL blood) dl 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.. 0 1990 b y The American S o c i e t y of Hematology.

ICOTINAMIDE adenine dinucleotide (NAD) synthetase (E.C. 6.3.5.1) catalyzes the final step in the Preiss-Handler pathway for NAD biosynthesis.' The enzyme transfers an amino group from glutamine to nicotinic acid adenine dinucleotide (NAAD) to form NAD in the presence of adenosine triphosphate (ATP), Mg2+, and K+.' NAD synthetase has been purified and characterized in yeast2and Escherichia coli,' and studied in cell-free crude extracts of Salmoaella typhimurium! To date, the direct measurement of NAD synthetase activity in human cell-free hemolysates has not been published. Incubation of intact human. RBC with nicotinic acid, glucose, inorganic phosphate, and glutamine results in substantial accumulation of NAD due to synthe~is.'.~ The presence of the intermediates ofNAD biosynthesis has been established in human red blood cells (RBC) under the above incubation conditions,' indicating that all of the NAD biosynthetic enzymes, including NAD synthetase, must be
From the Department of Medicine. Harbor-UCLA Medical f Center. University o California at Los Angeles School of Medicine, Torrance, CA. Submitted August 1,1989; accepted December I I , 1989. Supported by National Research Service Award HL 07364 (to C.R.Z.),Grant DK 14898 (to K.R.T.)from the National Institutes of Heahh. and grants from the Sickle Cell Disease Research Foundation of Los Angeles and the Research and Education Institute, Inc. Harbor-UCLA Medical Center (to C.R.Z.). Presented at the Annual Meeting of the American Society of Hemaiology. Washington, DC. December 7. I987 (Blood 70:57a. 1987). Address reprint requests to Charles R. Zerez, PhD, HarborUCLA Medical Center, 1124 West Carson St, CI-12, Torrance. CA 90502. The publication costs of this article were defrayed'inpart by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.section 1734 solely to indicate this fact. 0 1990 by The American Society of Hematology. 0006-@71/90/ 7507-0012$3.0O/O

present in human RBC. However, the classical studies of Preiss and Handler' demonstrated that when cell-free hemolysates of RBCs from outdated blood bank units are incubated with the immediate precursors of NAD (ie, nicotinic acid, phosphoribosylpyrophosphate(PRPP), Mg2+,K+, ATP, and glutamine), they cannot catalyze NAD synthesis, even though they are capable d NAAD synthesis. Recently, we were able to achieve NAD synthesis in liemolysates of freshlyobtained human RBC.' This suggests that NAD synthetase is active but labile in hemolysates and provides an explanation for the lack of NAD synthetase activity in cell-free hemolysates from stored RBC. Our interest in NAD synthetase resulted from our findings of an impaired rate of NAD synthesis in intact RBC from patients with pyruvate kinase (PK) deficiency: enolase deficiency: thalassemia," and sickle cell disease.'' Impaired NAD synthesis is due to the decreased' regeneration of ATP in PK-deficient RBC' and enolase-deficient RBC: and due to the decreased PRPP synthetase activity and decreased PRPP formation in thalassemic RBC.'* The mechanism for the impaired NAD synthesis in sickle RBC remains to be determined. In this report, we describe conditions that lead to greatly improved stability of NAD synthetase activity and describe a method for the partial purification of this enzyme from human erythrocytes. We also describe the kinetic properties of NAD synthetase to determine whether they can account for the impaired NAD synthesis in sickle RBC and to gain insight into the human erythrocyte enzyme. More importantly, we present data demonstrating that NAD synthetase is inactivated by lead and that enzyme activity is a sensitive indicator of lead exposure both in vitroand in vivo.
MATERIALS AND METHODS

Materials Sephadex G-200 was purchased from Pharmacia, Inc, Piscataway, NJ. Lead acetate (Pb [acetateld was purchased from Eastman Kodak Co, Rochester, NY. All other reagents were . purchased from Sigma Chemical Co, St Louis, MO.

1576

Blood, Vol 75, No 7 (April 1). 1990: pp 1576-1582

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NAD SYNTHETASE AND LEAD EXPOSURE

1577

Methods
Isolation o j erythrocytes. After obtaining informed consent, blood was obtained by routine venipuncture from normal individuals using heparin (15 U/mL whole blood) to prevent coagulation. A red cell-enriched fraction was prepared by passing whole blood through a column of a-cellulose and microcrystalline cellulose to deplete white cells and platelets, as described by Be~t1er.l~ RBC were washed three times with 0.15 mol/L NaCI. NAD synthetase purification. Unless otherwise indicated, all NAD synthetase purification procedures were performed at 20 to 25OC. Fifteen milliliters of packed fresh RBC was hemolyzed by dilution with 60 mL distilled water (1 part packed RBC to 4 parts water), and the resulting hemolysate was centrifuged at 39,000 x g for 20 minutes to remove stroma. A DEAE-cellulose column (1.5 x 6.0 cm) was equilibrated with a de-aerated solution containing 100 mmol/L KC1, 1.0 mmol/L EDTA, and 30 mmol/L Tris-HC1, pH 7.4 (NAD synthetase buffer). A total of 52 mL of the stroma-free hemolysate was applied onto the column in 13 4-mL aliquots. After the application of each aliquot, the column was washed twice with 10 mL of de-aerated NAD synthetase buffer. The eluate, which contained most of the hemoglobin (Hb) present in the applied stroma-free hemolysate, but none of the NAD synthetase activity, was discarded. The column was washed with 20 mL of a solution containing 150 mmol/L KCl, 1.0 mmol/L EDTA, and 30 mmol/L Tris-HC1, pH 7.4, which resulted in the elution of the remaining Hb. NAD synthetase was eluted with 20 mL of a solution containing 200 mmol/L KC1, 1.0 mmol/L EDTA, and 30 mmol/L Tris-HC1, pH 7.4. The eluate was adjusted with solid ammonium sulfate to 30% saturation, stirred for 30 minutes, and centrifuged at 30,000 x g for 20 minutes. The small pellet obtained was resuspended in 1.0 mL NAD synthetase buffer and dialyzed for 12 to 15 hours against 250 volumes of the same buffer. This two-step process resulted in a 29% yield and a 3,100-fold purification. The resulting partially purified preparation of NAD synthetase could be stored at 20 to 25OC for 1 month with little loss in activity. A summary of the purification is shown in Table 1. NAD synthetase assay. NAD synthetase activity was determined in a reaction mixture containing 30 mmol/L Tris-HC1, pH 7.4, 60 mmol/L KCl, 5.0 mmol/L L-glutamine, 2.0 mmol/L MgCI,, 1.O mmol/L NAAD, 2.0 mmol/L ATP, and 0.4 to 0.8 units (1 unit = I nmol/h) of NAD synthetase in a total volume of 100 pL. An identical reaction mixture lacking L-glutamine was used as the blank. The reaction was initiated by adding enzyme preparation and then incubating in a 37OC water bath for 30 minutes. The reaction was stopped by immersing the reaction mixture in a boiling water bath for 60 seconds. After cooling at 2 to 4C, coagulated protein was removed by centrifugation (Microcentrifuge model 235A, Fisher Scientific, Tustin, CA) for 30 seconds. A 20-pL aliquot of the resulting supernatant was used to measure NAD formation using our modificati~n~ the enzymatic cycling assay of Bernofsky and of Swan. NAD synthetase activity was calculated by subtracting the NAD content of the glutamine-deficient blanks from the NAD content of the complete reaction mixtures.
Table 1. Partial Purification of NAD Synthetase From Human
Erythrocytes Total Activity Yield Fraction Stroma-free hemolysate DEAE-cellulose Ammonium sulfate and dialysis
(gmol/h)

Molecular weight of NAD synthetase. Both the partially purified preparation and the stroma-free hemolysate were used to determine the molecular weight of NAD synthetase. All manipulations were performed at 20 to 25OC. Either 100 pL of the partially purified enzyme or 500 pL of stroma-free hemolysate were loaded onto a Sephadex G-200 column (1.6 x 32 cm) and then eluted at a rate of 0.24 mL/min with NAD synthetase buffer. The eluate was collected in 1.0-mL fractions and assayed for NAD synthetase activity. Molecular weight standards used were bovine serum albumin (66 Kd), yeast alcohol dehydrogenase (150 Kd), sweet potato &amylase (200 Kd), and horse apoferritin (443 Kd). The void volume was determined using Blue Dextran (2,000 Kd). Regulation and kinetics of NAD synthetase. The MichaelisMenten constant (Km) was determined for each substrate by varying the concentration of the substrate being tested while maintaining the other cosubstrates at their saturating concentrations. Kms and their standard deviations were calculated using a BASIC computer program based on the method of Wilkinson.I6The effect of lead and zinc on NAD synthetase activity was tested by using the acetate salt of Pb2+and the sulfate salt of Zn2+.To ensure that the anion of the metal in question did not affect NAD synthetase activity, we tested the effect of the sodium salt of the latter anions. Neither sodium acetate or sodium sulfate affected NAD synthetase activity. Protein determination. Protein concentration was determined with the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Richmond, CA) using bovine serum albumin as standard. Effect of lead exposure on NAD synthetase activity in intact RBC. Intact RBC were exposed to lead using a modification of the method of Paglia et al. Freshly obtained RBC (final packed cell volume = 20%) were incubated in a mixture containing 150 mmol/L NaCI, 1.0 mmol/L D-glucose, and either 50 pmol/L Pb (acetate), or 100 pmol/L Na (acetate) at 37OC for 30 minutes. RBC were then washed twice with at least 50 vol 150 mmol/L NaCl at 23OC using centrifugation. RBC were hemolyzed by dilution with 3 vol distilled water, and the resulting hemolysate was used to measure NAD synthetase activity, glucose-6-phosphate dehydrogenase (G6PD) activity, and pyrimidine 5-nucleotidase (PSN) activity. G6PD activity was determined as described by Beutler, and P5N activity was determined using the continuous spectrophotometric method of Zerez and Tanaka,18 with uridine monophosphate as substrate.
RESULTS

Stability of NAD Synthetase Activity

(%I
100 120

Specific Activity Purification (pmollh mgprotein) (fold)

1.12 1.35 0.33

0.00034 0.23 1.04

1 680 3,100

29

In crude hemolysate, N A D synthetase activity was quite labile, with nearly complete loss of activity after 2 4 hours at 4OC. After attempting a number of temperatures to improve enzyme stability, we found that room temperature (20 to 25OC) provided minimal loss of activity. We also found that the presence of 100 mmol/L KCl and 1.0 mmol/L E D T A increased enzyme stability dramatically. Interestingly, none of the enzymes substrates including NAAD, glutamine, or ATP, were found to have a n y stabilizing effects. N A D synthetase in crude hemolysate kept at room temperature, after dialysis against 100 mmol/L KCl, 1.0 mmol/L EDTA, and 30 mmol/L Tris-HC1, p H 7.4, had a half-life of about 10 days. T h e partially purified enzyme was considerably more stable under these conditions, with little loss in activity after 1 month.

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1578

ZEREZ, WONG, AND TANAKA

Partial Purification of NAD Synthetase

A summary of our N A D synthetase purification procedure is shown in Table 1. Because the enzyme constitutes a relatively small proportion of the nonhemoglobin protein in RBC, a large sample of hemolysate could be added to the DEAE-cellulose column a t 0.1 mol/L KC1 without interfering with N A D synthetase binding. As a result, a 680-fold purification was achieved when the enzyme was eluted with buffer containing 0.20 mol/L KCI. The greater than 100% yield after the first step was reproducible and may be caused by the removal of inhibitory factors, such as ZnZ+,from crude hemolysate. Further purification of N A D synthetase was obtained using ammonium sulfate fractionation. N A D synthetase precipitated a t 30% ammonium sulfate saturation. Because few other proteins are salted out a t this saturation, an additional 4.6-fold purification resulted, giving an overall 3,100-fold purification. This degree of purification did not result in a homogenous preparation of NAD synthetase. However, theenzyme was sufficiently pure to allow a test of potential regulatory metabolites without interference from H b and endogenous RBC metabolites. NAD Synthetase Molecular Weight (mol wt) and pH Optimum The mol wt of N A D synthetase was estimated using gel permeation chromatography on a Sephadex G-200 column. Using stroma-free hemolysate from three normal individuals, we obtained an apparent mol wt of 483 f 5 Kd (mean -c one SD) for N A D synthetase (Fig 1). Using the 3,100-fold purified preparation of N A D synthetase, we obtained an apparent mol wt of 480 Kd. Optimal NAD synthetase activity occurred a t the relatively broad pH range of 6.8 to 7.4 (Fig 2). NAD Synthetase Kinetic Constants Kinetic studies showed that saturation curves for all substrates were hyperbolic. The Km values for Mg2+, K',
I
I 1 1 1 1

D 2

70 .
PH

8.0

90 .

Fig 2. Effect of pH on partially purified NAD synthetase. Bis-Tris-HCI was used from pH 5.9 to 7.1 (0)and Tris-HCI was used from pH 7.1 to 8.9 (0).

0.3t \
I-

.\

ATP, NAAD, and glutamine are listed in Table 2. The Km for ammonia was 64.2 mmol/L (Table 2). To determine whether in vivo N A D synthetase activity is regulated by the availability of each of these substrates, we have also listed the intraerythrocytic or plasma concentrations of these substrates (Table 2). The intraerythrocytic concentration of NAAD has not been reported. Previous studies in this laboratory, in which erythrocytes were incubated with I4Cnicotinic acid, glucose, inorganic phosphate, and glutamine for 20 hours under conditions that lead to substantial increases in N A D content as a result of N A D synthesis, indicate that I4C-NAAD concentration is approximately 0.04 pmol/mL packed RBC.'' Under the same conditions of incubation, the concentration of I4C-NAD reaches 0.40 pmol/mL packed RBC." In freshly obtained normal erythrocytes, the concentration of total N A D (ie, NAD+ + NADH) rarely exceeds 0.1 pmol/mL packed RBC.19 Assuming that the in vitro ratio of NAAD to N A D is maintained in vivo, the NAAD concentration obtained from our in vitro labeling experiment would be an upper limit for the in vivo NAAD concentration in human erythrocytes. Thus, intraerythrocytic NAAD concentration is probably substantially less than 0.04 mmol/L (Table 2).

Regulation o NAD Synthetase f

A number of compounds were tested to determine whether they regulate N A D synthetase activity. The partially purified enzyme was not affected by the following pyridine nucleotide metabolites: nicotinic acid, nicotinic acid mononuTable 2. Kms of Partially Purified NAD Synthetase
lnnaerythrocytic Conceneation(mmd/Ll

Substate
~

Km (mmol/Ll

Reference for lntraerythrocvtic Concentration

MOLECULAR WEIGHT (Kd) Fig 1. Determination of the mol wt of NAD synthetase using Sephadex 6-200 chromatography. Mol wt standards used were bovine serum albumin (66 Kdl. yeast alcohol dehydrogenase (150 Kd). sweet potato B-amylase (200 Kd), and horse apoferritin 1443 Kd). NAD synthetase is indicated by the arrow.

NAAD ATP Mgzc Glutamine K+ NH,'

0.108 k 0.02% 0.154 2 0.027 1.36 & 0.31 2.17 k 0.42 8.32 f 1.16 64.2 & 10.5

<0.04 1.4 3.0 0.33 93 0.045

This study 20 20 20

20
26,27

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NAD SYNTHETASE AND LEAD EXPOSURE 1579

cleotide, nicotinamide mononucleotide,NADP, and NADPH. NAD synthetase was also unaffected by inorganic phosphate, ADP, adenosine monophosphate, flavin adenine dinucleotide, thiamin pyrophosphate, pyridoxal phosphate, lactate, alanine, 2,3-diphosphoglycerate, and either reduced or oxidized glutathione. However, slight inhibition occurred with inorganic pyrophosphate (PP,), pyruvate, 2-phosphoglyceric acid (ZPG), and phosphoenolpyruvate(PEP) (Table 3). The intraerythrocytic concentrations of pyruvate, PEP, and 2-PG are 53, 12, and 7.3 pmol/L, respectively;" whereas the intraerythrocytic concentration of PPi is < 30 pmol/L."
Efect o Lead and Zinc on NAD Synthetase Activity f

I I I I I I

I I IIII

Because the activity of NAD synthetase was stabilized by EDTA, we examined the effects of various divalent cations on NAD synthetase activity to uncover potential inhibitors of this enzyme. We found that NAD synthetase was inhibited by Pb2+ and Zn2+ ions. Concentrations of Pb2+ and Zn2+ necessary for 50% inhibition (Io5) of NAD synthetase activity were 1.3 and 2.0 pmol/L, respectively (Fig 3). Total inhibition of NAD synthetase activity was achieved at 5 pmol/L Pb2+and 10 pmol/L Zn2+(Fig 3).
Eflect of Lead Exposure on NAD Synthetase Activity in Intact RBC

Metol Cotion Concentroiion

(#MI

Fig 3. Effect of lead (0) and zinc (0)on partially purified NAD synthetase. The concentration of each metal cation necessary for 50% inhibition of activity under optimal assay conditions is indicated by the arrows.

The marked inhibtion of NAD synthetase activity by lead prompted an investigation of the effect of lead exposure on enzyme activity in intact RBC in vitro. A brief exposure of intact RBC to lead acetate followed by rigorous washing to remove lead indicated that GBPD, an enzyme that is insensitive to lead, was not affected by lead exposure (Table 4). Under the same conditions, P5N, an enzyme that is an accepted indicator of lead e x p ~ s u r e , ' ~ - * ~ ~ ~ ~ to 50% had a 30% decrease in activity after lead exposure (Table 4). In contrast, NAD synthetase activity was almost totally abolished by lead exposure under these conditions (Table 4).
Mechanism of Lead Inactivation o NAD Synthetase f Activity

activity (Table 5). However, exposure of intact RBC to higher lead concentrations (25 and 50 pmol/L) resulted in partial restoration of NAD synthetase activity after dialysis (Table 5). This suggests that there is a threshold of lead concentration above which NAD synthetase inactivation becomes irreversible. Similarly, dialysis of lead-treated hemolysate resulted in partial restoration of NAD synthetase activity (Table 5). The presence of reduced glutathione (GSH) during lead exposure of hemolysate did not prevent NAD synthetase inactivation (Table 5). However, the presence of EDTA during lead exposure resulted in nearly total preservation of NAD synthetase activity (Table 5).
NAD Synthetase Activity in a Patient With Lead Overburden

The marked reduction of NAD synthetase activity after lead exposure suggested that lead may act by inactivating the enzyme, and prompted studies to determine the mechanism of lead inactivation. Exposure of intact RBC to 10 pmol/L lead under the conditions described above, followed by exhaustive dialysis against the EDTA-containing NAD synthetase buffer resulted in full restoration of NAD synthetase
Table 3. Inhibition o Partially Purified NAD Synthetase f by Four Metabolites
Reference for

We also examined NAD synthetase activity in RBC from three patients with increased blood lead level. Mean NAD synthetase activity was higher in normal female volunteers than in normal male volunteers (Table 6). All patients with increased blood lead concentration had a decrease in NAD synthetase activity relative to their gender control group. NAD synthetase @Snity in the ptients with lead overburden appeared to vary inversely with blood lead level (Table 6). Furthermm;exhaustive dialysis of these patients' hemolysates against EDTA-containing b a e r increased NAD synthetase activity to levels higher than those in undialyzed hemolysates from normal males and females (Table 6).
Table 4. Effect of Lead Exposure on NAD Synthetase Activity in Intact Erythrocytes
Exp

Additions to Intact RBC+ Na(acetate) Pb(acetate1, Na(acetate) Pb(acetate),

GGPD
Lurplol/min

. g Hb)

P5N
(wol/h
1

NAD Synthetase
g HB)

(pmollh g Hb) 0.788 0.022 0.690 0.008

Relative Compound
No additions 2.0 mmd/L pyruvate 0.10 mmol/L PEP 1.O mmdlL 2-PG 1 .O mmolIL PP,
Activity

IntraerVthrocytic lntraerythrocytic Concentration (mmolR) Concentration

1 2

100 75 88 79 65

0.053 0.012 0.007 <0.03

20 20 20 21

7.73 7.45 9.54 10.3

11.1 5.65 9.35 6.66

"RBC were incubated with either 50 amol/L Pb(acetate), or 100 pmollL Na(acetate) at 37% for 3 0 minutes, and then washed rigorously to remove the lead (see Materialsand Methods).

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1580
Table 5. Mechanism of Lead Inactivation of N A D Synthetase
NAD Synthetase Activity (pmol/h g Hb)
Intact RBC.

ZEREZ, WONG. AND TANAKA

Hemolysatat
PB(Ac),

Na(Ac) Conditions
100 pmol/L
50 pmollL

Na(Acl
10 pmollL
10 umol/L

Pb(Ac), 5.0 umol/L

~~~~

25 pmol/L

Control Dialyzed$ PIUS GSH pius EDTA~~

0.754 0.782

0.148

0.613

0 0.767 -

0.702 0.746 0.791 0.851

0 0.263 0.044 0.680

Abbreviation: Ac, acetate. Intact R8C were incubated at 37C for 60 minutes with either Na(acetate1 or Pb(acetate),. washed rigorously, hemolyzed, and assayed for NAD synthetase activity as described (see Materials and Methods). tHemolysates (final Hb concentration = 8 g/dL) were treated with either Na(acetate1or Pb(acetate),, incubated at 37T for 60 minutes, and assayed for NAD synthetase activity as described (see Materials and Methods). $Hemolysates were dialyzed overnight against 1.000 EDTA-containing NAD synthetase buffer (see Materials and Methods) at 23OC. vol Same conditions as in footnote t except that hemolysateswere treated with 2.0 mmol/L reduced glutatione (GSH) in addition to either Na(acetate)or Pb(acetate1,. IlSame conditions as in footnote t except that hemolysateswere treated with 1 .O mmol/L EDTA in addition to either Na(acetate) or Pb(acetate),.

DISCUSSION

We have described conditions under which human erythrocyte NAD synthetase activity is sufficiently stable to allow the purification and characterization of this enzyme. Using these conditions, we have partially purified and examined properties of human erythrocyte NAD synthetase, including its mol wt, kinetic constants, and its possible regulation by various metabolites. The Km values for ATP, Mg2+, and K+ were all below their intraerythrocytic concentrations, suggesting that ATP, Mg2+, and K + play no roles in the regulation of human erythrocyte N A D synthetase. The intraerythrocytic concentrations of NAAD and glutamine are less than the Km of N A D synthetase for NAAD and glutamine, respectively. This suggests that the availability of NAAD and glutamine regulate NAD synthetase activity in vivo. The rate-limiting nature of glutamine is consistent with the findings of Preiss and Handler that glutamine supply is a rate limiting factor in human erythrocyte N A D synthesis. We also examined the ability of human NAD synthetase to use ammonia instead of glutamine. Although the enzyme can use ammonia, concentrations required for half-maximal activity are approximatqly 1,000-fold higher than those found in p l a ~ m a . * ~Therefore, glutamine, and not ammo.~ nia, is the physiologic aminQ group donor for human NAD
Table 6. NAD Synthetase Activity inpatients With Lead Overburden
Blood Lead Hemolysate (uddL) ~umol/L) Treatment

Sex Normal Patient 1

NAD Synthetase lumol/h I Hb) I

M (n = 9) <10 F (n = 8) t10 F 34

Patient 2 M Patient 3 F

66 72

t0.48 Untreated 0.849 2 0.190 t0.48 Untreated 1.04 f 0.20 1 6 Untreated . 0.609 Dialyzed 1.40 3.2 Untreated 0.026 Dialyzed 2.56 3.5 Untreated 0.018 Dialyzed 1.49

*Hemolysates were dialyzed overnight against 1,000 vol EDTAcontaining NAD synthetase buffer (see Materials and Methods) at 23C.

synthetase in vivo. This finding is consistent with the observation of Preiss and Handler that more N A D is synthesized in intact human erythrocytes in the presence of glutamine than in the presence of ammonia. Thus, the human enzyme, like the yeast enzyme,2 uses glutamine more effectively than ammonia. In contrast, bacterial N A D synthetases from E coli3and S typhimurium4 use ammonia preferentially. Yeast NAD synthetase is an oligomer of two different subunits with a total mol wt of 630 Kd. The S typhimurium enzyme is also an oligomer that contains at least two subunit^.^ The apparent mol wt of N A D synthetase which we have obtained (483 Kd) suggests that the human enzyme is smaller than the yeast enzyme. We have examined the effect of a number of metabolites to show possible regulators of N A D synthetase. Of the metabolites tested, slight inhibition was demonstrated only by pyruvate, PEP, 2-PG, and PP,. Because the inhibition of N A D synthetase required concentrations of the latter metabolites that are substantially higher than those found in normal erythrocytes, inhibition by pyruvate, PEP, 2-PG, and PP, was not deemed physiologically significant. These results suggest that the kinetic properties of N A D synthetase cannot account for the impaired rate of N A D synthesis in sickle RBC. N A D synthetase was markedly inhibited by Pb2+ and Zn+. This property of the enzyme may be responsible for the stabilizing effect of EDTA. Furthermore, it is possible that the rapid decay of NAD synthetase activity in freshly isolated intact RBC may be due, in part, to its inactivation by Zn2+, which is present in RBC at a concentration of 150 pmol/L. In fresh RBC, Zn2+,like other divalent cations, is mostly bound to anionic metabolites, such as nucleotides, which results in low concentrations of free Zn2+ and therefore readily detectable N A D synthetase activity. However, as the RBC suspension ages, the concentration of nucleotides and other anionic metabolites decreases, resulting in higher concentrations of free Zn2+.This may be the explanation for the decrease in NAD synthetase activity in stored RBC suspensions and for the lack of N A D synthetase activity in outdated blood bank units. The inhibition of NAD synthetase by lead suggested that

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NAD SYNTHETASE AND LEAD EXPOSURE
1581

enzyme activity may be an indicator of lead exposure in intact RBC. This was confirmed in our in vitro experiments (Table 4). More importantly, under the conditions of lead exposure that we used, P5N activity was only reduced by 30% to 50%, whereas N A D synthetase activity was almost totally abolished. This suggests that N A D synthetase activity is a more sensitive indicator of lead exposure than P5N activity, an accepted indicator of lead e x p ~ s u r e . , This ~ ~ ~~conclusion is supported by our finding of an N A D synthetase Io,,for Pb2+ of 1.3 pmol/L, compared with the P5N I , , for Pb2+ of approximately 10 pmol/L, as estimated from the data of Paglia et al. The ability of EDTA to nearly prevent lead-inactivation in hemolysate when EDTA is added concomitantly with lead suggests that lead inactivates N A D synthetase by binding to the enzyme. However, the failure of G S H to prevent lead-inactivation of NAD synthetase in hemolysate suggests that inactivation is not caused by the binding of lead to sulfhydryl groups on the enzyme. The classical enzymatic indicator of lead exposure is 6-aminolevulinic acid dehydratase (6-ALAD; E C 4.2.1.24).28 The inhibition of 6-ALAD by lead is responsible for the accumulation of plasma 6-aminolevulinic acid (&ALA) and the resulting increased urinary excretion of 6-ALA in patients with lead poisoning. Because the inactivation of 6-ALAD by lead is reversed by sulfhydryl reagents such as GSH29or dithi~threitol,~ has been suggested that leadit inactivation of 6-ALAD is mediated by the direct binding of lead to sulfhydryl groups on the enzyme. This is in contrast to the lead-incativation of N A D synthetase that appears not to involve sulfhydryl groups. The 6-ALAD Io,,for Pb2+can be

estimated from the in vivo data of Nakao et al to be 30 to 40 Pb2+/dL blood (ie, 1.4 to 1.9 pmol/L). We did not have the opportunity to determine an in vivo N A D synthetase I , , for Pb+ because of the paucity of lead-exposure patients in Southern California. However, if in vivo and in vitro I,,, values can be compared directly, our in vitro N A D synthetase Io,5 value of 1.3 pmol/L is similar to the in vivo 6-ALAD Io,, of 1.4 to 1.9 pmol/L. Thus, N A D synthetase and 6-ALAD may be equally sensitive to lead-inactivation. To provide evidence that N A D synthetase activity i w l s o a sensitive indicator of lead exposure in vivo, we examined N A D synthetase activity in hemolysate from patients with lead overburden. All patients with lead overburden had markedly decreased N A D synthetase activity that could be fully restored on dialysis (Table 6 ) .N A D synthetase activity restoration in these patients hemolysates suggests that both in vivo and in vitro lead-inactivation can be reversed by dialysis. Taken together, these results suggest that N A D synthetase activity is a sensitive indicator of lead exposure both in vitro and in vivo. Because nearly all metabolic pathways require N A D or N A D P as coenzymes, leadinactivation of N A D synthetase may be important in the pathogenesis of some of the clinical manifestations of lead poisoning.
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ACKNOWLEDGMENT

We are grateful to Sandra J. Lee for her expert technical assistance. We thank Drs Sergio Piomelli and Donald Williams for their help in obtaining b l o d samples from patients with lead overburden.

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