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1990 75: 1576-1582

CR Zerez, MD Wong and KR Tanaka

 enzyme activity is a sensitive indicator of lead exposuredinucleotide synthetase from human erythrocytes: evidence thatPartial purification and properties of nicotinamide adenine

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Partial Purification and Properties of Nicotinamide Adenine D inucleotideSynth etase From Human Erythrocytes: Evidence That Enzyme Activity I s a

Sensitive Indicator of Lead Exposure

By Charles R. Zerez, Mitche ll 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 extremekyunstable in crude hemolysate, with completdoss of activ-

it y occurring after 24 hours at 4°C. However, maintenance

of crude hemolysate at 20 t o 25°C in the presence of EDTA

and KCI increased NAD synthetase stability substantially

(half-life = 10 days). Using these conditions, NAD syn-

thetase was purified 3,100-fold with a 29% yield using

DEAE-cellulose column chromatography, ammonium sul-

fate fractionation, and dialysis. The apparent Michaelis-

Menten constants fo r 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, respec-

tively. The pH optimum ranged between 6.8 and 7.4, and

the molecular weight was estimated te be 483 f 5 Kd. The

ICOTINAMIDE adenine dinucleotide (NAD) syn-N thetase (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 yeast2 and

Escherich ia coli ,' and studied in cell-free crude extracts ofSalmoaella t y p h i m u r i u m ! 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 sub-stantial accumulation of NAD due to synthe~is .'.~ hepresence of the intermediates of NAD 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

Center. Unive rsityof California at LosAngeles 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. ) ro m the National Institutes

of Heahh. and grants from the Sickle Cell Disease ResearchFoundation of Los Angeles and the Research and EducationInstitute, Inc. Harbor-UCL A Medical Center (to C.R .Z .).

Presented at the Annual Meeting of the American Society ofHemaiology. Washington, DC. December 7. I987 (Blood 70:57a.

1987).Address reprint requests to Charles R. Zerez, PhD, Harbor-

UCLA Medical Center, 1124 West Carson St , CI-1 2, Torrance. CA

90502.The publication costs of this article were defraye d'inpart by page

charge payment. This article must therefore be hereby marked"advertisement" in accordance with 18 U.S .C. ection 1734 solely to

indicate thisfact .0 1990 by The American Society of Hematology.

000 6-@ 71/9 0/ 7507-001 2$3.0O/O

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 intactred blood cells wi th 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% t o 50% under these conditions.

More importantly, patients wi th lead overburden(34 o 72

pg Pb2+/dL blood) dl had markedly decreased NAD syn-

thetase activity. These data together with other results

suggest that erythrocyte NAD. synthetase activity is a

sensitive ndicator of lead exposure in humans..

0 1990b y TheA m e r i c a n S o c i e t y of H e m a t o l o g y .

present in human RBC. However, the classical studies of

Preiss and Handler' demonstrated that when cell-free hemoly-

sates 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 capabled 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 findingsof 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 bedetermined. 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 canaccount 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 vit roand in vivo.

MATERIALS AND METHODS

M a t e r i a l s

Sephadex G-200 was purchased from Pharmacia, Inc, Piscat-

away, 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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NA D S Y NTHE TAS E AN D LE AD E XP O S URE 1577

Molecular weight of NA D synthetase. Both the partially puri-

fied preparation and the stroma-free hemolysate were used to

determine the molecular weight of NAD synthetase. All manipula-

tions were performed a t 20 to 25OC. Either 100 pL of the partially

purified enzyme o r 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 wascollected in 1.0-mL fractions and assayed for NAD synthetase

activity. Molecular weight standards used were bovine serum albu-

min (66 Kd), yeast alcohol dehydrogenase (150 Kd), sweet potato

&amylase (200 Kd), and horse apoferritin (443 Kd). T he void

volume was determined u sing Blue Dextran (2,000 K d).

The Michaelis-

Menten constant (Km) was determined for each substrate by

varying the concentration of the substrate being tested while

maintaining the other cosubstrates at their saturating concentra-

tions. 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 Pb 2+and the sulfate salt of Zn2 +.To ensure

tha t 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 NA D synthetase

activity.

Protein concentration was determined

with the Bio-Rad protein assay reagent (Bio-Rad Laboratories,

Richmond, CA) using bovine serum albumin a s standard.

Effect of lead exposure on NAD synthetase activity in intact

RBC. Intact RBC were exposed to lead using a m odification 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 pmo l/L N a (ac etate) a t 37OC for 30 minutes. RBC

were then washed twice with at least 50 vol 150 mmol/L NaC l at

23OC using centrifugation. RBC were hemo lyzed 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.

G6P D activity was determined as described by Beutler,” and P 5N

activity was determined using the continuous spectrophotometric

method of Zere z and T anaka,18 with uridine mo nophosphate as

substrate.

Regulation and kinetics of NAD synthetase.

Protein determination.

Methods

After obtaining informed consent,

blood was obtained by routine venipuncture from no rmal individuals

using heparin (15 U /m L 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 B e~ t1 er .l ~ BC w erewashed three times with 0.15 mo l/L NaCI.

Unless otherwise indicated, all

NA D synthetase purification procedures were performed a t 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 x6.0 cm) w as equilibrated w ith 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. Afte r

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 K Cl , 1.0 mmol/L EDTA , and 30 mmol/L

Tris-HC 1, pH 7.4, which resulted in the elution of the remaining Hb.

NA D synth etase 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 fo r

20 minutes. The small pellet obtained was resuspended in 1.0 mL

NA D synthetase buffer and dialyzed for 12 to 15 hours against 250

volumes of th e same buffer. This two-step process resulted in a 29%

yield and a 3,100-fold purification. The resulting partially purified

preparation of NA D syntheta se could be stored at 20 t o 25OC for 1

month with little loss in activity. A summary of the purification is

shown in Table 1.NAD synthetase activity was deter-

mined 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 NA D synthetase in a total volume of 1 00 pL.

An identical reaction mixture lacking L-glutamine was used as the

blank. The reaction was initiated by adding enzyme preparation an d

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 4“C, coagulated protein

was removed by centrifugation (Microcentrifuge model 235A,

Fisher Scientific, Tustin, C A) for 30 seconds. A 20-pL aliquot of the

resulting supernatant was used to measure NAD formation using

our mod i f i c a t i~n ‘~f th e enzym atic cycling assay of Bernofsky and

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.

Isolation o j erythrocytes.

NA D synthetase purification.

NAD synthetase assay.

Table 1. Partial Purification of NAD S y n t h e t a s e From Human

E r y t h r o c y t e s

Total SpecificActivity Yield Activity Purification

Fraction (gmol/h) (% I (pmollh .mgprotein) (fold)

Stroma-free hemolysate 1.12 100 0.00034 1

DEAE-cellulose 1.35 120 0.23 680

Ammonium sulfate and

dialysis 0.33 29 1.04 3,100

RESULTS

Stability of NA D Synthetase Activity

In c r u d e h em o l ys a te , N A D synthetase activity was quite

l a b il e , w i th ne a r ly c omple te loss of a c t iv i t y a f t e r 2 4 h o u r s at

4OC. After a t t e m p t i n g a n u m b e r of temperatures to improveenzyme s tabi l i ty , we found that r o o m t e m p e r a t u r e ( 2 0 to

25OC) provided minimal loss of activity. We also found t h a t

th e presence of 100mmol/L K C l a n d 1.0 m m o l / L E D T A

increased e nz ym e s t a b i l i t y d ra ma t i c a l ly . Interestingly, none

of t h e enzyme’s s ubs t ra t e s inc lud ing NAAD, g lu ta mine , or

A T P , w e r e f o u n d to ha ve a ny s t a b i l i z ing effects. N A D

s yn the ta s e in c rude he molys a te ke p t at room t e m p e r a t u r e ,

a f te r d ia lys is aga ins t 100m m o l / L K C l , 1.0m m o l / L EDTA,

a n d 30mm ol /L Tr i s-HC 1 , p H 7.4, ha d a half- l ife of abou t 10

da ys . T he p a r t i a l ly pu r if i ed e nz yme wa s c ons ide ra b ly more

s tabl e under these condi t ions , wi th l i t t le loss in a c t iv i ty a f t e r

1m o n t h .

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1578

Partial Purification of N A D Synthetase

A su mmary 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 mo l/L KC1 without interfer-

ing 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+, ro m

crude hemolysate. Further purification of NAD synthetase

was obtained using ammonium sulfate fractionation. NAD

synthetase precipitated at 30% ammonium sulfate satura-

tion. Because few other proteins are salted out at this

satu ratio n, an ad dition al 4.6-fold purification resulted, giving

an overall 3,100-fold purification. T his degree of purification

did not result in a homogenous preparation of NAD syn-

thetase. However, thee nzy me was sufficiently pu re to allow a

test of potential regulatory metabolites without interferencefrom H b and endogenous R BC metabolites.

N A D Synthetase Molecular Weight (mol wt) and pH

Opt imum

The mol wt of NAD synthetase was estimated using gel

permeation chromatography on a Sephadex G-200 column.

Using stroma-free hemolysate from th ree normal individu-

als, we obtained an app arent 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 p H ran ge of 6.8 to

7.4 (Fig 2).

N A D Synthetase Kinetic Constants

Kinetic studies showed that saturation curves for all

substrates were hyperbolic. T he K m values for Mg2+,K',

ZEREZ, WONG, AND TANAKA

I I 1 1 1 1 I I I I I I I I.\0.3t \ 1

I- \

D I I I2 7.0 8.0 9.0

PH

Fig 2. Effect of pH on partially purified NAD synthetase.

Bis-Tris-HCI was used from pH 5.9 to 7.1 (0 ) nd Tris-HCI was

used from pH 7.1 to 8.9 (0) .

ATP, N AA D, and g lutamine are l isted in Ta b le 2. T h e K m

for ammonia was 64.2 mmol/L (Table 2). To determine

whether in vivo NA D synthetase activity is regulated by th e

availability of each of these sub strates, we have also listed th e

intraerythrocytic or plasma concentrations of these sub-

strates (Table 2). The intraerythrocytic concentration ofNAAD has not been reported. Previous studies in this

laboratory, in which erythrocytes were incubated with I4C-

nicotinic acid, glucose, inorganic phosphate, and glutamine

for 20 hours under conditions that lead to substantial

increases in NAD content as a result of N A D synthesis,

indicate that I4C-NAAD concentration is approximately

0.04 pmol/mL packed RBC.' ' Under th e same conditions of

incubation, the concentration of I4C-NAD reaches 0.40

pmol/m L packed RBC." In freshly obtained normal erythro-cytes, the concentration of total N A D (ie, N A D + + N A D H )

rarely exceeds 0.1 pmol/ mL packed RBC.19 Assuming t ha t

the in vitro ratio of NA AD 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, intraerythro-

cytic NAAD concentration is probably substantially less

than 0.04 mmol/L (Table 2) .

Regulationof N A D Synthetase

A number of compounds were tested to determin e whether

they regulate NAD synthetase activity. The partially puri-

fied enzyme was not affected by the following pyridinenucleotide metabolites: nicotinic acid, nicotinic acid mononu-

Table 2. Kms of Partially Purified NAD Synthetase

Referencefor

Substate Km (mmol/Ll Conceneation(mmd/Ll Concentration

lnnaerythrocytic lntraerythrocvtic

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 indicatedby the arrow.

~

NAAD 0.108 k 0.02% <0.04 This study

ATP 0.154 2 0.027 1.4 20

Glutamine 2.17 k 0.42 0.33 20

K + 8.32 f 1.16 93 20

NH,' 64.2 & 10.5 0.045 26,27

Mgzc 1.36 & 0.31 3.0 20

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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 dinucle-

otide, thiamin pyrophosphate, pyridoxal phosphate, lactate,

alanine, 2,3-diphosphoglycerate, and either reduced or oxi-

dized glutathione. However, slight inhibition occurred with

inorganic pyrophosphate (PP,), pyruvate, 2-phosphoglycericacid (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."

Ef e c t o Lead and Zinc on NA D Synthetase A ctivity

Because the activity of NAD synthetase was stabilized by

EDTA, we examined the effects of various divalent cationson 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 10pmol/L Zn2+ Fig 3).

Eflect of Lead Exposure on NA D Synthetase Activity in

Intact RBC

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 insensi-

tive to lead, was not affected by lead exposure (Table 4).

Under the same conditions, P5N, an enzyme that is anaccepted indicator of lead e x p ~ s u r e , ' ~ - * ~ ~ ~ ~ad a 30% to 50%

decrease in activity after lead exposure (Table 4). In con-

trast, NAD synthetase activity was almost totally abolished

by lead exposure under these conditions (Table 4).

Mechanism of Lead Inactivation of NA D Synthetase

Activity

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 10pmol/L

lead under the conditions described above, followed by

exhaustive dialysis against the EDTA-containing NAD syn-thetase buffer resulted in full restoration of NAD synthetase

Table 3. Inhibitionof Partially Purified NAD Synthetase

by Four Metabolites

Reference for

Compound Activity Concentration (mmolR) ConcentrationRelative IntraerVthrocytic lntraerythrocytic

- -o additions 100

2.0 mmd/L pyruvate 75 0.0 53 20

0. 10 mmol/L PEP 88 0.012 20

1 O mmdlL 2-PG 79 0.007 2 0

1 O mmolIL PP, 65 <0.03 21

I I I I I I I I I I I I I I I I I I I I I

Metol Cotion Concentroiion (#MI

Fig 3. Effect of lead (0 ) nd zinc (0 ) n partially purified NAD

synthetase. The concentration of each metal cation necessaryfor

50% inhibition of activity under optimal assay conditions is

indicated by the arrows.

activity (Table 5) . However, exposure of intact RBC tohigher 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 he-

molysate 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 pres-

ence of EDTA during lead exposure resulted in nearly total

preservation of NAD synthetase activity (Table 5) .

NA D Synthetase Activity in a Patient With LeadOverburden

We also examined NAD synthetase activity in RBC from

three patients with increased blood lead level. Mean NADsynthetase 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 pt ien ts with lead overbur-

den appeared to vary inversely with blood lead level (Table

6). Furthermm;exhaustive dialysis of these patients' hemoly-

sates against EDTA-containing b a e r increased NAD syn-

thetase 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

Additions to GGPD P5N NAD SynthetaseExp Intact RBC+ Lurplol/min . g Hb) (wol /h 1 g HB) (pmollh . g Hb)

1 Na(acetate) 7.73 11.1 0.788

2 Na(acetate) 9.54 9.35 0.690

Pb(acetate), 10.3 6.66 0.008

"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).

Pb(acetate1, 7.45 5.65 0.0 22

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1580 ZEREZ, WONG. AND TANAKA

Table 5. Mechanism of Lead Inactivation of NA D Synthetase

NAD Synthetase Activity (pmol/h . Hb)

Intact RBC. Hemolysatat

Na(Ac) PB(Ac), Na(Acl Pb(Ac),

Conditions 100 pmol/L 50 pmollL 25 pmol/L 10 pmollL 10 umol/L 5.0 umol/L~~~~

Control 0.754 0 0 0 0.702 0

Dialyzed$ 0.782 0.148 0.6 3 0.767 0.746 0.263

0.791 0.044IUS GSH§ - - - -0.85 0.680ius EDTA~~ - - - -

Abbreviation: Ac, a cetate.

‘Intact R8C were incubated at 37°C or 60minutes with either Na(acetate1 or Pb(acetate),. washed rigorously, hemolyzed, and assayed for N AD

tHemolysates (final Hb concentration = 8 /dL) were trea ted with either Na(acetate1 or Pb(acetate),, incubated at 37T or 60minutes, and assayed

$Hemolysates w ere dialyzed overnight against 1.000 ol EDTA -containing NAD synthetase buffer (see Mate rials and Me thods) at 23OC.

§Same conditions as in footnote t except that hemolysateswere treated with 2.0mmol/L reduced glutatione (GSH) in addition to either Na(ac etate) or

IlSame conditions as in fo otn ote t except that hemolysates were treated with 1 O mmol/L EDTA in addition to either Na (acetate ) or Pb(acetate),.

synthetase activity as described (see Materials and Methods).

for N AD s ynthetase activity as described (see Materials and M ethod s).

Pb(acetate1,.

DISCUSSIONW e have described conditions under which hu man erythro-

cyte N A D synth etase activity is sufficiently stable to allow

the purification and characterization of this enzyme. Using

these conditions, we have partially purified and examined

properties of h uman erythrocyte N A D synthetase, including

its mol wt, kinetic constants, and its possible regulation by

various m etabolites.

The Km values for ATP, Mg2+,and K+ were all below

their intraeryth rocytic concentrations,” suggesting tha t AT P,

M g2+ , and K + play no roles in the regulation of hum an

erythrocyte NA D synthetase. Th e intraerythrocytic concen-

trations of N AA D and glutamine are less than th e Km of

NAD synthetase for NAAD and glutamine, respectively.This suggests that th e availability of N AA D and glutamine

regulate N A D synthetase activity in vivo. Th e rate-limiting

natu re of gluta min e is consistent with t he findings of Preiss

and Handler’ t ha t glutam ine supply is a ra te limiting factor

in human erythrocyte N A D synthesis.

We also examined the abil i ty of human N AD synthetase

to use ammonia instead of glutamine. Although the enzyme

can use am monia, concentrations required for half-maximal

activity are approximatqly 1,000-fold higher than those

f o u n d i n p l a~m a .* ~ .~ ’herefore, glutamine, and not ammo-

nia, is the physiologic aminQgroup donor for human NAD

Table 6. NA D Synthetase Act ivity inpat ients

With Lead Overburden

Hemolysate NA D SynthetaseSex (uddL) ~umol/L) Treatment lumol/h . IHb)

Blood Lead

Normal M (n = 9) <10 t0.48 Untreated 0.849 0.190

Patient 1 F 34 1.6 Untreated 0.609

Patient2 M 66 3.2 Untreated 0.026

Patient 3 F 72 3.5 Untreated 0.018

F (n = 8) t10 t0.48 Untreated 1.04f 0.20

Dialyzed’ 1.40

Dialyzed’ 2.56

Dialyzed’ 1.49

*Hemolysates were dialyzed overnight against 1,000 ol EDTA-

containing NAD s ynthetase buffer (see Materials and Methods) at 23°C.

synthetase in vivo. This finding is consistent with t he observa-tion of Preiss and Handler’ t ha t more NA D is synthesized in

intact hum an e rythrocytes in the presence of glutamine than

in the presence of ammonia. Thus , the huma n enzyme, like

the yeast enzyme,2 uses glutam ine more effectively tha n

ammonia. In contrast, bacterial NAD synthetases from E

coli3and S typhimurium4use ammonia preferentially.

Yeast NAD synthetase is an oligomer of two different

subunits with a to tal mol wt of 630 Kd.’ The S typhimurium

enzyme is also an oligomer that contains at least two

subunit^.^ The ap parent mol wt of N A D synthetase which we

have obtained (483 Kd) suggests tha t the hum an enzyme is

smaller than th e yeast enzyme.

We have examined the effect of a num ber of metabolites to

show possible regulators of N A D synthetase. Of the m etabo-

lites tested, slight inhibition was dem onstrate d only by pyru-

vate, PEP, 2-PG, and PP,. Because the inhibition of NAD

synthetase required concentrations of the latter metabolites

that are substantially higher than those found in normal

erythrocytes, inhibition by pyruvate, P EP , 2-PG, and PP,wa s

not deemed physiologically significant. Th ese res ults suggest

that the kinetic properties of NAD synthetase cannot ac-

count for the impaired rate of NA D synthesis in sickle RBC .

N A D synthetase was markedly inhibited by Pb2+ and

Zn’+. This property of the enzym e 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 R BC may be d ue, in part, to its inactivation byZ n 2 + ,which is present in RBC at a concentration of 150

pmol/L.” In fresh RBC , Zn2+, ike other divalent cation s, is

mostly bound to anionic metabolites, such as nucleotides,

which results in low concentrations of free Z n2+ and there-

fore readily detectable N A D synthetase activity. However,

as the R B C suspension ages, the conce ntration of nucleotides

and other anionic metabolites decreases, resulting in higher

concentrations of f ree Zn2+ .This may be the explanation for

the decrease in NAD synthetase activity in stored RBC

suspensions a nd for the lack of N A D synthetase activity in

outdate d blood bank units.’

The inhibition of N A D synthetase by lead suggested th at

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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 tha t we used, P 5N activity was only reduced by

30% to 50%, whereas N A D synthetase activity was almost

totally abolished. T his suggests tha t N A D synthetase activ-

ity is a more sensitive indicator of lead exposure than P5Nact iv it y, an accep ted in di ca to r of l ead e x p ~ s u r e . ” , ~ ~ - ~ ~hi s

conclusion is suppo rted by our finding of an N A D synthetase

Io,, or Pb2+of 1. 3 pmol/L, compared with the P5N I , , fo r

Pb 2 + of approximately 10 pmol/L, as estimated from the

data of Paglia et al.” Th e ability of ED TA to nearly prevent

lead-inactivation in hemolysate when ED TA is add ed concom-

itantly with lead suggests that lead inactivates NAD syn-

thetase by binding to the enzyme. However, the failure of

G S H to prevent lead-inactivation of NA D 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 dehydr atase (6-A LAD; E C 4.2.1 24).2 8The inhibition of 6-ALAD by lead is responsible for the

accumulation of plasma 6-aminolevulinic acid (&A LA) a nd

the resulting increased urinary excretion of 6-ALA in pa-

tients with lead poisoning.” Because the ina ctivation of

6-ALAD by lead is reversed by sulfhydryl reagents such as

G SH 2 9or di th i~thre i to l ,~’t has been suggested that lead-

inactivation of 6-ALA D is mediated by the direct binding of

lead to sulfhydryl groups on the enz yme. This is in contrast to

the lead-incativation of N A D synthetase that app ears not to

involve sulfhydryl groups. Th e 6-ALA D Io,, or Pb2+can be

estimated from th e in vivo data of N aka o et al’’ to be 30 to 40

pg Pb 2+/ dL blood (ie, 1.4 to 1.9 pmol/L). We did not have

the opportunity to determ ine an in vivo NA D synthetase I , ,

for Pb’+ because of the paucity of lead-exp osure patien ts in

Southern California. However, if in vivo and in vitro I,,,

values can be compared directly, our in vitro NAD syn-

thetase Io,5value of 1.3 pmol/L is similar to the in vivo6-ALAD Io,, of 1.4 to 1.9 pmol/L. Thus, NA D synthetase

an d 6-ALAD m ay be equally sensitive to lead-inactivation.

To provide evidence that N A D synthetase activity iw ls 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 NA D synthetase activity that could be

fully restored on dialysis (Tab le 6 ) .NA D synthetase activity

restoration in these patients’ hemolysates suggests th at both

in vivo and in vitro lead-inactivation can be reversed by

dialysis. Taken together, these results suggest that NAD

synthetase activity is a sensitive indicator of lead exposure

both in vitro and in vivo. Because nearly all metabolicpathways require NAD or NADP as coenzymes, lead-

inactivation of NAD synthetase may be important in the

pathogenesis of some of the clinical manifestations of lead

poisoning.

ACKNOWLEDGMENT

We are grateful to Sandra J. Lee for her expert technical

assistance. We thank Drs Sergio Piomelli and Donald W illiams for

their help in obtaining b l o d samples from patients with lead

overburden.

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