BIOCHIMIE, 1975, 57, 325-335.

Human granulocyte 6 phosphogluconate dehydrogenase. Purification by elective elution with NADP +, immunological and kinetic properties.

D o m i n i q u e COTTREAU and P i e r r e BoIVIN. Axel KAHN ( * ~ ) , Anga!a MILANI (**), . loelle ).-IARIE,

UnilO 24 de I'INSERM et UniversitY, Paris Vll, Hdpital Bem,,.ion, 92110 Clichy, France.

(7-2-1975).

Summary. - - Human granulocyte 6 phosphoglueonate dehydrogenase has been totally purified f rom a single patient wi th chronic granulocytic leukaemia.

48 mg of protein, of specific activity 20 IU per mg of protein, have been obtained in the course of three different steps only. The overall yield was 30 p. cent and the purification was 100 folds.

Purified 6 phosphogluconate dehydrogenase was honmgeneous when tested in acry]amide and acrylamide SDS gel elcctrophoresis or in immunodiffusion.

The enzyme was immunological ly identical in red blood cells, blood platelets and normal leukocytes.

The fixation of both substrates,, NADP + and 6 phosphogl.uconate, seemed to proceed through a non ordered mechanism. NADPH was an inhibi tor strictly competit ive with respect to NADP + and non competit ive with respect to 6 phosphogluconate.

2-3 Diphosphoglyccrate seemed to be able to bind on. both the fixation sites of NADP ~ and 6 phosphogluconate. The inhibit ion by ATP was competit ive withe 6 pbosphogluconate and non competitive with NADP +.

6 phosphogluconate dehydrogenase was inactivated by SH reagents and was part ial ly protected against this inactivation by both substrates. Both substrates protected the enzyme again.st thermal inactivation.

The influence of ionic strength, pH and ions have been studied, and the results have been compared to those reported by other authors for erythrocyte enzyme.

I N T R O D U C T I O N .

H u m a n 6 p h o s p h o g l u c o n a t e d e h y d r o g e n a s e (D- g l u c o s e - l - p h o s p h a t e p h o s p h o i r a n s f e r a s e (decar - boxy la t ing ) EC 1.1.1.44) was first c o m p l e t e l y pu r i - f ied f r o m r e d b lood cells by P e a r c e and Rose- m e y e r [1]. S t a r t i ng w i t h about 30 l i te rs of b lood , these au tho r s o b t a i n e d 50 m g of h i g h l y pu r i f i ed 6 p h o s p h o g l u c o n a t e d e h y d r o g e n a s e (spec i f ic act i - v i ty : 10.0 I U / m g / p r o t e i n s ) in the cou r se of 9 dif- f e r en t s teps, w i t h an overa l l y i e ld of 6 p. cent .

W e d e s c r i b e the p u r i f i c a t i o n of the h u m a n leu- k o c y t e e n z y m e f r o m a p a t i e n t w i t h u n t r e a t e d c h r o n i c g r a n u l o c y t i c l eukaemia . T h i s p u r i f i c a t i o n was a c h i e v e d by e l ec t ive e lu t ion of the e n z y m e f r o m a C M - S e p h a d e x c o l u m n w i t h NADP +, as pre- v ious ly d e s c r i b e d fo r g lucose 6 p h o s p h a t e dehy - d r o g e n a s e [2]. Th is m e t h o d a l l o w e d us to ob ta in

(*) Charg6 de recherche h I'INSERM. Present address : H6pital Beaujon, 92d10 Cl,ichy, France.

(**) Faculty of biochemical sciences. Department of Hematology. Rosario, Argentine Republic.

To whom all correspondence should be addressed. This work was supported in part by a research grant

of the University Paris VII.

a h i g h l y pu r i f i ed 6 p h o s p h o g l u c o n a t e d e h y d r o - genase w i t h a spec i f i c a c t i v i t y of 20 I U / m g of p ro - t e in and an ove ra l l y i e ld of 30 p. cent . A m o n o - spec i f i c ant i - 6 - p h o s p h o g l u c o n a t e d e h y d r o g e n a s e a n t i b o d y has been o b t a i n e d f r o m rabb i t s and the i m m u n o l o g i c p r o p e r t i e s of 6 - p h o s p h o g l u c o n a t e d e h y d r o g e n a s e h a v e been s t u d i e d in pu r i f i ed pre- p a r a t i o n and c r u d e ex t r ac t s of h e m o p o i e t i c cells. The k ine t i c c h a r a c t e r i s t i c s of the l e u k o c y t e en- z y m e have been d e t e r m i n e d and c o m p a r e d w i t h those r e p o r t e d f o r e r y t h r o c y t e e n z y m e [1, 3, 41. The i m p o r t a n c e of the h e x o s e m o n o p h o s p h a t e p a t h w a y in the f u n c t i o n s of bo th l eukocy t e s and r ed b lood cel ls e m p h a s i z e s the i n t e r e s t of s u c h a s tudy of the << k e y - e n z y m e s >> of th is p a t h w a y , glu- cose 6 p h o s p h a t e d e h y d r o g e n a s e and 6 -phospho- g lucona t e d e h y d r o g e n a s e .

M A T E R I A L AND METHODS.

~{ATERIAL.

The l e u k a e m i c l eukocy t e s f r o m a p a t i e n t w i t h c h r o n i c g r a n u l o c y t i c l e u k a e m i a w e r e co l l ec t ed in th ree F e n w a l l bags for l e u k o p h e r e s i s , as p re - v ious ly r e p o r t e d for g lucose 6 p h o s p h a t e d e h y d r o -

326 A. K a h n and coll.

genase pur i f ica t ion [2]. The ion exchangers (DEAE Sephadex A50 and CM Sephadex C50). were sup- pl ied by Pharmacia . The substrates and effecto,rs of the enzymat ic react ions were suppl ied by Boehr inger Mannheim [6-phosphogluconate, nico- t inanl ide-adenine d inuc leo t ide phosphate oxidized (NAD,P ÷) and reduced (NADPH), adenosine tri- phosphate (ATP), 2-3 d iphosphoglycera te) ; phe- nazine methosulfate and te t razol ium salt came f rom Sigma-C.C., Acrylamide, b isacrylanf ide and sodium dodecylsulfate were furn ished by Eas tman Kodak, agarose by << L ' Indus t r ie Biologique Fran- caise >> and s tarch by Connaught. The enzymat ic react ions were read wi th a Zeiss PMQ II spectro- photometer and Aminco photof luor imeter , both related wi th a Servogor recorder . The absorpt ion at 2.8.0 nm of the eluale of the ch romatography columns was measured wi th a Beckman DB spcc- t rophotomete r also coupled to a recorder . The conduc t iv i ty of the buffers was measured wi th a Biolyon conductivi. ty-meters, and the pH wi th a Radiometer pH meter.

~IET'HODS.

All the steps of the pur i f ica t ion were car r ied out at A- 4°C. Unless o therwise ind ica t ion the buffers c o n t a i n e d : 10 -3 M. EDTA, 2 X 10 -3 M

mercap toe thanol and 2 X 10 -3 M e aminocapro ic acid. 6 phosl)hogluconate deTlydrogenase act ivi ty was measured in tris C1 0.'05 M buffer pH 8, 0.1 M KC1, 2 × 10 -4 M NADP ÷ and 6 X 10 -4 M 6-phos- phogluconate at 30 ° . On.e uni t represents the reduc t ion of 1 ~ mole of NADP + per minute at 3 0 ° .

The pro te in concent ra t ion was measured accor- ding to the Lowry ' s metho,d, wi th crysta l l ized beef a lbumin as s tandard [5].

The pur i ty of the enzymat ic p repara t ion was appra ised by immunologica l and e lec t rophore t ic methods :

- - Immunological methods.

An ant i -6-phosphogluconate dehydrogenase se- rum was obtained by in jec t ing two rabbits wi th 2 mg of the enzyme emuls ionned wi th an equal volume of complete F reund ' s adjuvant in intra- dermal inject ions at mul t ip le sites on the back (0.1 ml emulsion per location). This p rocedure was repeated 10 and 20 days after. On the 45th day, 2 mg of enzyme enlulsioned wi th F reund ' s adjuvant were injected in t ra-muscular ly and on the 55th day 1 mg was injected int ravei- nously. The rabbits were ki l led 5 days after and the blood was collected. The serum was heated

1 hour at 60 ° , then stored frozen ( - - 2 0 ° ) wi th sodium azide (0.02 p. cent - W/V).

This ant iserum was tested by immunodi f fus ion accord ing to the Ouchter lony 's method [6] against crude extracts and extracts at different stages of pur i f ica t ion . The immunoprec ip i t a t e l ine could be speci f ica ly stained for enzymic act ivi ty in a Tris- C1 0.05 M buffer pH 8 conta in ing 3 X 10-~ M 6-phosphogluconate, 10 -4 M NADP +, 0.1 m g / m l phenaz ine inethosulfate and 0.2 m g / m l tetrazo- l ium salt MTT. The neutra l iz ing t i ler of the anti- body was apprec ia ted by adding increas ing quan- tities of ant iserum to an enzyme prepara t ion of known activity. After incubat ion for 1 hour at 37 ° and 18 hours at 4 ° in 0.1 M phosphate buffer pH 7.3 conta in ing dit tf iothreitol 10 -~ M, di isopro- pyl f luorophosphate 2 × 10 -3 M and EDTA 2 X 10 3 the res idual act ivi ty was measured on the centr i fugat ion supernatant . In these condi t ions , 8 IU of enzyme were neutra l ized by 1 ml of anti- serum.

- - P o l g a c r y l a m i d e gel electrophoresis and so- dium dodecylsul[ate polgacrylamide gel electro- phoresis were made as previous ly repor ted for glucose 6 phosphate dehydrogenase pur i f ica t ion [2], the fo rmer in tris-Cl 0.05 M buffer pH 8.5 and the lat ter accord ing to Weber and Osborn [7].

- - Kinetic study.

The pure 6 phosphogluconate dehydrogenase was used for invest igat ion of the kinet ic proper - ties. The enzyme was s tored at + 4°G in tris-C1 0..05 buffer pH 8 conta in ing d i i sopropylf luorophos- phate 2 × 10 -3 M, EDTA 2 X 10 -3 M and dithio- threi tol 2 × 1,0-3 M. The solution was 100 p. cent saturated wi th ammonium s u l p h a t e ; the pro te in concent ra t ion was about 8 mg/ml .

In these condi t ions the enzyme remained fully act ive for several months. Before the kinet ic stu- dies the enzyme was di luted 10,0 folds in tris C1 0.0'5 M buffer pH 8 conta in ing EDTA × 10 -3 ,

mercap toe thanol 1.0 "3 M and crystal l ized beef a lbumin 2 nlg/ml , then was dialysed against 3 t imes 100 volumes of the same buffer (without albumin). For all the spec t rophotomet r ic measure- ments 50 !xl of an enzyme dilution, of w h i c h the maximal veloci ty was about 2 × 1.0 -3 IU, were added to the reac t ive mix ture in a cuvette of 1 ml. The t empera tu re was kept constant at 30 °.

The f luorometr ic measurements were made at 37 ° wi th 10 ,~1 of an enzyme dilut ion of maximal act ivi ty equal to 1-2 × 10~ IU, in 2 ml of reac t ive mixture.

BIOCHIMIE, 1975, 57, n ° 3.

Human granulocgte 6 phosphogluconate dehgdrogenase. 3 2 7

RESULTATS.

- - P u r i f i c a t i o n procedure .

1 - - T h e l e u k o e y t e s w e r e i s o l a t e d as p r e v i o u s l y i n d i c a t e d fo r g lucos e 6 p h o s p h a t e d e h y d r o g e n a s e p u r i f i c a t i o n I21, t h e n t h e y w e r e l y s e d i n s o d i u m p h o s p h a t e 5 × 10 -2 b u f f e r p H 6.4 s a t u r a t e d w i t h d i g i t o n i n a n d t he c e l l u l a r r e m a i n s w e r e e l i m i n - a t e d b y c e n t r i f u g a t i o n fo r 45 m i n u t e s at 15 000 g.

2 - - A m m o n i u m s u l p h a t e p r e c i p i t a t i o n :

A s a t u r a t e d (NH4)~ S O4 s o l u t i o n in t he p h o s - p h a t e 0.05 b u f f e r p H 6.4 w a s sh )wly ad, ded to t he l e u k o c y t e e x t r a c t up to 35 p. c e n t s a t u r a t i o n . T h e f i rs t p r e c i p i t a t e w a s r e m o v e d b y c e n t r i f u g a t i o n a n d t he s u p e r n a t a n t b r o u g h t to 55 p. c e n t s a t u r a - t i o n w i t h the s a t u r a t e d (N,H~) 2 S O~ s o l u t i o n . T h e 35 p. c e n t - 5 5 p. c e n t p r e c i p i t a t e c o n t a i n e d glu-

E " Conductivity

o o Cpa). - 4 0 0 0 "-. N 1 _

0.5

E

I

0 50 100 Fraction number

Fro. 1. - - Elution pattern of human leukocyte 6 phos- phogluconate dehydrogenase from a DEAE Sephadex column at pH 7.2. . . . . . . . . : eon4u.ctivity at 20°C.

: absorbanee at 280 nm. e ~ e : 6 phosphoglueona te dehydrogenase activity.

3.40,1) mg of p ro te ins were applied to a DEAE sepha- dex A 50 column (25 em X 3 cm) equi l ib ra ted wi th phospha te 5.10-aM buffer pH 7.2 conta in ing EDTA 10-3 M, s aminoeaproic aeid 2.10.-3 M and [3 mercapto- e thanol 2.11)-3 M. The column was washed (flow ra te = 36 m] per h.) wi th the same buffer, then the e lut ion was performed, wi th a l inea r gradient between 500. ml of the above phospha te buffer and 500 ml. of the same buffer conta in ing NaC1 0.01 M.

cose 6 p h o s p h a t e d e h y d r o g e n a s e a c t i v i t y a n d w a s s t o r ed . T h e s e c o n d su, p e r n a t a n t w a s b r o u g h t to 80 p. c e n t s a t u r a t i o n , w i t h s o l i d (NH4)2SO 4 a n d t he 55 p. c e n t - 80 p. c e n t p r e c i p i t a t e w a s r e m o v e d b y c e n t r i f u g a t i o n : i t c o n t a i n e d 80 to 90 p. c e n t of 6 p h o s p h o g l u c o n a t e d e h y d r o g e n a s e a c t i v i t y : t h i s p r e p a r a t i o n w a s c o l l e c t e d a n d e x t e n s i v e l y d i a -

l y s e d a g a i n s t s o d i u m p h o s p h a t e 5 × 10 -3 M b u f f e r p H 7.2 ( c o n d u c t i v i t y : 1 000 ~ at 20°).

3 - D E A E s e p h a d e x c o l u m c h r o m a t o g r a p h y (fig. 1).

T h e d i a l y s e d e x t r a c t (of w h i c h i o n i c s t r e n g h t w a s a b o u t 1 000 uz w a s a p p l i e d to a D E A E s e p h a - dex c o l u m n (3 × 25 cm) w h i c h w a s p r e v i o u s l y e q u i l i b r a t e d w i t h t h e d i a l y s i s buf fe r . T h e c o l u m n w a s w a s h e d w i t h 250 m l of the s a m e bu f fe r , t h e n t he e l u t i o n w a s p e r f o r m e d w i t h a l i n e a r g r a d i e n t b e t w e e n 500 m l of t h e p h o s p h a t e 5 × 10 -3 M b u f f e r p H 7.2 a n d 500 m l of t he s a m e b u f f e r con- t a i n i n g NaC1 0.01 M ( c o n d u c t i v i t y : 2 200 ~o) w i t h a f low r a t e of 36 m l / h ; 10 m l f r a c t i o n s w e r e c o l l e c t e d . F r a c t i o n s a m o u n t i n g to m o r e t h a n 90 p. c e n t of t he e n z y m i c a c t i v i t y w e r e p o o l e d a n d p r e c i p i t a t e d b y s o l i d (NH~)_,SO 4 to 80 p. c e n t s a t u r a t i o n . T h e p r e c i p i t a t e w a s c o l l e c t e d a n d dia- l y s e d a g a i n s t a s o d i u m p h o s p h a t e 0.01 M b u f f e r p H 5.8 ( c o n d u c t i v i t y : 19.85 ~ ) .

4 - - CM S e p h a d e x c h r o m a t o g r a p h y w i t h e lec- t ive e l u t i o n b y N A D P ÷ ( f igure 2)

T r e p r e p a r a t i o n w a s d e p o s i t e d on a CM s e p h a - dex C-50 co. lumn (2 × 20 cm) e q u i l i b r a t e d w i t h the p h o s p h a t e 0.01 M b u f f e r p H 5.8. T h e c o l u m n

Conductivity NAoP ÷ 2. lO?aM. (po) .u-

1" 6 0 0 0

~ N 6000 ~ • 30

L 0 20 50 60

Fraction number FIG. 2. - - Elution pattern of leukocyte 6 phospho-

gluconate dehydrogenase form a CM Sephadex column. ........ : conduct iv i ty at 20°C.

: absorbance at 280 nm. e - - e : 6 phosphog4uconate dehydrogenase activity.

216 mg of pro te ins were deposited on the top of a CM Scphadex C 50 column (20 × 2 cm) equi l ibra ted w i th a phospha te 0.01 M buffer pH 5.8 conta in ing EDTA 10-a M (3 mercap toe thanol 2.10-3 M and E aminocaproic acid 2.10-3. The column was washed (flow ra te : 25 ml per h) wi th 100 ml of t h i s phospha t e buffer, t hen wi th a l inear gradient between 200 ml of the above phos- pha te buffer and 200 ml of a phospha te 0.04 M buffer pH 5.8, also conta in ing EDTA, (3 mercap toe thano l and

aminoeaproic acid. Column was washed wi th th is la t te r buffer unt i l no absorbanee a 280 nm was obser- ved. Enzyme was eluted by adding NADP* 2.10 3 M to the phospha te 0.04 M buffer.

BIOCHIMIE, 1975, 57, n o 3.

328 A . K a h n a n d co l l .

w a s w a s h e d w i t h 100 ml of t he s a m e buf fe r , t h e n a l i n e a r g r a d i e n t w a s in . i t ia ted b e t w e e n 200 nfi of the above p h o s p h a t e bu f fe r a n d 200 ml of a p h o s - p h a t e 0.0.4 M. bu f f e r p H 5.8, ( c o n d u c t i v i t y : 4 570 ~o). The c o l u m n w a s w a s h e d w i t h th i s l a t t e r bu f f e r unt i l l no a b s o r b a n c e at 28,0 n m w a s obse r -

the w a s h i n g buf fer . The 6 p h o ~ p h o g l u e o n a t e d e h y - d r o g e n a s e ac t iv i ty w a s at its m a x i n l u m as s o o n as an incre,a,se of the a b s o r p t i o n at 280 n m a p p e a r e d in t he e luate , w h i c h w a s due to NADP +. It t h e n de- c r e a s e d e x p o n e n t i a l l y . F r a c t i o n s of 6 m l w e r e co l l ec ted . 80 p. cen t of the tota l e n z y m i c ac t i v i t y

TABLE I.

Proteins Activity Specific Accumulative I Yield (rag) (I-units) activity purification I

(IU/mg) (fold) ! (p. cent)

16.000 3.200 0.2 1 100 I,eukocyte crude extract . . . . . . . . . . . . . . . . . . .

Ammonium sulphate precipitation . . . . . . . . . . .

DEAE Sephadex column . . . . . . . . . . . . . . . . . . .

CM Sephadex column with elective elution . . .

3.460

216

48

2.400

1.400

960

0.7

6.5

20

3.5 75

32.5 44

100 30

v e d (flow ra te : 20 m l / h ) . The e lu t ion of 6 p h o s - p h o g l u c o n a t e d e h y d r o g e n a s e w a s c a u s e d by ad- d i n g NADP ÷ 2 X 1,0-3M at the p h o s p h a t e 0.04 M bu f f e r p H 5.8. NA, D!P + w a s a d d e d u n d e r t he f o r m of a so lu t i on of the s a m e c o n d u c t i v i t y as t ha t of

1 2 3

Fro. 3. - - Control of the pur i t y of the enzyme prepa- ration.

1 and 2 : Acrylamide gel eleetrophoresis (acrylamide 7.5 p. cent) in Tris' Cl buffer pH 8.5, 0.01 M for the gel and 0.05 M for the tank.

In 1, 20 i~g of proteins, were applied to the gel, and after the end of the migration, the gel was stained for the proteins, wi th amidohlack.

In 2, a dilution of enzymatic activity : 0.01 IU was deposited at the top of the gel. After the migrat ion this gel was stained for 6 phosphogl~ueonate dehydro- genase activity.

3, sodium dodeeyl sulphate gel electrophoresis, accor- ding to Weber and Osborn [7]. 2.0 ,~g of pro te ins were applied to the gel, which, af ter the migration, was stained wi th eoomas.ie blue.

BIOCHIMIE, 1975, 57, n ° 3.

w e r e f o u n d in five f r a c t i o n s ; t h e s e f r a c t i o n s w e r e c o l l e c t e d a n d v a c u u m d i a l y s e d a g a i n s t a s a t u r a t e d (NHd)2SO 4 t r i s C1 0.05 M b u f f e r pH 8, to f o r m a v o l u me of .about 6 ml. T h e n t h e e n z y m e w a s con - s e r v e d in th i s f o r m at d- 4°C.

Th i s p u r i f i c a t i o n p r o c e d u r e a l l o w e d us to o b t a i n 48 m g of p r o t e i n of a s p e c i f i c ac t iv i ty 20 I U / m g , w h i c h thus gave a to ta l y i e l d of 30 p. c e n t a n d a p u r i f i c a t i o n of abou t 100 folds . Table I s u m m a r i z e s the r e su l t s of the p u r i f i c a t i o n

p r o c e d u r e .

• ....

;:: 40: 2 0 , : 'i::

FIG. 4. - - I m m u n o d i f f u s i o n of h ighly pur i f i ed 6 phosphogluconate dehydrogenase againM the anti 6 phosphogluconate dehydrogenase rabi t t serum.

The upper wells were filled wi th purified enzyme (AG = antigen). The quant i ty of enzymatic protein applied to gel is indicated.

The lower wells containing rabi t ant iserum, pure and diluted at 1/2 (AS ----- ant iserum).

The gel was agarose 1 p, cent in phospha te 0.1 M buf- fer pH 7.2, containing sodium azide 0.0,2 p. cent (W/V).

H u m a n granu locy t e 6 p h o s p h o g l u c o n a t e dehydrogenase . 329

Criteria o[ purity.

- - P o l y a e r y l a m i d e gel e l e c t r o p h o r e s i s s h o w e d on ly one b a n d of p r o t e i n w h i c h c o r r e s p o n d e d to the ac t ive b a n d (fig. 3).

in d i f f e r en t aggrega t ion states, fo rms of w h i c h the d i f fus ion ra tes cou ld be d i f fe ren t .

An excess of a n t i b o d y c a n n o t to ta l ly i n a c t i v a t e d the enzyme , the e n z y m e a n t i b o d y c o m p l e x re t a in -

½

Enzymatic Activity

Fro. 5. - - hnmunodiffusion of the leukocyte crude extract against the anti 6 phosphogluco- hate dehydrogenase rabitt serum.

The upper wells were filled with 20 :~l of leukocyte crude extracts, pure and diluted at 1/2 (protein concentration of the non diluted extract : 5() mg/m]).

The lower wells contained 2.0 i~1 of the anti- serum, pure and diluted at 1/2.

The precipitat ion lines were stained for pro- teins wi th alnidoblack (upper picture) and for enzymatic activity (lower picture).

- - Also s o d i u m d o d e c y l s u l p h a t e p o l y a c r y l a m i d c gel e l e c t r o p h o r e s i s r e v e a l e d a s ing le p r o t e i n b a n d (fig. 3).

- The i m n m n o d i f f u s i o n e x p e r i m e n t gave o n l y one p r e c i p i t a t i o n l ine w i t h the i m m u n i z i n g ex t r ac t (fig. 4) as we l l as w i t h l e u k o c y t e c r u d e e x l r a c t (fig. 5).

lnzmunological properties.

- - T h e s ingle i m n m n o p r e c i p i t a t e l ines specif i - ca l ly s t a ined for 6 p h o s p h o g l u c o n a t e d e h y d r o - genase ac t i v i t y s h o w e d an aspec t of c o m p l e t e i d e n t i t y b e t w e e n ex t r ac t s of e r y t h r o c y t e s , l euko- ey tes and pl,atelets (fig. 6). In some c o n d i t i o n s m o r e one p r e c i p i t a t i o n l ine a p p e a r e d , w i t h anor - real , leukocyte e x t r a c t (fig. 5). Th i s even t w a s pe r - haps due to the e x i s t e n c e of s eve ra l e n z y m e fo rms

BIOCHIMIE, 1975, 57, n ° 3.

i ng about 20 p. cent of the first e n z y m a t i c ac t i - v i ty ; lh is d a t u m e x p l a i n s tha t the i n m m n o p r e c i - p i t a te kep t a p a r t of the e n z y m e ac t i v i t y and that it can be s p e c i f i c a l l y s ta ined .

KINETIC EXPERIMENTS.

1 ° E[fect o[ ions on enzymic properties.

The effect of va r i ous salts on the ac t i v i t y of 6 p h o s p h o g l u c o n a t e d e h y d r o g e n a s e at p H 8, is s h o w n in f igure 7. W i t h the m o n o v a l e n t ca t ions tes ted the re w a s a r i se in ac t i v i t y as the i on i c s t r eng th c o n t r i b u t i o n of the salt was i n c r e a s e d to 0.1. Th is w a s f o l l o w e d by a fal l in ac t i v i t y a t h i g h e r sal t c o n c e n t r a t i o n s . Na ÷ and K ÷ w e r e m o r e ef f ic ien t a c t i v a t o r t h a n NH~*.

W i t h the diva.l:ent ca t ions (M.g2*, Ca 2÷) the m a x i - mal ac t iv i ty was o b t a i n e d for a s tow ion i c s t r eng th

24

33,0 A. K a h n a n d coll .

(0.03) a n d t h e i n h i b i t i o n w a s m o r e i n t e n s i v e a n d

e a r l y t h a n w i t h t h e n i o n o v a l e n t c a t i o n s .

T h e c o m p a r i s o n b e t w e e n t h e e f fec t s of SO~Na 2

a n d C:,.Na on t im o n e h a n d , SO4Mg a n d C12Mg o n

the o t h e r h a n d i n d i c a t e d t h a t S O ( - a n i n h i b i t o r

i n f l u e n c e .

Fro. 6. - - Double immunodi f fus ion analysis of va- rious normal blood cell extracts against the anti 6 phosphoghtconate dehydrogenase rabilt serum.

A n t i s e r u m (20 ;~,1) di l~ted at 1/4 w a s placed in the center well. The ou te r wel ls contained, 20 :~tl of p la te le t ex t rac t (1), leukocyte lysa te (2) and h e m o l y s a t e at 1/2 (3).

The p rec ip i t a t ion l ines were specifically s ta ined for the 6 p h o s p h o g l u c o n a t e act ivi ty.

~--1.5

N 1 . . . . " ' -~

05 ~- :,'0 k :: k ' \ \ \ \

0 0.1 Q2 0.3 0.4 0.5

Ionic strength Fro. 7. - - Effect of ions on the activity of 6 phospho-

gluconate dehydrogenase. I,n Tr is Cl~ 0.02, M buffer pH 8 (i ~ 0.009) con ta in ing

NADP + 2.1.0-4 M and 6 p h o s p h o g l u e o n a t e 6.M~-4 M~, w i t h va r i ab le ionic s t r eng th c o n t r i b u t i o n of different sal ts . An ac t iv i ty of 1 was, t aken as t h a t in T r i s CI: buf fer w i t h o u t a n y salt.

[] ...... [:3 KCI • - - • Na~SO,~ I - - I NaCI 0 . - - . 0 Ca C1,_, & . . . . ~ NH~OH &-- . .& MgC12

. . . . . . . M g S O ~

BIOCHIMIE, 1975, 57, n ° 3.

W i t h v a r i a b l e c o n c e n t r a t i o n s of T r i s C1 b u f f e r

p H 8, t h e m a x i m a l a c t i v i t y w a s n o t e d f o r I : 0.025.

T h e e f f ec t s of i o n i c s t r e n g t h i n KC] o n M i e h a e l i s

c o n s t a n t f o r N A D P + a n d 6 p h o s p h o g l u c o n a t e a r e

, - , 6 0

I \ / ...o "01 . . . . . o_...o__.,:J

I i i i i i

0 0.1 0.2 0.3 0.4 0.5 Ionic strength

FiG. 8. - - Variation of Michaelis constants with pi t and with varying concentration of KCl.

The effects of ionic s~rength of KC1 w a s apprec ia ted in a Tr is C1 0.02 M buffer pH 8 con ta in ing increas ing concen t r a t ions of KC1. The influence of pH w a s s tudied be tween pH 7 and pH g in Tr i s C1 0.05 M buffer con- t a in ing KC1 0.1 M.

Km were de t c rmina t ed w i t h Hanes p lo t s ( s /V = f (s)). The concen t ra t ion of the non var iab le s u b s t r a t e w a s 2.10-4 M for NADP + and 6.10-4 M for 6 p h o s p h o g l u e o - na.te. m - - i : K m 6 p h o s p h o g l u c o n a t e = f (I) [] ---[3 : Km NADP + = f (I) © . . . . © : Km 6 phosphogl ,uconate = f (pH) & - - & : K m NADP + = f (pH).

In Vmax 4

1

i

r

i I I t

0 ~'5 6 7 8 ; I0 pH pKal = 6.66 pKa2 = 8.66

FIG. 9. - - Dependence of enzyme activity on pH. In a T r i s -G lyc ine -Phospha t e 0.1 M buffer ad ju s t ed

wi th HClor NaOH. n - - [ ] : ionic s t r eng th of buf fe r ad jus t ed to I = 0.05 & - - & : ionic s t r eng th ad ju s t ed to I ---- 0.1 w i t h NaC1 • - - • : ionic s t r eng th ad ju s t ed to I = 0.2 w i t h NaC1.

The ionic s t r e n g t h s were m e a s u r e d w i t h a conduct i - v i ty m e t e r at 20°C.

6 p h o s p h o g l u c o n a t e : 6.10-4 M and NADP ÷ = 2.10-4 M,

Human granulocgte 6 phosphogluconate dehgdrogenase. 331

fly

100

80

60

40

o

A

NADP + = lO-5 500 Vv

2.10 -5 • • 400

819 5 3OO

200 / / / / • 10C

o:s ; 6 phosphogluconate (10-5M)

B

6phosphogluconate = ~ 3.1o -I

/ 1 2 . 1 0 . 4

~ 13.10 "4

& 1 NADP + (lO-s M} ,

FIG. 10. Effect to NADP + on Mi- chaelis constant towards 6 phosphoglu- conate (A) and effect of 6 phosphoglu- eonate on Michaelis conslant towards XADP + (B).

The f ines a re L i n e w e a v e r B u r k p lo t s . The a s s a y s were in Tr i s -Cl 0.05 M b u f - fer pH 8 w i t h 0.1 M KC1. The K m fo r 6 p h o s p h o g l u e o n a t e a t d i f f e r en t con- c e n t r a t i o n s of NADP + were m e a s u r e d w i th a p h o t o f l u o r i m e t e r .

~=5o C:3

C) o~

NADP + ......_..-------

/ , , / / - " " - "

, . ~ " - - - - - - " - ~ o p h o s p h o glu con at e i // i

0 10 -4 6 .10-4M

[6 phosphoglueonate] [NADP+J

Fro. 11. -- Heat inact ivat ion-protect ion by 6 phos- phoglaconate and NADP ÷.

The e n z y m a t i c p r e p a r a t i o n w a s h e a t e d 45 m n a t 48°C in T r i s C1 0.05 M bu f f e r pH 8 c o n t a i n i n g EDTA 10-3 M

m e r c a p t o e t h a n o l 2.10-3 M a n d c r y s t a l l i z e d bee f a l b u - m i n ~ l n g / m l , w i t h d i f f e ren t c o n c e n t r a t i o n s of NADP + or 6 p h o s p h o g l n c o n a t e .

The r e s i d u a l ac t iv i ty w a s d e t e r m i n a t e d in t he u s u a l r eac t ion m i x t u r e .

s h o w n in f i g u r e 8. K i n - 6 p h o s p h o g h i c o n a t e w a s

m i n i m a l f o r I : 0.1, w h i l e K m - N A D P ~ w a s d n a f f e c -

t e d b y i o n i c s t r e n g t h u n t i l I : 0 .25. T h e n K i n

N A D P ~ i n c r e a s e d ,

2 <' E f f e c t of pH on act iv i tg .

T h e d e p e n d e n c e o f a c t i v i t y o n p H w a s s t u d i e d

a t c o n s t a n t i o n i c s l r e n g t h o f t h e T r i s - g l y c i n e -

p h o s p h a t e b u f f e r (I a d j u s t e d to 0.1 a n d 0.2 w i t h

NaC1) . O n e e x p e r i m e n t w a s m a d e w i t h o u t N a C I

b y a d j u s t i n g t h e b u f f e r c o m p o n e n t s to I : 0 . 0 5 . W i t h t h e t h r e e b u f f e r s y s t e m s t h e p H c u r v e s w e r e

i d e n t i c a l (fig. 9), w i t h a n o p t i m a l p H at 7.73. T h e

g r a p h i c r e p r e s e n t a t i o n l o g V . m a x = f ( p H ) al-

l o w e d u s to p o i n t 2 i o n i s a t i o n s p K at p H 6.66 a n d

8.66. T h e M i c h a e l i s c o n s t a n t s f o r N A I ) P ~ a n d

6 p h o s p h o g l u c o n a t e r e m a i n e d a h n o s t c o n s t a n t f r o m p H 7 to p H 8 (fig. 8).

1oo

75

= 5 0

-~ 30

2 0

lO

1 0 0 >. ._>

m -~ 5 o

B

c

L i i

5 10.10-6M 0 1 :2 pI hydroxymercudbenzoate 6 phosphogluconate or NADP ÷

Fro. 12. - - Inactivation of 6 Phosphogluconate dehgdrogenase by parahydroxymercuribenzoate and protection by the substrales.

(A) The e n z y m e p r e p a r a t i o n was i n c u b a t e d 15 m n at 23°C in T r i s C1 0.05 M bu f f e r pH 8 w i t h i n c r e a s i n g c o n c e n t r a t i o n s of p - h y d r o x y - m e r c u r i b e n z o a t e . T he i n a c t i v a t i o n r eac t i on was s topped by d i l u t i n g t he i n c u b a t i o n m i x t u r e in 20 v o l u m e s of the r e a c t i o n m i x t u r e , a n d t he r e s i d u a l a c t i v i t y w a s a s s a y e d .

(B) T he e n z y m e p r e p a r a t i o n w a s i n c u b a t e d 15 m n a t 23°C in Tris-C1 0.05 M b u f f e r pH 8, c o n t a i n i n g EDTA 10-3, p - H y d r o x y m e r - c u r i b e n z o a t e 5.10-6 M a n d v a r i o u s c o n c e n t r a t i o n of 6 P h o s p h o g l u - cona te or NADP +.

The p lo t i n d i c a t e d by << © >> r e p r e s e n t s the r e s i d u a l a c t i v i t y of the e n z y m e i n c u b a t e d in the c o n d i t i o n s desc r ibed above in pre - sence of NADP + 2.10-4 M a n d 6 p h o s p h o g l u c o n a t e 4.10 4 : in tile cou r se of t h i s i n c u b a t i o n , 1/5 o n l y of NADP * was r educed in NADPH. The r e s i d u a l ac t iv i ty w a s n~easured w i t h a f l uo rome te r .

10 -4 M / J

4

BIOCHIM1E, 1975, 57, n ° 3.

332 A. K a h n and coll.

3 ° Sequence of substrate addition, the Michae l i s c o n s t a n t fo r 6 p h o s p h o g l u c o n a t e

The Michae l i s c o n s t a n t s f o r N&DP + a n d 6 p h o s - w a s ind, e p e n d e n t of the NADP + c o n c e n t r a t i o n p h o g l u c o n a t e in Tr i s C1 0.0'5 buf fe r , KC1 0.1 M, (fig. 10). M o r e o v e r b o t h NADP + a lone a n d 6 p h o s -

1/v

40£

30C

2 O (

100

A NADPH , 1.6. lO-" SO0

/ Uv

93 . I0 -s 3 0 0

4 5 . t0 "5 2 0 0

17. 10 "~"

100

0.'25 NADP" (10-s M) - '

NADPH = 15.10 "4

= ~ B .10 "~

015 0.2 0 6.1 0'.2 d3 6 phosphogluconate (10 5 M)-'

Fro. 13. - - Effect of NADPH on the activity of 6 phosphogluconate dehy- drogenase.

A : effect of I~ADPH on the Km for NADP +.

B : effect of NADPH on the Km for 6 phosphogl'uconate.

The lines, are Lineweaver-Burk plots with respect to NADP + (A) and wi th respect to 6, p'hosphogluconate (B). The assays were in Tris C1 0.05 M buffer pH 8 wi th KC1 0.1 M, 6 phosphoglueo- nate 6.10-4 M in << A >>, N'ADP + 2.10-4 M in << B >>.

w e r e r e s p e c t i v e l y 18 ~ _+ 2 fo r t h e f o r m e r sub- s t r a f e a n d 38 ~tM __+ 2 f o r t h e l a t t e r one .

The Micbae l i s c o n s t a n t fo r NADP ÷ w a s i n d e p e n - d e n t of the 6 p h o s p h o g l u c o n a t e c o n c e n t r a t i o n a n d

® 1/v

2 - 3 D P G : 3.1o -3 30° F 2 - 3 D P G : I /

6,10 -3

200 3 ,10 -3 / 10-3

100 0

0.2 -0.1 0 0.1 0.3 -0.2 -0`I 0 0`I .

6 phosphogluconate C l o - S M ) -~

© @ 1/v

30O

200

100

-0 .4 -0,2

2-3 D P G : / 3.10 -3

-0 .4 -0 .2

NADP* (I0-5 M) -I

2 -3 D P G : 3.10_3

0~2 i 0.6

FIG. 14. - - Effect of 2-3 diphosphoglycerate on the activity of 6 phosphogluconate dehydrogenase, at various concentrations of both snbstrates. '2-3 diphosphoglycerate concentrat ions (2-3 BPG) are indicated.

The l ines are Lineweaver Burk plots wi th respect to 6 phosphoglueonate (A and: B) and NADP ÷ (C and D). The assays were in Trls Ct 0.05 Iv~ buffer pH 8, wi th KCI 0.1 M.

The concentrat ion of the non variable substrate was A : NADP ÷ 2.10-4M - - B : NADP + 4.10-5M - - C : 6 phosphogluconate 6.10-4 M - - D : 6 phosphogluconate 3.10,-5 M.

BIOCHIMIE, 1975, 57, n ° 3.

1)hogluconate a lone p ro , t ec ted the e n z y m e ag a i n s t t h e r m M i n a c t i v a t i o n (,fig. 11) or ag a i n s t t i le i n ac t i - va t ion b y P a r a h y d r o x y m e r c u r i b e n z o a t e a c i d [12]. T h e r e f o r e t hese resu,lts s eem to c o r r e s p o n d to an ind, e p e n d e n t f ixa t ion of the s u b s t r a t e s NADP ÷ a n d 6 p h o s p h o g l u c o n , a t e .

- - I n h i b i t i o n by products and various meta- bolites.

The i n h i b i t i o n by NAD~PH w a s c o m p e t i t i v e w i t h NADP + a n d n o n c o m p e t i t i v e w i t h 6 p h o s p h o g l u - c o n a t e (fig. 13). A c c o r d i n g to the D i x o n ' s r e p r e - s e n t a t i o n in T r i s G1 p H 8, t'he i n h i b i t i o n c o n s t a n t by N A D P H w i t h r e s p e c t to NADP + w a s 17 ~xM.

The inhibi ' . t ion b y 2-3 d i p h o s p h o g l y c e r a t e (fig. 14) w i t h r e s p e c t to N&DP ÷ w a s s t r i c t l y com- p e t i t i v e at h i g h 6 p h o s p h o g l u c o n a t e c o n c e n t r a t i o n a n d n o n compe td t ive ( o r m i x t e ) at l o w 6 p h o s p h o - g l u c o n a t e c o n c e n t r a t i o n . On the s a m e w a y , at h i g h NADP + c o n c e n t r a t i o n the 2-3 d i p h o s p h o g l y - c e r a t e i n h i b i t i o n w a s c o m p e t i t i v e w i t h 6 p h o s - p h o g l u c o n a t e , a n d w a s n o n c o m p e t i t i v e at l o w NADP ÷ c o n c e n t r a t i o n .

T h e i n h i b i t i o n c o n s t a n t s by 2-3 d i p h o s p h o g l y - c e r a t e m e a s u r e d a h i g h c o n c e n t r a t i o n in t he n o n v a r i a b l e s u b s t r a t e w a s r e s p e c t i v e l y 4 mM w i t h r e s p e c t to NADP + a n d 5 mM w i t h r e s p e c t to 6 p h o s p h o g l u c o n a t e ( D i x o n ' s r e p r e s e n t a t i o n ) .

ATP i n h i b i t i o n w a s m e a s u r e d by f l u o r o m e t r y ~t wery l o w c o n c e n t r a t i o n of n o n v a r i a b l e sub- s t r a t e s (NADP + 4 × 10-6 M a n d 6 p h o s p h o g l u c o - na te 4 × 10,5 M ) , i n Tr i s C1 0.05 M buf fe r p H 7.3 c o n t a i n i n g KCI 0.1 M a n d MsG1._, 0.4 × 10 -4 M a n d at 37 ° : in o t h e r c o n d i t i o n s (Tr i s CI p H 8, h i g h s u b s t r a t e c o n c e n t r a t i o n ) the i n h i b i t i o n w a s v e r y w e a k . In t h e s e eond,~tions A T P w a s a s t r i c t l y corn-

Human granulocgte 6 phosphoghtconate dehgdrogenase. 333

petit ive inh ib i tor wi th respect to 6 phosphogluco- hate ( K i : 1.7 raM} and non competi t ive with respect to NADP + (figure 15).

- - Inactivation by Parahgdroxgmercaribenzoate aud protection by the substrates.

Parahydroxymereur ibenzoa te acid was a potent inh ib i to r of 6 phosphogluconate dehydrogenase.

enzyme in the course of three steps only, with a high yield (30 p. cent). Thus the elective eIution of an enzyme by a substrate or a substrate analo- gue which specifically decreases the in terac t ions between exchanger and protein, seems to be an al ternative way to the true affinity chromatogra- phy. The prepara t ion obta ined was homogen according to the cr i ter ia of pur i ty we have cho-

400 1/v

300

2o0

lOO

0.8 0.4 0

A

ATP : 2 .lO -3

10_3

0

o'.. E8 6 phosphogluconate I10SM) -t

800{- 1/v i ATP =~ 2.10 -3

600 -/ ~ "l°-3 I / .

t y, 400 / .,~"

it' 2 0 + - ~/" ~A~- / 5"10-4

- 0.5 0 0.25 0.5 1 NADP* (]0-5 M) -~

Fro. 15. - - E[fect of ATP, All the measurements were made

with a photofluorimeter, at 37°C, in Tris C1 0.05 M buffer pH 7.3 containing KC1 0.1 M, MgCI_~ 4.103M and NADP + 4.10-~ M in A, 6 phosphogluconate 4.10-~ M in B.

The lines arc Lineweaver Burk plots with respect to 6 phosphoglueonate (A) and with respect to NADP + (B). The ATP concentrations are indicated.

Each substrate incomplete ly protected the enzyme against this inact ivat ion. Both substrates together were more potent protectors titan each one of them separately (fig. 12).

DISCUSSION.

The r ichness in enzymes of anaerobic glycolysis of grannlocytes of pat ients wi th hyper leukocyt ie chronic granulocyt ie lenkaemia has always been pointed out for the pur i f ica t ion of glucose 6 phos- phate dehydrogenase [2] : here we were able to pur i fy about 50 mg of pure enzyme front a single donor. In effect the leukocytic concent ra t ion of 6 phosphoglueonate dehydrogenase was very high, ahou't 3 to 4 t imes higher than the glueose 6 phos- phate dehydrogenase concent ra t ion (0.01 mg of the former and 0.0'03 mg of the lalter per mg of prote in in the leukolysate).

The procedure of elective elution with NADP- is like the method we have descr ibed for glucose 6 phosphate dehydrogenase p u r i f i c a t i o n : Here also there seemed to be a compet i t ion between the ionic groups of the resin and free NADP" for the l ink ing to 6 phosphogluconate dehydroge- n a s e ; at the ionic s t renglh chosen the first type of in terac t ion (enzyme-exchanger) was sufficiently repressed so that the second type predominated . Consequent ly elution caused by NADP + was maxi- mum as soon as it appeared in the eluate and then it decreased exponential ly . This elective elution method allowed us to obtain a highly pur i f ied

BIOCHIMIE, 1975, 57, n ° 3.

sen; its specific activity was twice higher than the one of the p repara t ion obtained by Pearce and Rosemeyer [1, 3]. Yet these authors d idn ' t measure enzyme activity at optimal pH, but at pH 9 (and at 25° ) ; the precise comparison between "these results is therefore impossible.

In imnmnodif fus ion, leukocyte 6 phosphoglu- conate dehydrogenase shows a complete ident i ty react ion wi th erythroeyte enzyme. Fur thermore it is always known that this enzyme is coded by the same gene in all the ceils [8]. Nevertheless post- t ransla t ional feainres can modify enzyme propert ies in every tissue. These post- t ranslat ional features (especially enzyme forms of different aggregation states) can explain the existence of several prec ip i ta t ion lines between the ant ibody and the crude extracts of normal leukocytes. Also these post t rans la t ional modif icat ions perhaps account for the differences of some kinet ic pro- perties noted between the leukocyte enzyme and tile erythrocyte enzyme that Yoshida [4], Pearce and Rosmneyer have previously s t u d i e d : espe- cially the Michaelis constant for 6 phosphogluco- hate is twice higher for leukocyte enzyme than for erythrocyte enzyme according to Yoshida [4] and Pearce and Rosemeyer [1].

The opt inmm pH for leukocyte 6 phosphogluco- nate dehydrogenase is 7.73 in Tris-glycine-phos- phate buffer pH 8 I = 0.05 ; 0.1 or 0.2 ; it was a few lower than for erythrocyte enzyme: 8.3 in Tris C1 buffer for highly pur i f ied erythrocyte enzyme r l] , 8.1 in Tris C1 0.05 M buffer pH 8 for

334 A. Kahn and coll.

p a r t i a l l y p u r i f i e d e r y t h r o c y t e e n z y m e [9]. In con- t rast , t he effect of ions is a lmos t the same in the P e a r c e ' s s t udy and in o u r s .

Our resu l t s i n d i c a t e t ha i h u m a n l e u k o c y t e 6 p h o s p h o g l n c o n a t e d e h y d r o g e n a s e u t i l i zed a n o n o r d e r e d m e c h a n i s m of subs t ra te f i x a t i o n : b o t h NADP + and 6 p h o s p h o g l u e o n a t e can be l i n k e d to the enzyme , each f ixa t ion b e i n g i n d e p e n d e n t f r o m the o ther . Thus e a c h subs t ra te can p r o t e c t the e n z y m e aga ins t i n a c t i v a t i o n by hea t o r p a r a h y - d r o x y m e r c u r i b e n z o a t e , and b o t h subs t ra tes toge- t h e r p r o t e c t the e n z y m e m o r e t h a n each subs t ra te a lone.

In a g r e e m e n t wi,th Yosh ida [4] w e f o u n d tha i the i n h i b i t i o n by N A D P H is c o m p e t i t i v e w i t h NAD'P +, w i l h an i n h i b i t i o n c o n s t a n t of 17 !tM in Tr i s C1 0.05 M p H 8 and non c o m p e t i t i v e w i t h 6 p h o s p h o g l u c o n a t e . Also ATP inh ib i t s 6 phos- p h o g l u c o n a t e d e h y d r o g e n a s e c o m p e t i t i v e l y w i t h 6 p h o s p h o g l u c o n a t e and non c o m p e t i t i v e l y w i t h NADP +.

Our resul t s w i t h r e g a r d to the e n z y m e inh ib i - t ion by 2-3 d i p h o s p h o g l y c e r a t e are at v a r i a n c e w i t h those of Yosh ida : th is a u t h o r f o u n d tha t i n h i b i t i o n by 2-3 diphcxspho.glycerate was c o m - pe t i t i ve w i t h 6 p h o s p h o g l u c o n a t e and was non c o m p e t i t i v e w i t h NADP +. In con t r a s t we h a v e f o u n d tha t 2-3 d i p h o s p h o g l y c e r a t e can be l i n k e d to b a t h the f ixa t ion s i tes of N A D P ÷ a n d the one of 6 p h o s p h o g l u e o n a t e : c o n s e q u e n t l y the m o d e of inhibi , t ion s.eemed to be c o m p e t i t i v e w i t h bo th subs t ra tes w h e n the u n v a r i a b l e subs t ra te w a s at h i g h c o n c e n t r a t i o n and s.eemed to be non compe t i - t ive w i t h bo th subs t ra tes w h e n the u n v a r i a b l e subs t ra te was at l o w c o n c e n t r a t i o n . I t is i n t e r - e s t ing to p o i n t oui tha t NAI)PH, A T P and 2-3 di- p h o s p h o g l y c e r a t e h a v e i n h i b i t o r effects at p h y s i o - log ica l c o n c e n t r a t i o n s in e r y t h r o c y t e s . In con t r a s t , 2-3 d iphosphog lyce ra , t e is p r e s e n t at l o w c o n c e n - t r a t i on in l eukocy tes , and thus it does not seem to p l a y a p r o e m i n e n t p a r t in these cel ls .

T h e l e u k o c y t e 6 phosphogluconate d e h y d r o g e - nase was i n a e t i v a t e d by p - h y d r o x y m e r c u r i b e n - zoa te (a SH reagen t ) in a s i m i l a r w a y to yeas t [10i o r s t e a r o t h e r m o p h i l u s [11] enzyme . Both 6 phos- p h o g l u c o n a t e and NADP- s h o w e d a pa r t i a l p r o - t e e t i ve effect on the i n a c t i v a t i o n . These resu l t s i n d i c a t e the p r e s e n c e of s u l f b y d r i l g roups n e a r the ac t ive site. More than one SH-group mus t be i n t e r e s t e d by th is i n a c t i v a t i o n because the func - t ion Log r e s i d u a l / i n i t i a l a c t i v i t y ~ f ( p - h y d r o x y - m e r c u r i b e n z o ~ t e c o n c e n t r a t i o n ) was no t l i nea r , as p r e v i o u l s y r e p o r t e d for e r y l h r o c y t e g lucose 6 p h o p s h a t e d e h y d r o g e n a s e [12].

BIOCH1MIE, 1975, 57, n" 3.

In conc lu s ion , th is w o r k d e s c r i b e s a n e w and easy m e t h o d for total p u r i f i c a t i o n of h u m a n leu- kocy te 6 p h o s p h o g l u c o n a l e d e h y d r o g e n a s e f r o m a s ingle pa t i en t w i t h c h r o n i c g r a n u l o c y t i c l eukae- mia .

The s tudy in d i f f e ren t t i ssues of a p u r i f i e d e n z y m e coded by a s ingle gene in all the ce l ls a l lows 1o def ine wha t , in its p r o p e r t i e s , is gene t i - cally d e t e r m i n a t e d o r is due to pos t t r a n s l a t i o n a l p h e n o m e n a . These pos t s y n t h e t i c a l t e ra t ions , mo- l e cu l a r ag ing or m o d i f i c a t i o n s spec i f i c of a g iven t issue, can m o d i f y the c a t a l y t i c p r o p e r t i e s of -m e n z y m e in d i f f e r en t w a y in e a c h t i ssue [13, 14i. The d i s c r e p a n c i e s b e t w e e n the p r o p e r t i e s of ery- t h r o c y t e and l e u k o c y t e fi phosp :hog lucona te d e h y - d r o g e n a s e can be e x p l a i n e d in th is m a n n e r .

Acknowledgments.

We are very grateful to Dr J. F. Bernard for refering the patient with chronic granuloeytie leukemia to us, and to M'rs N. Lemaire for the typing of the manuscript.

R~suM~.

La 6 phosphogluconate leucocytaire humaine a ~t6 totalement puriti6e h part i r ties globules blancs leued- miques d'un mal,ade at teint de ]euc~mie my61oide ehro- nique.

48 Ing de prot6ine d'activit~ sp@ifique, 30 IU/mg, ont dt~ obtenues au terme de seulelnent trois dtapes distinetes. Le rendement total dtait de 30 p. cent et la purification de 100 fois.

Le 6-PGD purifide est homog6ne en 6lectrophor6se sur acrylamide et acrylamide SDS et en immunod, iffusion.

L'enzyme est i lnlnunologiquement identique dans les h~maties, ]es plaquettes et les leucocytes.

La fixation des suhstrats (NADP + et 6-PG) semhle inddpendante. Le NADPH est un inhibi teur comp6tit if vis-h-vis du NADP + et non comp~titif vis-h-vis du 6-PG.

Le 2,,3 DPG semble capable de se fixer h l 'enzyme h la fois au niveau des sites du 6-PG et du NADP ÷. L'ATP est un inhibi teur comp~tit if vis-h-vis du 6-PG et non comp6titif vis-h-vis du NADP +.

La 6-PGD est inactivde par les rgactifs d.es groupes sulfhydriles et est par t ie l lement protdgde contre cette inactivation par l 'un et l 'autre substrat. De m~me, les deux substrats protbgent l 'enzyme contre la ddnatu- rat ion thermique.

L'influence de Ia force ionique, du pH et des ions ont 6t~ ~tudids et les r~sultats eompards h ceux rap- port6s par d 'autres auteurs pour l 'enzyme ~rythro- cytaire.

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BIOCHIM1E, 1975, 57, n ° 3.