Characterization of human leukocyte β-glucuronidase

BIOCHIMIE, 1978, 60, 543-546.

Characterization of human leukocyte $-glucuronidase. J.-P. P ~ R I N and P. JOLLIES <>.

Laboratoire des Prot~ines, Universitd de Paris V, 45 rue des Saints-Pbres, 75270 Paris Cedex 06.

(17-2-1978).

Introduction. ~-Glucuroni,dase (EC 3.2.1.31) plays a p r o m i n e n t role in acid m u c o p o l y s a c c h a r i d e (MPS) catabolism [1]. A deficiency in this e n z y m e p r o d u c e s a disease c h a r u c t e r i z e d by the i n t r a c e l l u l a r a c c u m u lation of ~ P S a n d t h e i r ex~cessive urina~ry excretion [2]. The uptake of exogenous 0-g~ucuronidase f r o m tissue culture m e d i u m by fibroblasts c u l t u r e d f r o m patients w i t h this d e f i c i e n c y c o r r e c t s t h e i r i n c a p a c i t y to catabolise MPS [3, 4, 5]. L e u k o c y t e lysates f r o m an h o m o z y g o u s deficient patient cont a i n e d no ~-ghrcuroni4ase a c t i v i t y w h e r e a s an heterozygous c a r r i e r p r o v i d e d l e u k o c y t e lysates w i t h an e n z y m e level i n t e r m e d i a t e b e t w e e n the c o n t r o l and the :~-glucuronidase d,ef,icie.nt patient. Glaser and Sly [6] p r e s e n t e d e v i d e n c e that l e u k o c y t e lysates fr(~m h e a l t h y subjects w e r e a v e r y reliable source of ~ - g l u c u r o n i d a s e for c a r r i e r detection. Detailed c h a r a c t e r i z a t i o n of h u m a n l e u k o c y t e ~-glucuronidase seemed thus neces.sary. P r e v i o u s investigations h a v e been d e v o t e d to hum.an l i v e r [7], p l a c e n t a [8] an'd m o r e r e c e n t l y s y n o v i a l fluid [9] ~ g l u c u r o n i d a s e s . H o w e v e r the p u r i f i c a t i o n and c h a r a c t e r i z a t i o n of h u m a n l e u k o c y t e ,~-glucur o n i d a s e h a v e n e v e r been r e p o r t e d . The p r e s e n c e of m u l t i p l e forms of I~-glucuronidase w a s c h a r a c t e rized in several m a m m a l i a n tissues such as bovine, mouse, rat an, d r a b b i t [10, 11, 12, 13, 1.4]. Such a h e t e r o g e n e i t y w a s also r e p o r t e d for the e n z y m e f r o m some h u m a n tissues as shov~-n by both c o l u m n c h r o m a t o g r a p h y [15, 16] a n d i s o e l e c t r i c focusing [17]. The p r e s e n t s t u d y deals w i t h the p u r i f i c a t i o n of the saline e x t r a c t a b l e ~ - g l u c u r o n i d a s e f r o m h u m a n leukocytes and some of its catalytic and m o l e c u l a r properties. T h e i r k n o w l e d g e a l l o w e d to establish some c o m p a r i s o n s w i t h o t h e r p r e v i o u s l y partia'lly purified h u m a n .and n.on-human (5-glucuronidases.

Materials and Methods. Reagents : Phcnolphtalein-glucuronic acid was obtained from Sigma. Sephadex G-200, Sepharose 6 B, CM-Sephadex C-50, DEAE-Sephadex A-50 were purcha<> To whom all correspondence should be addressed.

sed from Pharmacia. All other reagents (analytical grade) were from Merck or Prolabo. Enzyme source : leukocytes from healthy humans were provided by the (( Centre de Transfusion Sanguine des Armdes >>, F-92141 Clamart, France. Leukocyte suspensions prepared according to Maupin [18] and Maupin and Chary [19] contained 11)9 cells/ml i n Hanks solution. Assay conditions : the standard method of assay for ~-glucuronidase activity was derived from those described by Fishman et al. [20] and Stahl and Touster [21] : 1~)-8 ~ phenolphtalein-glueuronide was incubated in 5.10-2 1~. sodium acetate buffer, pH' 5.0, in the presence of various amounts of the enzyme in a total volume of 1 ml. A~ter 30 min incubation at 37°C the reaction was stopped by adding 5 ml of a 0.0¢2 M glycine, 0.2 M NaC1, pH 10.4 buffer. The liberated amount of phenolphtalein was evaluated at 54~0 nm. Under these conditions 1 unit of enzyme activity was defined as the amount of enzyme which liberates 1 nM phenolphtalein from phenolphtalein-glueuronide per hour. Protein determination : proteins were usually detected at 280 nm with a Beckman Acta III spectrophotometer in cuvettes of 1 em path ; they were quantitatively evaluated with the Folin reagent [22] using hen egg-white lysozyme as standard.

Results. PURIFICATION OF THE ENZYME. The p u r i f i c a t i o n p r o c e d u r e i n c l u d e d 6 steps and is s u m m a r i z e d in table I. Step 1 : ~-glucuronidase w a s e x t r a c t e d f r o m leukocytes by r u p t u r i n g the ceils for 15 sec w i t h an U l t r a - t u r r a x m i x e r in a 0.05 M Tris-HC1, 0.1 M NaC1, pH 7.4 buffer .at 4°C f o l l o w e d by a centrifug a t i o n at 3000 r p m f o r 20 rain in an I n t e r n a t i o n a l PR-2 centrifuge, r o t o r n ° 850 (1 vol of b u f f e r / 1 vol of l e u k o c y t e suspension). After t h r e e successive e x t r a c t i o n s the s u p e r n a t a n t s w e r e p o o l e d ; they c o n t a i n e d 90 p e r cent of the a c t i v i t y of the h o m o genate. F r o m 8 p r e p a r a t i o n s it w a s o b s e r v e d that 2000 units (average value) of e n z y m e activity w e r e e x t r a c t e d p e r ml of leukocytes suspension.

J.-P. P ~ r i n a n d P. Jollbs.

544

(0 to 0.25 M, m i x i n g c h a m b e r 500 m l ) . T h e a c t i v e luateria'l was then dialysed against distilled water to b e u s e d f o r e n z y m e s t u d i e s .

Step 2 : a f t e r d i a l y s i s a g a i n s t t h e e x t r a c t i o n buffer, the active material was precipitated by a m m o n i u m s u l f a t e (55 p e r c e n t ) . T h e p r e c i p i t a t e

TABLE I.

Human leukocyte ~-glucuronidase purification. Step

1. Extraction, s u p e r n a t a n t 2. (NH~)eSOI precipitation, solubilisation 3. pH 5.5 precipitation, supernatant 4. CM-Sephadex chromatography 5. Sephadex G-200 filtration 6. DEAE-Sephadex chromatography

Activity (units)

Proteins (mg)

Specific activity lunits/mg)

Purification

Yield (per cent}

563 000 550 O00

4 970 1 350

113 407

1

3.6

100 97.8

545 OOO

1 045

521

4.1

96.0

490 000

113.5

4 320

38

87.0

425 000

18

23 600

209

75.5

53 000

470

45 8

260 000

4.9

w a s w a s h e d t w o t i m e s w i t h 50 m l of a 0.05 M Tris-HC1, 0.1 M NaC1, p H 7.4 s o l u t i o n c o n t a i n i n g 55 p e r c e n t a m m o n i u m s u l f a t e . A f t e r e e n t r i f u g a t i o n t h e p e l l e t w a s s o l u b i l i z e d w i t h 75 m l of t h e s a m e b u f f e r d e v o i d of a m m o n i u m s u l f a t e .

Step 3 : t h e m a t e r i a l s o l u b i l i z e d at p H 7.4 w a s s u b m i t - t e d t o a n e x t e n s i v e d i a l y s i s a g a i n s t a 0.0.5 M s o d i u m a c e t a t e , 0.05 M N,aC1, p H 5.5 b u f f e r . A n i n a c t i v e p,reci,pitate w a s d i s . c a r d e d b y c e n t r i f u g a t i o n (3000 r p m ; 20 r a i n ) . Step 4 : t h e a c t i v e s u p e r n a t e n t 'was s u b m i t t e d to an ion exchange chromatography on a CM-Sephad e x ,C-50 . c o l u m n (30 c m X 2.5 c m ) e q u i l i b r a t e d w i t h t h e 0.05 M s o d i u m a c e t a t e , 0.05 M NaC1, p H 5.5 b u f f e r . T h e e n z y m e w a s f i r s t r e t a i n e d o n t h e eo'lu.mn a n d l a t e r o n e l u t e d w i t ' h ,a l i n e a r NaCI g r a d i e n t (0.05 M t o 0.25 M, u d x i n g c h a m b e r 500 m l ) as a s i n g l e a c t i v e p e a k . T h e m a t e r i a l w a s t h e n c o n c e n t r a t e d o n a XM-50 m e m b r a n e i n a n A m i e o n c e l l to .a v o l u m e b e l o w 10 ml. Step 5 : a gel f i l t r a t i o n o n a S ' e p h a d e x G-200 c o l u m n (110 c m × 3 e m ) e q u i l i b r a t e d w i t h a 0.'05 M Tris-HC1, 0.1 M NaC1, p H 7.4 b u f f e r a l l o w e d to . c h a r a c t e r i z e a s i n g l e .active p e a k . T h e a c t i v e m a t e r i a l w a s . d i a l y z e d a g a i n s t a 0'.05 M Tris-HC1, p H 8.0 baaffer. Step 6 : t h e e n z y m e w a s a d s o r b e d o n a D E A E S e p , h a d e x A-50 . c o l u m n (25 c m X 2.5 c m ) e q u i l i b r a t e d w i t h a 0.05 M Tris-H.C1, p H 8.0 b u f f e r a n d e l u t e d as a s i n g l e p e a k w i t h a l i n e a r NaC1 g r a d i e n t BIOCHIMIE, 1978, 60, n ° 5.

0,50"

E

,~0.25'

0.05 4

5

pit

6

7

8

FI6. 1. - - Dependance of the activity. The enzyme activity was d e t e r m i n e d in a pH zone r a n g i n g f r o m 3 to 8 in t h r e e different buffers : O - - O 0.05IV[ citric acid - - 0.1 M d i s o d i u m phosphate ; O - - O 0.05 M Tris - - 0.05 M acetate ; / x - - A 0.1 M sodium acetate. Activity was expressed b y a b s o r p t i o n m e a s u r e m e n t s of the l i b e r a t e d p h e n o l p h t a l e i n at 540 n m as described in the Methods. All the assays have been carried out w i t h 205 u n i t s of enzyme ( s t a n d a r d conditions).

Human

545

leukocyte ~-glucuronidase.

ENZYME PROPERTIES.

p H dependance of the e n z y m i c a c t i v i t y : the o p t i m u m pH was d e t e r m i n e d w i t h three different buffer solutions at 37°C (fig. 1). I n a 0.1 M s o d i u m acetate buffer, the o p t i m u m pH was 4.4 ; a s i m i l a r value (4.25-4.30)was established i n a 0.05 M Tris0.05 M acetate buffer. I n 0.05 M c i t r i c acid-0.1 M d i s o d i u m phos~phate buffer, the o p t i m u m pH was shifted t o w a r d s 5.15. I n the latter buffer system at pH 5.0, a 34 p e r cent i n h i b i t i o n of the a c t i v i t y was observed w h e n c o m p a r e d to the activity determined i n the acetate system. I n the Tris-acetate buffer the i n h i b i t i o n was smaller (27 per cent).

E f f e c t of temperature : the effect of temperature on the catalytic activity has been m e a s u r e d w i t h o u t p r i o r i n c u b a t i o n of the enzyme at each temperature. F i g u r e 2 shows the v a r i a t i o n of the release of p h e n o l p h t a l e i n from p h e n o l p h t a l e i n g l u c u r o n i d e i n the s t a n d a r d assay conditions. The activity raised with i n c r e a s i n g t e m p e r a t u r e s from 5 ° to 60°C ; above 60°C the r e a c t i o n was i n h i b i t e d p r o b a b l y because of the d e n a t u r a t i o n of the enzyme.

0.50,

TABLE If. Effect of some salts on the actioit?l of human leukocyte ~-ylucuronidase. Experimental details are given zn the text. Salt

Activity (per cent) (')

50 per cent inhibition ('*)

E_ 0.25 o

CuC12

34

0.5 10-2 M

CuSO~ HgCl~

28 0

0 . 4 10 -~ M ~ 10 -3 M

Zn(CHaC00)~ Pb(CH~C00)~

88 80

5.2 10-~ M 3.8 10-2 M

(*) Percentage of the activity in the presence of a 10-2 M concentration of each salt. (**) Concentration of each salt sufficient for a 50 per cent inhibition, 100 per cent of activity was measured in standard assay conditions. No inhibition was observed with NaC1, Na~SO~, NAN03, NaI-L..PO~,KC1, LiCl, CaCla, M.gCl~and MgSO~.

E f f e c t of some salts on lhe a c t i v i t y : i n s o d i u m acetate buffers of pH 5.0 r a n g i n g from 0.025 M to 0.2 M, the a,ctivity r e m a i n e d c o n s t a n t ; therefore all the e x p e r i m e n t s achieved i n o r d e r to test the specific effect of other salts on the activity have been c a r r i e d ou i n a 0.1 M s o d i u m acetate, laH 5.0 buffer following an i n c u b a t i o n of the enzyme for 20 rain at 3'7°C before m e a s u r i n g the residual activity. Our results are r e p o r t e d i n table II. The presence of chloride, sulfate or n i t r a t e did not affect the c a t a l y t i c activity of the enzyme. Among the various cations tested n e i t h e r the alkali n o r the alkaline-earth metals were i n h i b i t o r s . On the other h a n d metallic ions p r e s e n t e d a v a r i a b l e i n h i b i t o r y effect ; m e r c u r y was the most po~verfull i n h i b i t o r . The low i n h i b i t o r y effect observed for d i s o d i u m phosphate was i n agreement w i t h the already reported observation c o n c e r n i n g the citrate-phosphate buffer. BIOCHIMIE, 1978, 60, n ° 5.

•

IO

,

.

20

50

.

.

40 50 T(oC)

.

60

70

i0

FIG. 2. Temperature dependance of the actioitg. Activity was expressed by absorption measurements of the liberated phenolphtalein at 544)nm as described in the Methods. -

-

E f f e c t of substrate and s a c c h a r o - l A - l a c t o n e concentrations : at pH 5.0 i n a 0.1 M sodium acetate buffer, the r e a c t i o n appeared to follow the Michaelis-Menten kinetics. The K~t value for p h e n o l p h t a l e i n - g l u c u r o n i d e was 0.8 _ 0.05. 10 .3 M. Saccharo1,4dactone b e h a v e d as a competitive i n h i b i t o r w i t h a II~ value of 1.5 _+ 0.05. 10~6 M at 9 H 5.0 i n a 0.1 M s o d i u m acetate buffer. Molecular w e i g h t of 6-glucuronidase. The molec u l a r weight of h u m a n lettkacyte ~-glucuronidase was d e t e r m i n e d b y gel filtration on a Sepharose 6 B c o l u m n (110 c m × 1.5 cm ; e l u a n t 0.05 M Tris-HCl ; 0.1 M NaC1, pH 7.4 buffer) on a sample o b t a i n e d after step n ° 4 : it was 315,000. The follow i n g m a r k e r s were e m p l o y e d for the c a l i b r a t i o n c u r v e : f e r r i t i n e (480,000), cata~lase (240,000) a n d aldolase (147,000).

546

J.-P. Pdrin and P, JolI~s.

Discussion.

M a m m a l i a n ,~-glucuronidase has been d e s c r i b e d as a l y s o s o m a l e n z y m e ; h o w e v e r its p r e s e n c e in a different f o r m in t h e m i c r o s o m a l f r a c t i o n has been r e p o r t e d f o r s o m e m u r i n e and r a b b i t tissues [23, 24, 25, 26]. Studies devoted to rat l i v e r lysosomal and m i c r o s o m a l !~-glucuronidases h a v e s h o w n that the latter c o u l d be released by t r e a t m e n t s w i t h detergents [23] w h e r e a s the f o r m e r w e r e e x t r a c t e d by o s m o t i c shock [21]. Since 90 p e r cent of the a c t i v i t y of the h o m o g e n a t e is r e c o v e r e d after the c e n t r i f u g a t i o n of the h u m a n leukocytes extracts, it is most likely that the e n z y m e w e o b t a i n e d orig i n a t e d f r o m both lysosomes and m i c r o s o m e s (if both f o r m s of the e n z y m e exist). H o w e v e r any attempt to c h a r a c t e r i z e these t w o forms in our extracts on p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s in the Davis [27] system was unsuccessful (unpublish e d results) ; e v e r y e x t r a c t gave rise to a b r o a d active band. Some r e p o r t s on rat l i v e r [28, 29] and rabbit l i v e r [26] ~-glucuronidases i n d i c a t e d that m i c r o s o m a l and l y s o s o m a l f(>rms of the e n z y m e are v e r y si.milar on inmmno'logic an'd c a t a l y t i c points of view. Thus it did not seem n e c e s s a r y to us, in this p r i m a r y c h a r a c t e r i z a t i o n of the catalytic p r o p e r t i e s of t h e h u m a n l e u k o c y t e f3-glucuronidase, to give some p a r t i c u l a r attention to the subceHular o r i g i n of the enzyme. The Ku value of h u m a n l e u k o c y t e .~-glucuronidase for phenolp~htalein-glucuronide, 0.8. 10-3 M, agrees e x a c t l y w i t h the values r e p o r t e d for placental [8] a n d s y n o v i a l fulid [93 h u m a n ,~-glucur o n i d a s e s but was t w o times h i g h e r t h a n that rep o r t e d for the h u m a n l i v e r e n z y m e [7]. S a c c h a r o 1,4-1actone e x h i b i t e d a c o m p e t i t i v e i n h i b i t o r y effect ; the iK~ value for h u m a n l e u k o c y t e ~-glucur o n i d a s e was h o w e v e r d i f f e r e n t f r o m those r e p o r ted for other m a m m a l i a n ~-glucuronidases [25, 263. The pH and t e m p e r a t u r e a c t i v i t y curves w e r e in a g r e e m e n t w i t h results p u b l i s h e d for h u m a n l i v e r ~-glucuroni, dase [7]. T h e e n z y m e ~was i n a c t i v a t e d at t e m p e r a t u r e s above 60°,C a n d p h o s p h a t e - c i t r a t e buffers w e r e i n h i b i t o r s w h e n c o m p a r e d to acetate buffers. T h e m o l e c u l a r w e i g h t of 315,000 for the a c t i v e e n z y m e ,calculated after gel f i l t r a t i o n on Sepharose 6 B w a s in a g r e e m e n t w i t h the values r e p o r ted for the m o u s e k i d n e y (300,000) [253, r a b b i t l i v e r (300,000) [14], r a t ~liver lysosomes (280,000) [21] a n d r a b b i t b r a i n (266,000) [303 e n z y m e s but s e e m e d s o m e w h a t h i g h e r t h a n the value r e p o r t e d for h u m a n l i v e r ,~-glucuronidase (218,000) [7]. BIOCHIMIE, 1978, 60, n ° 5.

This d i s c r e p a n c y m i g h t be due to differences in the t e c h n i q u e s used for the m o l e c u l a r w e i g h t determinations.

Acknowledgements. The authors t h a n k Miss M, Rougeot for s k i l f u l technical assistance and Dr, J. Saint-Blancard for donating n u m e r o u s h u m a n leukocyte samples. J.-P, P, is Chargd de Recherche 5 I'I.N.S,E.R.M. This research was s u p p o r t e d by the I.N.S.E,R.M. (troupe U-116), the C.N.R~. (E.R. N ° 102) and the Fortdalton pour la Recherche Mgdicale Fran~aise.

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