Isolation and partial purification of a glutathione peroxidase from pig blood platelets

A C T A U N I V E R S I T A T I S L O D Z I E N S I S

FOLIA BIOCHIMICA ET BIOPHYSICA 11, 1996

Halina M . Żbikowska, Barbara Wachowicz

ISOLATION AND PARTIAL PURIFICATION OF A GLUTATHIONE PEROXIDASE

FROM PIG BLOOD PLATELETS

An enzyme, glutathione peroxidase from pig platelets by the method of Mad- dipati and M amett (1987) was isolated and partially characterized. The purifica­ tion procedure involved ammonium sulphate precipitation, hydrophobic chromato­ graphy on phenyl- Sepharose CL-4B and anion exchange chrom atography. SDS-electrophoresis of the enzyme obtained from the final chromatographic step revealed a 23 K D a subunit. It was established that selenium -dependent GSH-Px in platelets was predominant and showed about 86% of the total glutathione peroxidase activity. Sodium selenite enhanced the activity of the enzyme whereas copper ions, N-ethylmaleimide and dsplatin were the inhibitors of the GSH-Px activity.

1. INTRODUCTION

G lutathione peroxidase (G SH -Px) activity was found in various m am ­ m alian tissues and cells [I, 2]. A lthough it has also been isolated from different sources [3, 4, 5] little d a ta exist on platelet G S H -Px. Blood platelets contain a large am ount o f selenium (0.5-0.7 /zg/g wet weight) [6, 7, 8] and the selenium-dependent glutathione peroxidase is m ore active in these cells than in red blood cells [1, 3, 9]. G S H -P x assay can be used for determ ination o f selenium content in hum an beings and anim als [I]. G S H -Px is involved in the m etabolism o f fatty acid hydroperoxides and cyclic endoperoxides formed respectively, via the lipoxygenase and the cyclooxygenase pathw ays [10, 11]. In view o f the im portant role o f G S H -P x in platelet m etabolism we have undertaken isolation and purification o f the enzyme from pig platelets to study its physical and kinetic properties. O ur prelim inary results are presented in this paper.

2. MATERIALS AND METHODS

The enzyme was isolated from washed pig blood platelets as described by M a d d i p a t i and M a r n e 11 [4] for the purfication o f hum an plasm a G S H -P x. All purfication steps were performed at 4°C. C rude extracts of the enzyme were obtained by three cycles of freezing (-20°C) and thaw ing (37°C) of pig platelets. Briefly, purfication procedure included precipitation o f the platelet cytosol protein with ammonium sulfate (20-60% saturation). Am m onium sulfate fraction dissolved in buffer A (0.1 M T ris-H C l, 0.2 M am m onium sulphate, 5 m M ED TA , 1 mM GSH , pH 7.2) was then applied on the column with phenyl-Sepharose C L -4 B and washed extensively until the 280 nm absorbance o f the eluate was equal to th at of the buffer. Afterw ards the column was subjected to a linear gradient elution between 0.1-0.01 M T ris-H C l, pH 7.2. Active fractions (2 ml) were pooled and co n cen trated (YM - 10 m em brane); the buffer w as changed to 0.2 M Tris-H C l, 5 mM EDTA, 1 mM GSH, pH 8.0 (buffer B). The concentrated peroxidase was then applied to a D E A E-Sephadex A -50 column. A fter washing the column with about 200 ml of buffer B the enzyme was eluted with a linear gradient, between 0.2 M -0.4 M T ris-H C l, pH 8.0, both containing 5 mM ED TA and 1 m M GSH. Peroxidase acive fractions (1.6 m l) were pooled and concentrated to about 5 ml. G lutathione peroxidase activity tow ards cumene and t-b u ty l hydroperoxides was m easured as described by Paglia and Valentine in the presence o f sodium selenite (100 /iM ), C u S 0 4 (20 m M ), NEM (100 m M ), cisplatinum (30 ¡ M ) and com pared with control [12], Protein was estimated according to L o w r y et al. [13], S D S-P A G E was performed according to the procedure of L a e m m l i [14] using 14% acrylamide gel.

3. RESULTS AND DISCUSSION

A n enzyme with a glutathione peroxidase activity was isolated from pig blood platelets and partially purfied. The enzyme was similar to G S H -P x obtained from hum an platelets by Ram os M artinez et al [3], The typical elution pattern of the phenyl-Sepharose CL-4B column is presented in Fig. 1. The profile shows only the gradient elution o f the column. H ydrophobic interaction chrom atography on phenyl-Sepharose C L -4B proved to be a key step th at eliminated m ost o f the contam inating protein.

Fig. 2 shows the anion exchange chrom atography of concentrated active fractions on D E A E-Sephadex A -50. The peroxidase was eluted as a single peak which m ay suggest the absence o f multiple forms o f the platelet

A b s o rb a n c e at 28 0 nm A b s or b an c e at 28 0 nm Absorbance Activity 0.70 0.60 0.50 I CO 0.40 _c 0.30 >» > o 0.20 < 0.10 0.00 10 15 20 No fraction

1. Chromatography on phenyl-Sepharose CL-4B. The profile shows only the gradient elution of the column. Details are given under Materials and Methods

Absorbance ■ - A ctivity .E ut E >. > O < No fraction

enzyme. A summ ary of the purification steps of G S H -P x activity from pig blood platelets is shown in T able 1.

T a b l e 1 Purification of pig platelet glutathione peroxidase

Purification step Protein (mg) Total activity (units) Specific activity (units/mg) Recovery (%) Purification (-fold) Crude extract 91.1 16.61 0.18 100 20-60% (NH4)2S 0 4 13.2 8.64 0.65 52 3.6 Phenyl-Sepharose 5.6 8.14 1.46 49 8.1 DEAE-Sephadex A-50 0.9 7.73 8.60 46 48.0

D uring our experiments we om itted gel filtration on Sephadex G -200, comm only used in purfication o f G S H -Px, because the recovery o f the enzyme was very low. The m olecular m ass of the platelet G S H -P x subunit determ ined by SD S-PA G E on 14% acrylamide gel in the presence of /J-m ercaptoethanol was about 23 kD a (Fig. 3).

kDa 9 4 67 f — — 60 kDa 30

mmm

Fig. 3. SDS-PAGE of purified pig platelet GSH-Px. Lines: 1) LM W -standards (phosphorylase b, 94 kDa; bovine serum albumin, 67 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; soybean trypsin inhibitor, 20.1 kDa; a-lactalbumin, 14.4 kDa); 2) platelet GSH-Px after purfication on phenyl-Sepharose, reduced, 3) non-reduced and 4) platelet GSH-Px after the

Using two different substrates (cumene and t-butyl hydroperoxides) in estim ation o f G S H -P x activity we assessed th a t in platelets the sele­ nium -dependent G S H -P x was predom inant and exhibited abo ut 86% of the total activity. Sodium selenite enhanced the G S H -P x activity. A t the selenite concentration o f 100 /zM an about 20% increase was achieved. C opper ions (C u S 0 4), N -ethylm aleim ide (NEM ) and cis-platinum reduced the activity of platelet G S H -P x (Fig. 4).

Fig. 4. The effect of different factors on the activity of pig blood platelet glutathione peroxidase

F o r the enzyme (isolated in the presence o f PM SF) K m value calculated from Lineweaver-Burk plots were: 5.9 mM for cumene hydroperoxide, 10 m M for t-b u ty l hydroperoxide, 0.833 m M for hydrogen peroxide and 0.375 m M for GSH .

F u rth er studies on characterization o f S G H -P x from pig platelets are in progress.

4. REFERENCES

[1] C a r m a g n o l F., S i n e t P. M., J e r o m e H. (1983), Biochim. Biophys. Acta, 759, 49-57. [2] A a s e t h J., F r e y H., G l a t t r e E., N o r h e i m G., R i n g s t a d J., T h o m a s s e n Y.

(1990), Biol. Trace Element Res., 24, 147-152.

[3] R a m o s M a r t i n e z J. J., L a u n a y J . M., D r e u x C . (1980), Thromb. Res., 19, 73—83. [4] M a d d i p a t i K. R., M a r n e t t L. J. (1987), J. Biol. Chem., 262, 17398-17403.

[5] T a k a h a s h i K., A v i s s a r N., W h i t i n J., C o h e n H. (1987), Arch. Biochem. Biophys., 256, 677-686. [6] K a s p e r e k K., I y e n g a r G. V., K i e m J., B o r b e r g H., F e i n e n d e g e n L. E. (1979), Clin. Chem., 25, 711-715. [7] K i r e m i d i j a n - S c h u m a c h e r L., S t o t s k y G. (1987), Environment Res., 42, 227-303. [8] W a c h o w i c z B. (1989), „Kosmos” , 38, 319-329.

[9] N e v e J., V e r t o n g e n F., C a p e l P. (1988), Amer. J. Clin. Nutr., 48, 139-143. [10] V e r i c e l E., R e y C., C a l z a d a C., H a o n d P., C h a p u y P. H., L a g a r d e M.

(1992), „Prostaglandis” , 43, 75-85.

[11] H a t z e l m a n A., S c h a t z M., U l l r i c h V. (1989), Eur. J. Biochem., 180, 527-533. [12] P a g l i a D. E., V a l e n t i n e V. N. (1967), J. Lab. Clin. Med., 70, 158-169.

[13] L o w r y O. H., R o s e b r o u g h N. J., F a r r A. L., R a n d a l l R. J. (1951), J. Biol. Chem., 193, 265-275.

[14] L a e m m l i U. K. (1970), „Nature” , 227, 680-685.

Came in editorial office Department of Biochemistry

„Folia biochimica et biophysica” Institute of Biochemistry

4.07.1994 University of Lodz

Poland

Halina M. Żbikowska, Barbara Wachowicz

IZOLOW ANIE I CZĘŚCIOW E OCZYSZCZANIE PEROKSYDAZY GLUT ATIO NO W E.I Z PŁYTEK KRWI ŚWINI

Metodą Maddipati i Mamett (1987) wyizolowano i częściowo scharakteryzowano peroksydazę glutationową z płytek krwi świni. Proces oczyszczania przebiegał kolejno poprzez wysalanie siarczanem amonu, hydrofobową chromatografię na złożu Phenyl-Sepharose CL-4B oraz chromatografię jonowymienną. Elektroforeza w żelu poliakryloamidowym z SDS (w obecności /3-merkaptoetanolu) enzymu otrzymanego w ostatnim etapie oczyszczania ujawniła obecność podjednostki o masie cząsteczkowej 23 kDa. Wykazano, że w płytkach dominującą formą G SH-Px jest selenozależna peroksydaza glutationowa, która wykazuje ok. 86% całkowitej aktywności tego enzymu. Selenin sodowy powodował wzrost aktywności otrzymanego enzymu, podczas gdy jony miedzi, NEM i cisplatyna była inhibitorami peroksydazy glutationowej.