The peroxidatic and catalatic activity of catalase in normal and acatalasemic mouse liver

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Biochimica et Biophysica Acta, 633 (1980) 317--322 © Elsevier/North-Holland Biomedical Press

BBA 29463

THE P E R O X I D A T I C AND CATALATIC ACTIVITY OF CATALASE IN N O R M A L AND ACATALASEMIC MOUSE L I V E R

SATISH K. SRIVASTAVA and NASEEM H. ANSARI

Department of Human Biological Chemistry and Genetics, Division of Human Genetics, C3-14 Child Health Center, The University of Texas Medical Branch, Galveston, TX 77550 (U.S.A.) (Received July 24th, 1980)

Key words: Peroxidatic activity; Catalatic activity; Acatalesemia; Catalase; (Mouse liver)

Summary Monomeric, dimeric and tetrameric forms of mouse liver catalase have been shown to express peroxidatic activity while the tetrameric form expresses the catalatic activity. Autosomally inherited acatalasemia, produced by X-ray irradiation of mice results in almost complete loss of catalatic activity of catalase b u t has no effect on the peroxidatic activity. Liver catalase from normal and acatalasemic mice was purified by following the catalatic and peroxidatic activity, respectively. Antiserum produced in rabbit against catalase from normal mouse completely precipitated the catalatic and peroxidatic activity from normal liver, and peroxidatic activity from the acatalasemic liver homogenate. Similar results were obtained when antiserum against peroxidase from acatalasemic mice was used. These studies indicate that acatalasemia in mice is due to a structural gene mutation which leads to synthesis of structurally altered catalase subunits. The altered subunits express peroxidatic activity b u t do not combine to form a tetramer which expresses catalatic activity.

Introduction

Catalase was first described in mammalian tissues by Thenard in 1818 b u t the role of this enzyme has never been well established. It has been postulated by various investigators [1,2] that catalase exerts a dual function dependent u p o n the steady state concentration of H202. At higher concentrations of H202 it expresses catalatic activity, whereas at lower concentrations of H202 and in the presence of a hydrogen d o n o r it expresses peroxidatic activity [3--5]. However, the conditions under which peroxidatic activity is expressed

318 and the physiological significance of this activity have never been systematically studied. The demonstration of acatalasemia in several Japanese families, and subsequently in European families, provided the first good model for studying the role of this enzyme. Subsequently, the production of a catalase-deficient strain of mice from a wild strain (Cs a) by X-ray irradiation provided a good animal model for the study of catalase deficiency. Like human acatalasemia, the acatalasemic mice have 1--3% of residual catalatic activity. Besides this experimental model, ducks have also been shown to be catalase-deficient [6]. Based on immunological studies using antiserum raised against mouse hemolysates, Feinstein et al. [ 7], reported that acatalasemia in mice is due to a structural gene mutation. On the other hand, acatalasia in man has been proposed to be due to 'controller gene disease' rather than a structural gene defect [8]. In the present studies, we have demonstrated that in acatalasemic mice structurally altered catalase subunits are synthesized. These subunits express peroxidatic activity b u t do n o t combine to form a tetramer which expresses catalatic activity. Materials and Methods L,fl-3,4-Dihydroxyphenylalanine and 3-amino-l,2,4,triazole were purchased from Sigma Chemical Co., St. Louis, MO. All other chemicals were of reagent grade. The Ouchterlony plates were purchased from Hyland Laboratories, CA. Two strains of mice, normal (C3H-Cs~-ANL) and acatalasemic (C3H-Csb-ANL) were obtained from Argonne National Laboratory, Argonne, IL. The acatalasemic strain of mice was produced by exposing the mice (C3H-Csa-ANL) to X-ray irradiation [9,10] which resulted in a b o u t a 99% loss of erythrocyte catalase activity. Preparation o f tissue ex tract. The mice were anesthetized with ether, and the liver was dissected o u t and stored frozen until needed. The liver was homogenized at 4°C under a continuous stream of nitrogen in 50 mM potassium phosphate buffer, pH 7,4 using a Potter-Elvehjem glass homogenizer to make a 10% homogenate. The homogenate was centrifuged at 10 000 X g for 45 min at 4°C using a Sorvall centrifuge, Model RC-5. The supernatants were used for the purification of catalase and for immunotitration. Enzyme determinations. The enzyme determinations were performed at 37°C using a Gilford Recording spectrophotometer, Model 2400-2. Catalatic activity o f catalase. The catalatic activity of catalase was assayed by following the rate of decomposition of H202 at 230 nm [11]. 1 ml of reaction mixture containing 50 mM Tris-HC1 buffer, pH 8.0, with 5 mM EDTA, and 9 mM H20: was incubated for 10 min at 37°C, and the reaction was started by adding the enzyme. The decrease in absorbance was monitored at 230 nm for 3 min. 1 unit of enzyme is defined.as the a m o u n t which brings a b o u t the cleavage of 1 # m o l of H202/min. Peroxidatic activity o f catalase. The peroxidatic activity was determined by following the formation of dopaquinone from L fl-3,4-dihydroxyphenylalanine at 470 nm [5]. 1 ml of reaction mixture containing 100 mM potassium phosphate buffer, pH 7.0, 1 mM H202 and 1 mM L,/3-3,4-dihydroxyphenylalanine was incubated at 37°C for 10 min. The reaction was started by adding the

319 enzyme and the increase in absorbance was measured at 470 nm. A unit of enzyme is defined as the a m o u n t which causes a change of 1.0 absorbance unit/ minute at 470 nm. Inhibition by 3-amino-l,2,4-triazole. Inhibition of the catalatic and peroxidatic activities was performed using the purified or the partially purified (hemoglobin-free) enzyme preparations. The enzymes were preincubated at 37°C with 50 mM final concentration of 3-amino-1,2,4-triazole for 10 min. The reaction was started by the addition of 9 mM H:O2 for catalatic activity and 1 mM H20: for the peroxidatic activity. Purification of the antigens. The 10 000 × g supernatant was mixed with 50 ml of DEAE-cellulose (DE-52) equilibrated with 1.5 mM potassium phosphate buffer, pH 6.9. The enzymes were eluted with 100 mM phosphate buffer, pH 6.9. The eluate was brought to 70% saturation by adding solid ammonium sulphate. The precipitate was dissolved in 1.5 mM phosphate buffer, pH 6.9, extensively dialyzed against the same buffer and centrifuged before passing through a DE-52 column (1.5 × 30 cm; flow rate, 40 ml/h) and 4-ml fractions were collected. The column was washed with 150 ml of the equilibrating buffer and the enzymes were eluted with a linear gradient of 0--100 n,M phosphate buffer, pH 6.9. The fractions having enzyme activity were dialyzed against 10 mM sodium acetate, pH 4.8, and passed through a CM-ceUulose (CM-52) column (2.5 × 30 cm; flow rate, 40 ml/h). The resin was pre-equilibrated with the dialyzing buffer. The column was washed with 200 ml of the equilibrating buffer and eluted with a linear gradient of 200 ml each of 50 mM potassium phosphate, pH 6.8, and I 0 0 mM sodium acetate, pH 4.8. The fractions containing the enzyme activity were pooled, concentrated by ultrafiltration using Amicon ultrafiltration equipment and passed through a Sephadex G-150 column by upward flow at 18 ml/h. The fractions containing enzyme activity were pooled. Aliquots were used for immunizing t h e rabbit and for polyacrylamide gel electrophoresis to assess the purity of the enzymes. Polyacrylamide gel electrophoresis was performed as described by Davis [12]. Protein was determined b y the m e t h o d of Bradford [13]. Immunological studies. The immunization of rabbits was achieved by mixing 1.5 ml of protein solution containing a b o u t 100 pg protein with equal a m o u n t of Freund's complete adjuvant, vortexing, and injecting half of it intradermally at 10 places in the back of the rabbit. The remainder was divided into two equal portions and t w o booster doses were injected 15 days apart after the first injection. The rabbits were subsequently bled through the ear vein and the serum was separated and heat-inactivated as described previously [14]. The serum was filtered through a Millipore filter and stored in sterile containers. The double immunodiffusion studies were performed as described earlier. In all cases the antiserum was placed in the center well and the antigens in the outer well. F o r immunotitration studies, freshly prepared 1 0 0 0 0 × g supernatant of 10% liver homogenates was used. The incubation mixture in a total volume of 0.4 ml contained 0.02 ml of 0.3% IgG in 10 mM potassium phosphate, pH 7.0, and approx. 100 mU of peroxidatic activity from the tissues of both acatalasemic and normal mice or a b o u t 250 U of catalase activity in the case of normal mice. Varying amounts of antiserum were used and the volume was made up

320 with 10 mM potassium phosphate, pH 7.0. The samples were saturated with N: and incubated overnight at 4°C. They were centrifuged at 10 000 X g for 45 min and the supernatants were assayed for enzyme activity. Molecular weight determination. For molecular weight determination Sephadex G-150 and G-200 columns (1.6 X 60 cm) were calibrated using c y t o c h r o m e c, ribonuclease, ovalbumin, hemoglobin and aldolase at an upward flow rate (18.0 ml/h), 2-ml fractions. Blue dextran was passed to determine the void volume. Results

The catalase and peroxidase were purified from a b o u t 4.0 g of liver by following the catalatic or peroxidatic activity. The yield of catalase from normal liver was a b o u t 300 pg protein and that of peroxidase from the acatalasemic liver was a b o u t 200 pg. Both of the enzymes were apparently homogeneous because only one protein band was observed in polyacrylamide gel electrophoresis at pH 8.6. Both the proteins had the peroxidatic activity but only the protein purified from the normal liver had the catalatic activity. The molecular weight of the protein from acatalasemic mice was 68 000 and that from normal mice was a b o u t 240 000. In double immunodiffusion studies, antisera raised against the peroxidase from acatalasemic mouse liver and catalase from normal liver gave precipitin lines with liver supernatants from normal as well as acatalasemic mice {Fig. 1). Complete precipitation of catalatic and peroxidatic activities from normal liver and peroxidatic activity from catalase-deficient liver was observed in the immunotitration experiments using antiserum against catalase from normal mouse (Fig. 2). Similar results were obtained when antiserum against peroxidase was used. To precipitate the same a m o u n t of peroxidatic activity from

Fig. 1. T h e i m m u n o d i f f u s i o n s t u d i e s w e r e c a r r i e d o u t as d e s c r i b e d i n t h e t e x t . O u t e r w e l l s 1 a n d 3 c o n t a i n e d 1 0 ~1 o f n o r m a l m o u s e l i v e r h o m o g e n a t e a n d wells 2 a n d 4 c o n t a i n e d a e a t a l a s e m i c m o u s e liver homogenate. Left center well contained anti-mouse l i v e r c a t a l a s e a n d r i g h t c e n t e r well c o n t a i n e d a n t i mouse liver peroxidase antiserum.

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Fig. 2. T h e i n c u b a t i o n m i x t u r e s , t o t a l v o l u m e 4 0 0 pl, c o n t a i n e d 1 0 0 - - 1 2 0 m U o f p e r o x i d a t i c a c t i v i t y o r 2 5 0 U o f c a t a l a t i c a c t i v i t y , 2 0 #1 o f 0 . 3 % h u m a n 7 - g l o b u l i n f r a c t i o n o b t a i n e d f r o m S i g m a C h e m i c a l Co. with various amounts of anticatalase antiserum and 10 mM phosphate buffer, pH 7.0. The samples were incubated overnight at 4°C, centrifuged at 10000 × g for 45 min and supernatants were assayed for e n z y m e ( p e r o x i d a t i c a n d c a t a l a t i c ) a c t i v i t y . N o r m a l liver p e r o x i d a t i c a c t i v i t y (X ×) a n d c a t a l a t i c a c t i v i t y (o o) a n d a c a t a l a s e m i c liver p e r o x i d a t i c a c t i v i t y (m m).

acatalasemic samples the a m o u n t of anticatalase antiserum required was a b o u t four times greater than for the normal samples (Fig. 2). Aminotriazole (50 mM), a known inhibitor of catalase, completely inhibited the catalatic and peroxidatic activities of normal and peroxidatic activity of acatalasemic mouse liver. Discussion Catalase is known to have both catalatic and peroxidatic activities. However, the conditions under which it expresses t h e peroxidatic activity and its physiological significance are n o t clearly defined. Our previous studies have indicated that acatalasemic mice have almost normal levels of peroxidatic activity, and normal red cell life spa~ and are able to take care of oxidative stress by drugs, such as phenylhydrazine which is known to generate H202 and superoxide anion, in the same way as normal mice [15]. In addition, the a m o u n t of peroxidatic activity in the normal and acatalasemic mice tissues was comparable and significant amounts of peroxidatic activity is present in ducks which are genetically acatalasemic and have no apparent hemolytic anemia. These studies would indicate that the catalatic activity of catalase is n o t important. In the present studies using antiserum raised against purified catalase from normal mouse liver and peroxidase from acatalasemic mouse liver, we have

322 demonstrated that both the antiserum in double immunodiffusion studies precipitate catalase from normal mouse liver and peroxidase from acatalasemic mouse liver. Furthermore, the immunotitration studies demonstrate that anticatalase antiserum prepared against normal mouse liver catalase precipitates the peroxidatic activity from acatalasemic mouse liver and the antiperoxidase antiserum completely precipitates the catalase activity from normal mouse tissues. This would indicate that catalase protein which has peroxidatic activity but no catalatic activity, is indeed, synthesized in acatalasemic mice. It is therefore proposed that autosomally inherited acatalasemia in mice is due to a structural gene mutation. This probably results in the synthesis of structurally altered catalase subunits. The alteration is such that the peroxidatic activity site is not affected. The altered monomers of catalase do no.t combine to form tetrameric catalase which has catalatic activity. The peroxidatic activity, on the other hand, is expressed by monomeric and dimeric as well as tetrameric catalase. The requirement of almost four times as much anticatalase antiserum to precipitate the same a m o u n t of peroxidatic activity from the normal mouse liver homogenate as compared to acatalasemic mouse further substantiates this interpretation. It is obvious that the precipitation of monomers from acatalasemic mouse liver peroxidase will require more antiserum than the precipitation of monomeric, dimeric and tetrameric catalase which express peroxidatic activity.

Acknowledgments This work was supported in part by The National Institutes of Health grants EY 01677 and NS 14966 01A1.

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