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Isolation of glyoxalase II from two different compartments of rat liver mitochondria. Kinetic and immunochemical characterization of the enzymes
Biochimica et Biophysica Acta, 993 (1989) 7-11
7
Elsevier BBAGEN 23180
Isolation of glyoxalase II from two different compartments of rat liver mitochondria. Kinetic and immunochemical characterization of the enzymes V i n c e n z o T a l e s a ~.3 L a s s e U o t i l a ~.2, M a r t t i K o i v u s a l o a, G i o v a n n i P r l n c i p a t o 3, Elvio Giovannini 3 and Gabriella Rosi 3 Departments of t Medical Chemistry and : Clinical Chemistry. University of Helsinki. Helsinki (Finland) and 3 Department of Experimental Medicine. Division of Cell and Molecular Biology, University o] Perugia, Perugia (Italy)
(Received 6 January 1989) (Revised manuscriptreceived25 April 1989)
Key words: Glyoxalase11; Mitochondrion;Kinetic constant; Immunochemicalcharacterization;(Rat liver) Two separate pools of glyoxalase II were demonstrated in rat liver mitochondria, one in the intermembrane space and the other in the matrix. The enzyme was purified from both sources by affinity chromatography on S(earbobenzoxy)ghitathione-Affi-Gel 10. From both crude and purified preparations polyacrylamide gel-electrophoresis resolved multiple forms of glyoxalase II, two from the intermembrane space and five from the matrix. Among the thioesters of glutathione tested as substrates, S-D-lactoylglutathione was hydrolyzed most efficiently by the enzymes from both sources. Significant differences were observed in the speeificities between the intermembrane space and matrix enzymes with S-acetoacetylglutathione, S-acetylglutathione, S-propionylglutathione and S-succinylglutathione as substrates. Pure glyoxalase I! from rat liver eytosol was chemically polymerized and used as antigen. Antibodies were raised in rabbits and the antiserum was used for comparison of the two purified mitochondrial enzymes with cytosolic glyoxalase !I by immunoblotling. The enzyme purified from the intermembrane space cross-reacted with the antiserum, but the matrix glyoxalase I! did not. The results give ev!dence for the presence in rat liver mitochondria of two species of glyoxalase II with differing characteristics. Only the enzyme from the intermembrane space appears to resemble the cytosolic glyoxalase 11 forms.
Introduction Glyoxalase I (EC 4.4.1.5) and glyoxalase II (EC 3.1.2.6) constitute the well-established glyoxalase system ubiquitously present in mammalian cell cytosol [1,2]. This system converts 2-ketoaldehydes to the corresponding 2-hydroxycarboxylic acids and is able to form a main line of cellular defense against these cytotoxic compounds [3]. 2-Ketoaldehydes can be formed en,zlogenously from various metabolites, e.g, from aminoacetone [4], from ketone bodies [5], or from dihydroxyacetone phosphate by methytglyoxal synthase [6,7]. The activity of the glyoxalase system changes during cell cycle [8] and under some pathologica! conditions
Abbreviations: DTNB, 5,5'-dithiobis(2-nitrobenzoicacid); SDS, sodium dodecyl sulfate. Correspondence: V. Talesa, Dipartimento di Medicina Sperimentale, Sezione d] Biologiacellulare e molecolare. Via del Giochetto, 06100 Perugia, Italy.
[9,10], b-'t it is glyoxalase I1 which shows the widest differences in activity. Principato et al. [11,12] found a decreased activity of glyoxalase 1I in regenerating rat liver and chicken liver during embryo development. Another possible function of the glyoxalase system lies in modulating the intracellular levels of S-Dlactoylglutathione. This thioester is biologically active, having an effect on histamine release from leukocytes [13], microtubule assembly [14!, pathology of diabetes [9] and in vitro control of leukemia cell proliferation [8]. In spite of the above results suggesting that the glyoxalase enzymes can play important roles in cell metabolism, their physiological functions are far from understood. New perspectives may arise from our recent demonstration of glyoxalase 11 in rat liver mitochondria [15]. These organelles contained as much as 10-15% of the total activity in rat liver. Mitochondrial glyoxalase 1I consists of fivc forms, three of which differ from the cytosolic enzymes in isoelectric point values and in electrophoretic mobilities [15]. Preliminary data on the intramitochondrial distribution of.glyoxalase 11
0304-4165/89/$03.50 © 1989 ElsevierScience PublishersB.V. (Biomedica! Division)
8 indicated that the activity is found in both the matrix and the intermembrane space [15]. In this paper we report purification and some properties of glyoxalase II fro.~_ these two mitochondrial compartments. Materials and Methods
Materials GSH, DTNB, bovine serum albumin, d i # t o n i n and 1-cyclohexyl-3(2-morpholinoet hyl)carbodiimide methop-toluene sulfonate were purchased from Sigma (U.S.A.), Freund's adjuvants from Gibco (U.S.A.), peroxidaseantiperoxidase complex from Dakopatts (Denmark) and Affi-Gel 10 from Bio-Rad (U.S.A.). The thioesters of glutathione used were synthesized and purified as previously described [16]. S-(Carbobenzoxy)glutathione was synthesized following the method of Hsu and Norton [17] and coupled to Affi-Gel 10 according to the instructions of the manufacturer of the gel. Other chemicals used were analytical grade products from various sources. All solutions were made up in twice-distilled water,
Purification of mitochondrial glyoxalase lI Livers (about 60 g) from ten 2-month-old rats of both sexes were used. Preparation and sublractionation of the mitochondria into matrix and intermembrane space were carried out following the method of Greenawalt [18]. The amount of digitonin added to the isolated mitochondria was 0.12 mg per mg of mitochondrial protein. Glyoxalase II was purified from the mitochondrial matrix and the intermembrane space according to a modification of the method previously reported [15], by using S-(carbobenzoxy)glutathione coupled to Affi-Gel 10 in the affinity chromatography step.
Preparation of antibodies against cytosolic glyoxalase 11 Glyoxalase II was purified from rat liver cytosol as two separate forms ( a and fl) as described [19]. In order to increase the immunogenic properties of the enzyme, the mixture of the a and fl forms was chemically polymerized as follows. 1-Cyclohexyl-'~-(2-morpholinoethyl)carbodiimide metho-p-to!uene sulfonate (20 mg) was incubated for 18 h at 5 ° C with 4 lug of enzyme dissolved in 5 ml of 10 mM Tris-HCl buffer (pH 8.0). Unreacted carbodiimide was removed on a Sephadex G-25 column (1.5 × 20 cm) equilibrated and eluted with 10 mM Tris-HCl buffer (pH 8.0). The protein peak containing polymerized glyoxalase II (4 ml) was recovered and used for immunization. Four male rabbits of local breed (6 months old) were used, each weighing about 2.5 kg at the beginning of the treatment. PrimaI2," immunization was performed using 1 ml of polymerized glyoxalase II for each rabbit. The immunogen was emulsified in 1 ml of complete Freund's
adjuvant and the mixture was then i~ected into about 50 intradermal sites on the back of the animals. Booster injections were performed at intervals of 14 days using half of the amount of immunogen used in the primary immunization. The immuncgen was emulsified in 1 ml of incomplete Freund's adjuvant and given subcutaneously into four back site~. Antibody titer was assayed by immunoblotting at intervals of 14 days starting from 7 days after the first booster treatment. Binding cap'mity of each antiserum was evaluated from serial dilutions in the presence of 10 #g of purified cytosolic glyoxalase II.
Otlier methods Glyoxalase II activity was assayed by following the decrease of absorbance due to thioester hydrolysis at 240 nm, or at 412 nm by titrating the formation of GSH in the presence of 0.2 mM DTNB [15]. S-D-Lactoyl-, S-acetoacetyl-, S-succinyi-, S-propionyl- and Sacetylglutathione were used as substrates. Molar absorbances at 240 nm in the assay for each substrate were taken from Ref. 16. One enzyme unit (IU) catalyzes the hydrolysis of 1 #mol of S-D-lactoylglutathio.oe/min at the saturating substrate concentration and 20 ° C. Disc gel electrophoresis was performed in 7.5% polyacrylamide gels as described [20] and the gels were stained for enzymatic activity [21]. Polyacrylamide electrophoresis in the presence of SDS (12.5% gels) was performed according to Laemmli [22], immunoblotting was performed according to Towbin [23] and the immunological detection of glyoxalase II was done by using antiserum to cytosolic glyoxalase II in the peroxidase-antiperoxidase complex method [24]. The amount of purified glyoxalase II used was 0.5 IU. Protein was assayed by the method of Lowry et al. [25] or by the biuret reaction [26]. Bovine serum albumin was used as the standard.
Results Glyoxalase II was found in muluple locations in rat liver mitochondria. Most of the activity (64%) was localized in the matrix but an additional significant amount (28%) in the intermembrane space, in accordance with earlier results [15]. Table I shows the purification procedure for glyoxalase II from the two mitochondrial compartments. The same procedure could be used for the enzyme from both the matrix and the intermembrane space. The use of S-(carbobenzoxy)glutathione as affinity-ligand resulted in a high degree of purification. Final specific activity was not obtained for the intermembrane space enzyme due to too low protein content (undetectable) in the purified preparations. The purifications achieved were comparable to those obtained by a slightly different method from whole
9 TABLE I Purification procedure for glyoxalase 11 from rat liver mitochondrial matrix (.4) and intermembrane space (B)
Subfractioaation of the purified mitochondria was performed as described [15,18]. The purifications were carried out from the matrix and intermembrane space fractions as described for whole mitochondria [15] with slight modifications given in text. Glyoxalase il activity was assayed by following the decrease of absorbance at 240 nm due to the enzymatic hydrolysis of S-D-lactoylglutathione. Five concentrations of S-D-lactoylglutathione (0.05-0.40 mM) were used, and Vmax values were calculated from the linear double-reciprocal plots obtained, n.d., not detectable. Purification step
Protein (mg)
Activity (IU)
Specific activity (IU/mg)
Purification factor
Yield (%)
A. Matrix enzyme 100000 x g supernatant Acetone 30-80% S-(Carbobenzoxy)glutathione-A ffi-gel-10
120 53.0 0.13
58.0 54.0 22.0
0.483 1.02 169
1 2.1 350
100 9? 38
B. lntermembrane space enzyme Digitonin supernatant Acetone 30-80% S-(Carbobenzoxy)glutathione-A ffi-Gel- 10
52.9 10.2 n.d.
24.9 24.0 13.0
0.471 2.35 n.d.
1 5 n.d.
100 96 52
mitochondria [15]. T h r o u g h o u t the p u r i f i c a t i o n processes, electrophoresis with e n z y m a t i c staining w a s p e r f o r m e d to detect possible m o d i f i c a t i o n s in glyoxalase II pattern. These could be excluded, since the s a m e pattern, t w o e n z y m e f o r m s f r o m the i n t e r m e m b r a n e s p a c e a n d five f o r m s f r o m the matrix, w a s always resolved in electrophoresis for b o t h c r u d e a n d purified glyoxalase II p r e p a r a t i o n s (Fig. 1). All the i m m u n i z e d r a b b i t s p r o d u c e d a s i g n i f i c a m a m o u n t of a n t i b o d i e s to cytosolic glyoxalase II. T h e a n t i s e r u m with the highest a n t i b o d y titer s h o w e d binding capacity at a dilution range of 1 : 4 0 0 to 1 : 8 0 0 . H i g h e s t values a p p e a r e d 5 - 7 weeks after the p r i m a r y i n u n u n i z a t i o n . G l y o x a l a s e II purified f r o m the t w o
A
B
o
it
m i t o c h o n d r i a l c o m p a r t m e n t s as well as the e n z y m e isolated f r o m cytoso] [19] were studied u s i n g the a n t i s e r u m in the i m m u n o b l o t t i n g technique after S D S electrophoresis. T w o e n z y m e f o r m s were resolved f r o m cytosolic glyoxalase lI, a s t r o n g o r e ( M r = 29000) a n d a w e a k o n e ( M r = 27000) (Fig. 2). T h e e n z y m e f r o m the i n t e r m e m b r a n e space s h o w e d a cross-reaction w i t h the cytosolic a n t i s e r u m . A single e n z y m e f o r m w a s visualized w h i c h h a d a mobility identica] to that of the m a j o r cytosolic f o r m (Fig. 2). In contrast, n o cross-reaction at all w a s o b t a i n e d for the m a t r i x glyoxalase II (Fig. 2). C o n t r o l s p e r f o r m e d w i t h p r e i m m u n e s e r u m were always negative. T h e specificities of the purified e n z y m e s f r o m the i n t e r m e m b r a n e space a n d the m a t r i x were investigated
B
C
w
Fig. 1. Polyacrylarmde disc gel-electrophoresis according to gel system 1 of Maurer [20] of the enzymes from the mitochondrial intermembrane space (gel A) and the mitochondrial matrix (gel B). The gels were stained for glyoxalase 11 activity with $-D-lac~.oylglutathione as the substrate as described by Uotila [21]. The anode is at the bottom of the figure. The position of the tracking dye (Bromophenol blue) is marked by a copper wire.
Fig. 2. lmmunobloning of purified glyoxalase 11 from rat liver cytosol (A), mitochondrial intermembrane space (B) and mitochondrial matrix (C). Slab-gel electrophoresis was performed in 12.5% polyacrylamide in the presence of SDS with the anode at the bottom of the figure. The gel was subsequently subjected to electrophoretic blotting as described in text. hnmunological detection of glyuxalase II by antiserum to rat liver cytosolic glyoxalase I1 was carried out by the peroxidase-antiperoxidase method [24].
10 TABLE II Kinetic constants for the substrates of purified glyoxalase 11 from intermembrane space and matrix of rat liver mitochondria
Activity assays were performed at 240 nm (substrate concentration range 0.05-0.40 raM). Relative Vm~x values with S-n-lactoylglutathione as the substrate have been set to 100. Vma~ and Km values are given as mean±S.D. The last two columns give the statistical comparisons (P values in t-test) of the relative Vm~~ as well as the K m values obtained for a particular sub,irate for the intermembrane space and matrix enzymes, respectively. The values given in parentheses after each substrate indicate the number of independent experiments performed, n.s.. no significant difference. Substrate s-D- Lactoylglutathione S-Acetoacetylglutathione S-Succinylglutathione S-Propionylglutathione S-Acetylglutathione
(10) (10) (6) (9) (7)
Intermembrane space enzyme Vm~ (%) K m (mM) 100 0.5985:0.287 93.2±4.7 0.413+0.157 24.2 :t:8.0 0.186±0.108 30.7+3.2 0.792±0.333 17.6±0.9 0.603±0.326
and compared using five glutathione thiol esters as the substrates. S-D-Lactoylglutathione gave the highest relative V,~, value as well as the highest Vmax/K m ratio for the enzymes from both sources (Table II). The enzymes isolated from the intermembrane space and from the matrix showed statistically significant differences when their relative Vmax and K m values, in comparison to S-D-Lactoylglutathione, were c o m p a r e d for the other four substrates (Table II). All other substrates tested showed, in comparison to S-D-lactoylglutathione, a higher relative Vm~ value for the matrix enzyme than for the intermembrane space enzyme. SAcetoacetylglutathione and S-succinylglutathione showed, in comparison to S-o-lactoylglutathione, a higher Kn~ value for the matrix enzyme than for the intermembrane space enzyme. Acetylcoenzyme A was not used as the substrate by the enzyme from either compartment. Discussion
Polymorphism of glyoxalase II has been reported from the soluble fraction of liver [19,27], brain [19] and red blood cells [21]. In rat liver the activity is distributed between cytosol and mitochondria, in both of which multiple forms of the enzyme have been detected [15]. Glyoxalase II was isolated in this work from the intermembrane space and matrix of purified mitochondria. The former gave two and the latter five electrophoretic forms, under non-denaturating conditions. Using whole mitochondria, a pattern of five forms of glyoxalase II was detected earlier by electrophoresis and isoelectric focusing [15]. These findings are in agreement, since the forms from the intermeplbrane space were electrophoretically indistinguishable from two forms of" the matrix.Under the native elcctrophoretic conditions, the two enzyme forms from lhe intermembrane space also had similar mobilities to cytosolic glyoxalase II forms [19]. The immunoblotting experiments performed also indicate similarity between intermembrane space and cytosolic glyoxalase 1I ue-
Matrix enzyme Vm~ (%) 100 52.6±9.6 38.6±6.3 41.6±8.3 24.8±3.1
K m (mM) 0.3845:0.230 0.634±0.254 0.535+0.268 1.200±0.648 0.600±0.276
P ( Vmax)
P (Kn~)
<0.02 <0.05 < 0.01 < 0.01
n.s. <0.05 <0.05 n.s. n.s.
cause o f the positive cross-reaction of the former with the antiserum to cytosolic glyoxalase II; in addition, the major cytosolic enzyme form and the intermembrane space enzyme had identical mobilities in SDS electrophoresis (Fig. 2). However, because considerably less glyoxalase II antigen was visualized from the intermembrahe space enzyme preparation than from the cytosolic one (Fig. 2), it appears that either the cross-reaction of the former enzyme was incomplete or only part of the intermembrane space enzyme was visualized in the immunoblotting performed. In contrast, ~o cross-reaction with antibodies to cytosolic glyoxalase II was observed for the enzyme from mitochondriai matrix. Therefore, although two of the five matrix enzyme forms observed under native electrophoretic conditions had mobilities identical to those the enzyme forms from cytosol and intermembrane space, all the matrix enzyme forms clearly differ from the cytosolic enzyme forms, and there must be at least a partial difference between the activities from mitochondrial matrix and i n t e r m e m b r a n e space. Thus, liver mitochondria contain at least two pools of glyoxalase II which are immunologically different. Differences between matrix and intermembrane space enzymes are also evident from the specificity studies which were p e r f o r m e d with different glutathione thiol esters as substrates. Both enzyme pools are specific for thioesters of glutathione .and no activity was found using acetylcoenzyme A as the substrate. S-DLactoylglu:athione war the best substrate for lhe two pools of mitochondrial glyoxalase II. The substrate specificity observed in this work for the intermembrane space enzyme resembles that found earlier for the cytosolic enzyme forms [15], whereas that observed for the matrix enzyme is close to the specificity of the enzyme purified from whole mitochondria [15]. These data are in accordance with the results of the immunoblotting experiments. Since our purified mitochondrial preparations did not show any glyoxalase II activity as long as the
mitochondrial m e m b r a n e s remained intact [15], it is unlikely that cytosolic glyoxalase II was included as a contaminant in the mitochondrial intermembrane space preparatiorls used. However, the possibility of a very strong absorption of cytosolic glyoxalase II to the mitochondrial m e m b r a n e cannot completely be ruled out. Regarding the origin of mitochondrial glyoxalase II, it is unlikely that these enzymes are encoded by mitochondrial D N A , since the products of mitochondrial genome are known [28]. Therefore the enzymes are probably expressed by the nuclear genome and undergo post-translational changes during translocation into the '.'gitochondrion. Possible ubiquitous distribution of glyoxalase II in mitochondria is indicated by the presence of this enzyme in these organelles from other animal sources and spinach leaves (unpublished data). Until now, no data have been published to show the presence of the whole glyoxalase system in mitochondria, but our preliminary experiments suggest that glyoxalase I does not exist in rat liver mitochondria. The significance of the presertce of glyoxalase lI in rat liver both in the cytosol and in two c o m p a r t m e n t s of the mitochondrion is not yet clear. The cytosolic enzymes have a n u m b e r of similar properties when investigated from many different species [27]. Similar comparative studies of the mitochondrial glyoxalase II activities appear appropriate for in future studies.
References 1 Racker. E. (1951) J. Biol. Chem. 190, 685-696. 2 Crook, E.M. and Law, K. (1952) Biochem. J. 52, 492-499. 3 Mannervik, B. (1980) in Enzymatic Basis of Detoxification, Voi. II (Jakoby. W.B., ed.), pp. 263-273, Academic Press. New York.
4 Effiott, W.H. (1960) Nature 185, 467-468. 5 Casazza, J.P., Felver, M.E. and Vcech, R.L. (1984) J. Biol. Chem. 259, 231-236. 6 Sato, J., Wang, Y.M. and Van Eys, J. (1980) J. Biol. Chem. 255, 2046-2050. 7 Ray, S. and Ray, M. (1981) J. Biol. Chem. 256, 6230-6233. 8 Thornalley, P.J. and Tisdale, M.3. (1988} Lcukaemia Res., in press. 9 Thornalley, P.J. 0988) in CRC Handbook on Glut,~,'hione Metabolism (Vi~a, J., ed.). in press, CRC, Cleveland, OH. 10 Jerzykowski, T., Winter, R., Matuszewski, W. and Piskorska, D. {1978} Int. J. Biochem. 9, 853-860. 11 Principato, G.B., Locci, P., Rosi, G., Talesa. V. and Giovannini, E. (1983) Biochem. Int. 6. 249-255. 12 Principato, G.B., Bodo, M., Biagioni. M.G., Rosi, G. and Liotti, F.S. (1982) Acta Embryol. Morphol. Exp. 3, 173-179. 13 Gillespie, E. (1979) Nature 277, 135-137. 14 Giltespie, E. (1978) J. Immunol. 121,923-925. 15 Talesa, V., Uotila, L., Koivusalo. M, Prineipato. G., Giovannini. E. and Rosi, G. (1988} Biochim. Biophys. Acta 955, 103-110. 16 Uotila, L. (1977) Methods Enzymol. 77, 424-430. 17 Hsu, Y.R. and Norton. S.J. (1983) J. Med. Chem. 26, 1784-1785. 18 Greenawalt, J.W. (1974) Methods Enzymol. 31. 310-323. 19 Principato. G., Rosi. G., Talesa, V., Giovannini, E. and Uotila, L. (1987) Biochim. Biophys. Acta 911,349-355. 20 Maurer, H.R, (1968) Disk-Electrophoresis, p. 42, Walter de Gruyter, Berlin. 21 Uotila, L. (1979) Biochim. Biophys. Acta 580, 277-288. 22 Laemmli, U.K. (1970) Nature 227. 680-685. 23 Towbin. H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354. 24 Glass. W.F., II, Brigs. R.C. and Hnilica. L.S. {1981) Science 211, 70-72. 25 Lowry. O.H., Rosebrough, N.J.. Farr, A.L. and Randall, R.J. (1951} J. Biol. Chem. 193, 265-275. 26 Goa, J. (1953) Scand. J. Clin. Lab. Invest. 5, 218-233. 27 Principato, G., Rosi, G., Talesa, V,, Giovannini. E. and Norton. S.J. (1987) Enzyme 37. 164-167. 28 Tzagoloff, A. and Myers, A.M. (1986) Annu. Rev. Biochem. 55, 249-285.
7
Elsevier BBAGEN 23180
Isolation of glyoxalase II from two different compartments of rat liver mitochondria. Kinetic and immunochemical characterization of the enzymes V i n c e n z o T a l e s a ~.3 L a s s e U o t i l a ~.2, M a r t t i K o i v u s a l o a, G i o v a n n i P r l n c i p a t o 3, Elvio Giovannini 3 and Gabriella Rosi 3 Departments of t Medical Chemistry and : Clinical Chemistry. University of Helsinki. Helsinki (Finland) and 3 Department of Experimental Medicine. Division of Cell and Molecular Biology, University o] Perugia, Perugia (Italy)
(Received 6 January 1989) (Revised manuscriptreceived25 April 1989)
Key words: Glyoxalase11; Mitochondrion;Kinetic constant; Immunochemicalcharacterization;(Rat liver) Two separate pools of glyoxalase II were demonstrated in rat liver mitochondria, one in the intermembrane space and the other in the matrix. The enzyme was purified from both sources by affinity chromatography on S(earbobenzoxy)ghitathione-Affi-Gel 10. From both crude and purified preparations polyacrylamide gel-electrophoresis resolved multiple forms of glyoxalase II, two from the intermembrane space and five from the matrix. Among the thioesters of glutathione tested as substrates, S-D-lactoylglutathione was hydrolyzed most efficiently by the enzymes from both sources. Significant differences were observed in the speeificities between the intermembrane space and matrix enzymes with S-acetoacetylglutathione, S-acetylglutathione, S-propionylglutathione and S-succinylglutathione as substrates. Pure glyoxalase I! from rat liver eytosol was chemically polymerized and used as antigen. Antibodies were raised in rabbits and the antiserum was used for comparison of the two purified mitochondrial enzymes with cytosolic glyoxalase !I by immunoblotling. The enzyme purified from the intermembrane space cross-reacted with the antiserum, but the matrix glyoxalase I! did not. The results give ev!dence for the presence in rat liver mitochondria of two species of glyoxalase II with differing characteristics. Only the enzyme from the intermembrane space appears to resemble the cytosolic glyoxalase 11 forms.
Introduction Glyoxalase I (EC 4.4.1.5) and glyoxalase II (EC 3.1.2.6) constitute the well-established glyoxalase system ubiquitously present in mammalian cell cytosol [1,2]. This system converts 2-ketoaldehydes to the corresponding 2-hydroxycarboxylic acids and is able to form a main line of cellular defense against these cytotoxic compounds [3]. 2-Ketoaldehydes can be formed en,zlogenously from various metabolites, e.g, from aminoacetone [4], from ketone bodies [5], or from dihydroxyacetone phosphate by methytglyoxal synthase [6,7]. The activity of the glyoxalase system changes during cell cycle [8] and under some pathologica! conditions
Abbreviations: DTNB, 5,5'-dithiobis(2-nitrobenzoicacid); SDS, sodium dodecyl sulfate. Correspondence: V. Talesa, Dipartimento di Medicina Sperimentale, Sezione d] Biologiacellulare e molecolare. Via del Giochetto, 06100 Perugia, Italy.
[9,10], b-'t it is glyoxalase I1 which shows the widest differences in activity. Principato et al. [11,12] found a decreased activity of glyoxalase 1I in regenerating rat liver and chicken liver during embryo development. Another possible function of the glyoxalase system lies in modulating the intracellular levels of S-Dlactoylglutathione. This thioester is biologically active, having an effect on histamine release from leukocytes [13], microtubule assembly [14!, pathology of diabetes [9] and in vitro control of leukemia cell proliferation [8]. In spite of the above results suggesting that the glyoxalase enzymes can play important roles in cell metabolism, their physiological functions are far from understood. New perspectives may arise from our recent demonstration of glyoxalase 11 in rat liver mitochondria [15]. These organelles contained as much as 10-15% of the total activity in rat liver. Mitochondrial glyoxalase 1I consists of fivc forms, three of which differ from the cytosolic enzymes in isoelectric point values and in electrophoretic mobilities [15]. Preliminary data on the intramitochondrial distribution of.glyoxalase 11
0304-4165/89/$03.50 © 1989 ElsevierScience PublishersB.V. (Biomedica! Division)
8 indicated that the activity is found in both the matrix and the intermembrane space [15]. In this paper we report purification and some properties of glyoxalase II fro.~_ these two mitochondrial compartments. Materials and Methods
Materials GSH, DTNB, bovine serum albumin, d i # t o n i n and 1-cyclohexyl-3(2-morpholinoet hyl)carbodiimide methop-toluene sulfonate were purchased from Sigma (U.S.A.), Freund's adjuvants from Gibco (U.S.A.), peroxidaseantiperoxidase complex from Dakopatts (Denmark) and Affi-Gel 10 from Bio-Rad (U.S.A.). The thioesters of glutathione used were synthesized and purified as previously described [16]. S-(Carbobenzoxy)glutathione was synthesized following the method of Hsu and Norton [17] and coupled to Affi-Gel 10 according to the instructions of the manufacturer of the gel. Other chemicals used were analytical grade products from various sources. All solutions were made up in twice-distilled water,
Purification of mitochondrial glyoxalase lI Livers (about 60 g) from ten 2-month-old rats of both sexes were used. Preparation and sublractionation of the mitochondria into matrix and intermembrane space were carried out following the method of Greenawalt [18]. The amount of digitonin added to the isolated mitochondria was 0.12 mg per mg of mitochondrial protein. Glyoxalase II was purified from the mitochondrial matrix and the intermembrane space according to a modification of the method previously reported [15], by using S-(carbobenzoxy)glutathione coupled to Affi-Gel 10 in the affinity chromatography step.
Preparation of antibodies against cytosolic glyoxalase 11 Glyoxalase II was purified from rat liver cytosol as two separate forms ( a and fl) as described [19]. In order to increase the immunogenic properties of the enzyme, the mixture of the a and fl forms was chemically polymerized as follows. 1-Cyclohexyl-'~-(2-morpholinoethyl)carbodiimide metho-p-to!uene sulfonate (20 mg) was incubated for 18 h at 5 ° C with 4 lug of enzyme dissolved in 5 ml of 10 mM Tris-HCl buffer (pH 8.0). Unreacted carbodiimide was removed on a Sephadex G-25 column (1.5 × 20 cm) equilibrated and eluted with 10 mM Tris-HCl buffer (pH 8.0). The protein peak containing polymerized glyoxalase II (4 ml) was recovered and used for immunization. Four male rabbits of local breed (6 months old) were used, each weighing about 2.5 kg at the beginning of the treatment. PrimaI2," immunization was performed using 1 ml of polymerized glyoxalase II for each rabbit. The immunogen was emulsified in 1 ml of complete Freund's
adjuvant and the mixture was then i~ected into about 50 intradermal sites on the back of the animals. Booster injections were performed at intervals of 14 days using half of the amount of immunogen used in the primary immunization. The immuncgen was emulsified in 1 ml of incomplete Freund's adjuvant and given subcutaneously into four back site~. Antibody titer was assayed by immunoblotting at intervals of 14 days starting from 7 days after the first booster treatment. Binding cap'mity of each antiserum was evaluated from serial dilutions in the presence of 10 #g of purified cytosolic glyoxalase II.
Otlier methods Glyoxalase II activity was assayed by following the decrease of absorbance due to thioester hydrolysis at 240 nm, or at 412 nm by titrating the formation of GSH in the presence of 0.2 mM DTNB [15]. S-D-Lactoyl-, S-acetoacetyl-, S-succinyi-, S-propionyl- and Sacetylglutathione were used as substrates. Molar absorbances at 240 nm in the assay for each substrate were taken from Ref. 16. One enzyme unit (IU) catalyzes the hydrolysis of 1 #mol of S-D-lactoylglutathio.oe/min at the saturating substrate concentration and 20 ° C. Disc gel electrophoresis was performed in 7.5% polyacrylamide gels as described [20] and the gels were stained for enzymatic activity [21]. Polyacrylamide electrophoresis in the presence of SDS (12.5% gels) was performed according to Laemmli [22], immunoblotting was performed according to Towbin [23] and the immunological detection of glyoxalase II was done by using antiserum to cytosolic glyoxalase II in the peroxidase-antiperoxidase complex method [24]. The amount of purified glyoxalase II used was 0.5 IU. Protein was assayed by the method of Lowry et al. [25] or by the biuret reaction [26]. Bovine serum albumin was used as the standard.
Results Glyoxalase II was found in muluple locations in rat liver mitochondria. Most of the activity (64%) was localized in the matrix but an additional significant amount (28%) in the intermembrane space, in accordance with earlier results [15]. Table I shows the purification procedure for glyoxalase II from the two mitochondrial compartments. The same procedure could be used for the enzyme from both the matrix and the intermembrane space. The use of S-(carbobenzoxy)glutathione as affinity-ligand resulted in a high degree of purification. Final specific activity was not obtained for the intermembrane space enzyme due to too low protein content (undetectable) in the purified preparations. The purifications achieved were comparable to those obtained by a slightly different method from whole
9 TABLE I Purification procedure for glyoxalase 11 from rat liver mitochondrial matrix (.4) and intermembrane space (B)
Subfractioaation of the purified mitochondria was performed as described [15,18]. The purifications were carried out from the matrix and intermembrane space fractions as described for whole mitochondria [15] with slight modifications given in text. Glyoxalase il activity was assayed by following the decrease of absorbance at 240 nm due to the enzymatic hydrolysis of S-D-lactoylglutathione. Five concentrations of S-D-lactoylglutathione (0.05-0.40 mM) were used, and Vmax values were calculated from the linear double-reciprocal plots obtained, n.d., not detectable. Purification step
Protein (mg)
Activity (IU)
Specific activity (IU/mg)
Purification factor
Yield (%)
A. Matrix enzyme 100000 x g supernatant Acetone 30-80% S-(Carbobenzoxy)glutathione-A ffi-gel-10
120 53.0 0.13
58.0 54.0 22.0
0.483 1.02 169
1 2.1 350
100 9? 38
B. lntermembrane space enzyme Digitonin supernatant Acetone 30-80% S-(Carbobenzoxy)glutathione-A ffi-Gel- 10
52.9 10.2 n.d.
24.9 24.0 13.0
0.471 2.35 n.d.
1 5 n.d.
100 96 52
mitochondria [15]. T h r o u g h o u t the p u r i f i c a t i o n processes, electrophoresis with e n z y m a t i c staining w a s p e r f o r m e d to detect possible m o d i f i c a t i o n s in glyoxalase II pattern. These could be excluded, since the s a m e pattern, t w o e n z y m e f o r m s f r o m the i n t e r m e m b r a n e s p a c e a n d five f o r m s f r o m the matrix, w a s always resolved in electrophoresis for b o t h c r u d e a n d purified glyoxalase II p r e p a r a t i o n s (Fig. 1). All the i m m u n i z e d r a b b i t s p r o d u c e d a s i g n i f i c a m a m o u n t of a n t i b o d i e s to cytosolic glyoxalase II. T h e a n t i s e r u m with the highest a n t i b o d y titer s h o w e d binding capacity at a dilution range of 1 : 4 0 0 to 1 : 8 0 0 . H i g h e s t values a p p e a r e d 5 - 7 weeks after the p r i m a r y i n u n u n i z a t i o n . G l y o x a l a s e II purified f r o m the t w o
A
B
o
it
m i t o c h o n d r i a l c o m p a r t m e n t s as well as the e n z y m e isolated f r o m cytoso] [19] were studied u s i n g the a n t i s e r u m in the i m m u n o b l o t t i n g technique after S D S electrophoresis. T w o e n z y m e f o r m s were resolved f r o m cytosolic glyoxalase lI, a s t r o n g o r e ( M r = 29000) a n d a w e a k o n e ( M r = 27000) (Fig. 2). T h e e n z y m e f r o m the i n t e r m e m b r a n e space s h o w e d a cross-reaction w i t h the cytosolic a n t i s e r u m . A single e n z y m e f o r m w a s visualized w h i c h h a d a mobility identica] to that of the m a j o r cytosolic f o r m (Fig. 2). In contrast, n o cross-reaction at all w a s o b t a i n e d for the m a t r i x glyoxalase II (Fig. 2). C o n t r o l s p e r f o r m e d w i t h p r e i m m u n e s e r u m were always negative. T h e specificities of the purified e n z y m e s f r o m the i n t e r m e m b r a n e space a n d the m a t r i x were investigated
B
C
w
Fig. 1. Polyacrylarmde disc gel-electrophoresis according to gel system 1 of Maurer [20] of the enzymes from the mitochondrial intermembrane space (gel A) and the mitochondrial matrix (gel B). The gels were stained for glyoxalase 11 activity with $-D-lac~.oylglutathione as the substrate as described by Uotila [21]. The anode is at the bottom of the figure. The position of the tracking dye (Bromophenol blue) is marked by a copper wire.
Fig. 2. lmmunobloning of purified glyoxalase 11 from rat liver cytosol (A), mitochondrial intermembrane space (B) and mitochondrial matrix (C). Slab-gel electrophoresis was performed in 12.5% polyacrylamide in the presence of SDS with the anode at the bottom of the figure. The gel was subsequently subjected to electrophoretic blotting as described in text. hnmunological detection of glyuxalase II by antiserum to rat liver cytosolic glyoxalase I1 was carried out by the peroxidase-antiperoxidase method [24].
10 TABLE II Kinetic constants for the substrates of purified glyoxalase 11 from intermembrane space and matrix of rat liver mitochondria
Activity assays were performed at 240 nm (substrate concentration range 0.05-0.40 raM). Relative Vm~x values with S-n-lactoylglutathione as the substrate have been set to 100. Vma~ and Km values are given as mean±S.D. The last two columns give the statistical comparisons (P values in t-test) of the relative Vm~~ as well as the K m values obtained for a particular sub,irate for the intermembrane space and matrix enzymes, respectively. The values given in parentheses after each substrate indicate the number of independent experiments performed, n.s.. no significant difference. Substrate s-D- Lactoylglutathione S-Acetoacetylglutathione S-Succinylglutathione S-Propionylglutathione S-Acetylglutathione
(10) (10) (6) (9) (7)
Intermembrane space enzyme Vm~ (%) K m (mM) 100 0.5985:0.287 93.2±4.7 0.413+0.157 24.2 :t:8.0 0.186±0.108 30.7+3.2 0.792±0.333 17.6±0.9 0.603±0.326
and compared using five glutathione thiol esters as the substrates. S-D-Lactoylglutathione gave the highest relative V,~, value as well as the highest Vmax/K m ratio for the enzymes from both sources (Table II). The enzymes isolated from the intermembrane space and from the matrix showed statistically significant differences when their relative Vmax and K m values, in comparison to S-D-Lactoylglutathione, were c o m p a r e d for the other four substrates (Table II). All other substrates tested showed, in comparison to S-D-lactoylglutathione, a higher relative Vm~ value for the matrix enzyme than for the intermembrane space enzyme. SAcetoacetylglutathione and S-succinylglutathione showed, in comparison to S-o-lactoylglutathione, a higher Kn~ value for the matrix enzyme than for the intermembrane space enzyme. Acetylcoenzyme A was not used as the substrate by the enzyme from either compartment. Discussion
Polymorphism of glyoxalase II has been reported from the soluble fraction of liver [19,27], brain [19] and red blood cells [21]. In rat liver the activity is distributed between cytosol and mitochondria, in both of which multiple forms of the enzyme have been detected [15]. Glyoxalase II was isolated in this work from the intermembrane space and matrix of purified mitochondria. The former gave two and the latter five electrophoretic forms, under non-denaturating conditions. Using whole mitochondria, a pattern of five forms of glyoxalase II was detected earlier by electrophoresis and isoelectric focusing [15]. These findings are in agreement, since the forms from the intermeplbrane space were electrophoretically indistinguishable from two forms of" the matrix.Under the native elcctrophoretic conditions, the two enzyme forms from lhe intermembrane space also had similar mobilities to cytosolic glyoxalase II forms [19]. The immunoblotting experiments performed also indicate similarity between intermembrane space and cytosolic glyoxalase 1I ue-
Matrix enzyme Vm~ (%) 100 52.6±9.6 38.6±6.3 41.6±8.3 24.8±3.1
K m (mM) 0.3845:0.230 0.634±0.254 0.535+0.268 1.200±0.648 0.600±0.276
P ( Vmax)
P (Kn~)
<0.02 <0.05 < 0.01 < 0.01
n.s. <0.05 <0.05 n.s. n.s.
cause o f the positive cross-reaction of the former with the antiserum to cytosolic glyoxalase II; in addition, the major cytosolic enzyme form and the intermembrane space enzyme had identical mobilities in SDS electrophoresis (Fig. 2). However, because considerably less glyoxalase II antigen was visualized from the intermembrahe space enzyme preparation than from the cytosolic one (Fig. 2), it appears that either the cross-reaction of the former enzyme was incomplete or only part of the intermembrane space enzyme was visualized in the immunoblotting performed. In contrast, ~o cross-reaction with antibodies to cytosolic glyoxalase II was observed for the enzyme from mitochondriai matrix. Therefore, although two of the five matrix enzyme forms observed under native electrophoretic conditions had mobilities identical to those the enzyme forms from cytosol and intermembrane space, all the matrix enzyme forms clearly differ from the cytosolic enzyme forms, and there must be at least a partial difference between the activities from mitochondrial matrix and i n t e r m e m b r a n e space. Thus, liver mitochondria contain at least two pools of glyoxalase II which are immunologically different. Differences between matrix and intermembrane space enzymes are also evident from the specificity studies which were p e r f o r m e d with different glutathione thiol esters as substrates. Both enzyme pools are specific for thioesters of glutathione .and no activity was found using acetylcoenzyme A as the substrate. S-DLactoylglu:athione war the best substrate for lhe two pools of mitochondrial glyoxalase II. The substrate specificity observed in this work for the intermembrane space enzyme resembles that found earlier for the cytosolic enzyme forms [15], whereas that observed for the matrix enzyme is close to the specificity of the enzyme purified from whole mitochondria [15]. These data are in accordance with the results of the immunoblotting experiments. Since our purified mitochondrial preparations did not show any glyoxalase II activity as long as the
mitochondrial m e m b r a n e s remained intact [15], it is unlikely that cytosolic glyoxalase II was included as a contaminant in the mitochondrial intermembrane space preparatiorls used. However, the possibility of a very strong absorption of cytosolic glyoxalase II to the mitochondrial m e m b r a n e cannot completely be ruled out. Regarding the origin of mitochondrial glyoxalase II, it is unlikely that these enzymes are encoded by mitochondrial D N A , since the products of mitochondrial genome are known [28]. Therefore the enzymes are probably expressed by the nuclear genome and undergo post-translational changes during translocation into the '.'gitochondrion. Possible ubiquitous distribution of glyoxalase II in mitochondria is indicated by the presence of this enzyme in these organelles from other animal sources and spinach leaves (unpublished data). Until now, no data have been published to show the presence of the whole glyoxalase system in mitochondria, but our preliminary experiments suggest that glyoxalase I does not exist in rat liver mitochondria. The significance of the presertce of glyoxalase lI in rat liver both in the cytosol and in two c o m p a r t m e n t s of the mitochondrion is not yet clear. The cytosolic enzymes have a n u m b e r of similar properties when investigated from many different species [27]. Similar comparative studies of the mitochondrial glyoxalase II activities appear appropriate for in future studies.
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