277
Biochimica et Biophysics Acta, 580 (1979) @ Elsevier/North-Holland Biomedical Press
277-288
BBA 38287
GLUTATHIONE
THIOL ESTERASES
FRACTIONATION FOCUSING
LASSE
OF HUMAN RED BLOOD CELLS
BY GEL ELECTROPHORESIS
AND ISOELECTRIC
UOTILA
Department
of Medical Chemistry,
(Received December (Revised manuscript
University of Helsinki, Helsinki (Finland)
28th, 1978) received April 3rd, 1979)
Key words: Glutathione
thiol esterase; Glyoxalasc II; (Human erythrocyte;
Fractionation)
Summary The number and the substrate specificities of glutathione thiol esterases of human red blood cells have been investigated by gel electrophoresis and isoelectric focusing and staining methods devised for the location of these enzymes on gels. Several glutathione thiol esterase forms, both unspecific (with respect to the S-acyl group of the substrate) and specific were found. Electrophoresis on both polyacrylamide and agarose gels resolved three enzyme components with apparently similar substrate specificity. Isoelectric focusing in liquid column separated two unspecific thiol esterase components with S-lactoylglutathione (pZ = 8.4) and S-propionylglutathione (pZ = 8.1) as the best substrates, respectively, and two specific enzymes, S-formylglutathione hydrolase (pZ = 5.2) and S-succinylglutathione hydrolase (pZ = 9.0). Isoelectric focusing on polyacrylamide gel resolved nine unspecific glutathione thiol esterase bands (between pH values 7.0 and 8.4). Partially purified glyoxalase II (S-2EC 3.1.2.6) from erythrocytes or liver hydroxyacylglutathione hydrolase, still gave three components on electrophoresis and several activity bands on gel electrofocusing. These results indicate that human red cells contain at least four separate glutathione thiol esterases. Glyoxalase II, one of these enzymes, apparently occurs in multiple forms. These were neither influenced by pretreatment of the samples with neuraminidase or thiols nor were interconvertible during the fractionations.
Abbreviations: diphenyl
TEMED.
tetrazolium
N,N,N’,N’-tetramethylethylenediamine.
bromide.
MTT,
3-(4,5_dimethylthiazolyl-2)-2.5-
278
Introduction Glyoxalase I (S-lactoyl-glutathione methylglyoxal-lyase (isomerizing), EC 4.4.1.5) and glyoxalase II (S-2-hydroxyacylglutathione hydrolase, EC 3.1.2.6) form the glyoxalase activity ubiquitously found in living cells [ 11. A genetically determined polymorphism of glyoxalase I from human red blood cells has been described [2]. Little is known about red cell glyoxalase II. One recent report has even claimed that glyoxalase II is lacking in red cells [ 31. A highly purified preparation of this enzyme has only been achieved from human liver [ 41. During the studies of liver glyoxalase II two new specific glutathione thiol esterases, S-formylglutathione hydrolase and S-succinylglutathione hydrolase were found [ 51. Both have later been purified to homogeneity [ 6,7]. In the present study the occurrence of glutathione thiol esterases in human red cells has been studied by gel electrophoresis and isoelectric focusing. Staining methods have been devised for the location of these enzymes on gels. Several enzyme components, both specific and unspecific with respect to the S-acyl group of the substrate have been resolved. Materials and Methods Chemicals. 3-(4,5-Dimethyl thiazolyl-2)2,5-diphenyl tetrazolium bromide, 2,6-dichlorophenol indophenol, D-lactate dehydrogenase (EC 1.1.1.28 from Lactobacillus leichmannii) and glutamate-pyruvate transaminase (EC 2.6.1.2) were purchased from Sigma. Acrylamide (specially purified) was obtained from BDH, NJ’-methylenebisacrylamide and N,N,N’,N’-tetramethylethylenediamine (TEMED) from Eastman. Agarose (Indubiose A-37 or A-45) was a product of 1’Industrie biologique Francaise. Ampholytes for isoelectric focusing (40% solutions (w/v) of AmpholineR, pH 3.5-10 and 7-9) and thin-layer electrofocusing gel slabs (Ampholine PAGplates” No. 1804-101, pH 3.5-9.5) were purchased from LKB. The glutathione thiol esters used were prepared by the methods described earlier [ 51. Preparation of red cell hemolysates. Blood samples in 0.31% (w/v) sodium citrate were centrifuged and the plasma and the buffy coat removed. Distilled water (1.5 ~01s.) was added to the washed red cells and after stirring for 45 min at 4” C the hemolysate was centrifuged at 23 000 X g for 10 min. The supernatant was either used immediately or with unchanged results after storage for several weeks at -70°C. Agarose gel electrophoresis. Agarose gels (160 X 65 X 1.5 mm) were run at 4°C. In an acidic system the electrode buffer was 200 mM potassium phosphate, pH 6.2. The gels (l%, w/v) were prepared in l/10 diluted electrode buffer. The samples (about 3 ~1) were applied near the anode into small slots made with a razor blade. The electrophoresis was continued horizontally for 18 h with a potential difference of 8 V/cm between the electrodes. In an alkaline system the electrode buffer was 661 mM Tris/83 mM citric acid pH 9.1. The gel (l%, w/v) was prepared in l/10 diluted electrode buffer. Samples were applied near the cathode. Otherwise the running conditions were similar to those in the acidic system. Polyacrylamide gel electrophoresis. Polyacrylamide slab gels (82 X 82 X 2.7
279
mm) were prepared by the gel slab-casting apparatus of Pharmacia (GSC-8) and run with the Pharmacia gel electrophoresis apparatus (GE 4) at 10°C. In an acidic system the mixture for the gels (7%, w/v) contained seven parts of a 20% (w/v) acrylamide solution (acrylamide and bisacrylamide, 96.5 : 3.5, w/w), two parts 0.4% (v/v) TEMED in 0.135 M acetate buffer, pH 5.5, two parts 0.9% (w/v) ammonium persulfate and nine parts 0.135 M acetate buffer, pH 5.5. The electrode buffer was a 1 : 3 dilution of the acetate buffer mentioned above. The electrophoresis was continued for 20 h at 25 mA/gel. For an alkaline system similar slab gels were prepared according to the small-pore gel system 1 of Maurer [ 81. The electrophoresis was continued for 3 h at 30 mA/gel. Isoelectric focusing on polyacrylamide gel. Some experiments were done on the LKB thin-layer electrofocusing gels, pH 3.5-9.5. Better results were, however, achieved by self-prepared gels (LKB application note No. 306). The gel polymerization mixtures contained 10 ml 29.1% (w/v) acrylamide, 10 ml 0.9% (w/v) bisacrylamide and 37 ml of a solution of 7.5 g sucrose in water. Additionally the mixture for broad pH range contained 3.0 ml Ampholine (pH 3.510) and 0.6 ml 0.004% riboflavin (photopolymerization) and the mixture for narrow pH range 0.03 ml TEMED, 2.4 ml Ampholine (pH 7-9), 0.6 ml Ampholine (pH 3.5-10) and 1.5 ml 2% (w/v) ammonium persulfate. The final ampholyte concentration was 2% (w/v). The anode and cathode solutions were, respectively, 0.2% (v/v) H3POJ and 0.4% (v/v) ethylenediamine. The experiments were continued for 3-4 h on the 2117 Multiphor apparatus with tap water cooling and the following maximum settings on the power supply (LKB, No.2103): power 25 W, voltage 900 V (broad pH range) or 1200 V (narrow pH range) and current 40 mA. Isoelectric focusing in liquid column. The isoelectric focusing column of LKB (model 8101, volume 110 ml) was used. The pH gradient was created with 1% (w/v) Ampholine, pH 7-9, in a sucrose gradient. The gradient solutions contained 1 mM dithiothreitol. The anode (top) solution contained 0.1 ml concentrated sulfuric acid and 20.0 ml H20, and the cathode (bottom) solution 11.0 g sucrose, 14.0 ml HZ0 and 1.0 ml 2 M NaOH. The focusing was continued for 48 h with a voltage of 300 V in the beginning and 600 V in the end. Activities for glutathione thiol esters and pH (4°C) were then measured for each 2 ml fraction. Location of glutathione thiol esteruses on gels. The staining solution used in most cases contained 0.1 M Tris-HCl buffer, pH 8.0, 1 mM glutathione thiol ester, 0.1 mg/ml 2,6-dichlorophenol indophenol and 0.5 mg/ml MTT in 0.7% agar (Bacto-Agar, Difco). The agar solution was kept separately at 50°C before use. In an alternative method the staining solution contained 0.1 M Tris-HCI buffer, pH 8.0, 1 mM S-lactoylglutathione, 3.5 mM NAD’, D-(-)lactate dehydrogenase (8 U/ml), 0.1 mg/ml phenazine methosulfate, 0.5 mg/ml MTT, glutamate-pyruvate transaminase (3.2 U/ml) and 50 mM L-glutamate. The solution was applied on the gel in 0.7% agar. The electrophoresis gels were before staining incubated three times for 5 min in a large volume of 0.1 M Tris-HCl buffer, pH 8.0. Partial purification of glyoxalase II from human red cells. The red cell hemolysate (1 : 5) was dialyzed against 10 mM Tris-HCl buffer, pH 8.0, containing 2 mM 2-mercaptoethanol and then applied to a DEAE-Sephacel column equili-
280
brated with the same solution. The enzyme was slowly eluted by the equilibration buffer whereas most other proteins, among them hemoglobin, were bound. Gel chromatography. A Sephadex G-100 column (2.5 X 70 cm) was equilibrated and eluted with 10 mM Tris-HCl buffer, pH 7.6, containing 200 mM NaCl. The column was calibrated with five standards and the void volume determined by blue dextran. The apparent molecular weights of unknown proteins were determined from a plot of VJV, versus log M, [9], constructed from the results with standards. Treatment with neuraminidase. Hemolysates were incubated for 24 h at 4°C in 20 mM sodium phosphate buffer, pH 6.8, containing 1 mM Ca*’ and 50 U/ml neuraminidase (Calbiochem, from Vibrio cholerue, EC 3.2.1.18). Determination of enzyme activity and protein. Enzyme activity assays were done on a spectrophotometer at 25°C. The disappearance of the thiol esters was measured at 240 nm [4,5] or glutathione formation was recorded in the presence of one of the following agents at 0.2 mM: 5,5’-dithiobis(2nitrobenzoate) (E = 13 600 M-’ - cm-‘, h = 412 nm), 2,2’-dithiodipyridine (E = 7060 M-l * cm-‘, 343 nm) or 4,4’-dithiodipyridine (E = 19 800 M-’ * cm-‘, 324 nm). One enzyme unit catalyzed the hydrolysis of 1 pmol of the substrate with highest velocity under the conditions used. Protein measurements were done by the Lowry method [lo] and for partially purified preparations also fluorometrically [ 111. Results Electrophoresis of glutathione thiol esterases Three separate glutathione thiol esterase components which moved slowly towards the cathode were constantly found in electrophoresis of hemolysates on both agarose and polyacrylamide (Fig. 1) gel in acidic pH. Hemoglobin moved on both gels faster than the enzyme bands. The fastest enzyme component was the strongest one and the slowest the weakest. The highest amount (120) of separate hemolysate samples were analysed by the agarose technique. No variants from that described above were found with any of the techniques used. All bands were visualized with either S-lactoglutathione, S-propidnylglutathione or S-acetylglutathione as the substrate. The reactivity of these compounds decreased in the order mentioned above. The three enzyme components had similar apparent specificity for the glutathione thiol esters studied which indicates that they all represent unspecific glutathione thiol e&erase(s). Reelectrophoresis experiments were also carried out on polyacrylamide gel. Only part of the gel was stained and the separated enzyme components cut from the unstained gel part. After homogenization these were used as samples for new electrophoresis on a similar gel. Both of the two fastest enzyme components gave only the original enzyme form (Fig. 1). The slowest (and weakest) band could not be visualized in reelectrophoresis. In the alkaline electrophoresis systems described three unspecific glutathione thiol esterase components were obtained on both agarose and polyacrylamide gel. The enzyme bands moved towards the anode, again slower than hemoglobin. The strongest enzyme component had now the slowest velocity. The sub-
281
Fig. 1. Polyacrylamide gel electrophoresis of red cell glutathione thiol esterases and reelectrophoresis of the separated enzyme forms (pH 5.5). The sample marked H was a hemolysate (activity applied to the gel 0.18 unit with S-lactoylglutathione as the substrate). The blue enzyme components (I-III) are marked by reman numerals to separate them from the red hemoglobin band (Hb). The other samples were the separated enzyme forms I, II and III, as marked in the figure. which were cut after electrophoresis from unstained gel and then rerun under the same conditions in the gel shown. The faint activity band obtained from form II is marked by an arrow.
&rate specificity of the enzyme components was similar to that found in the acidic systems. No enzyme with the specificity of S-formylglutathione hydrolase which can be visualized with S-acetylglutathione and S-propionylglutathione (see below) was found on electrophoreses. In the acidic systems the latter enzyme did not move towards the cathode and in the alkaline systems it was apparently inactivated. Isoelectric
focusing
on polyacrylamide
gel
Isoelectric focusing on the gel with a broad pH gradient resolved bands between pH values 7.0 and 8.4 with either S-lactoylglutathione S-propionylglutathione (Fig. 2b) or S-acetylglutathione (Fig. 2c) strate. The most intense band was the most alkaline one (~1 8.4). bands were on its acidic side and one further band on both sides of
six enzyme (Fig. Za), as the subThree other hemoglobin
282
Jib
Jib
Fig. 2. Polyacrylamide gel electrofocusing of glutathione thiol esterases. (a-c). Gels with a broad PH gradient (3.5-10) stained with S-lactoylglutathione (a), SJPropionylglutathione (b) and S-acetylglutathione (c). (a and b) both show the staining result for two hemolysates and for a partially purified glyoxalase II preparation from liver [41. (c) for two hemolysatei and for purified S-formylglutathione hydrolase from liver [6]. Unspecific glutathione thiol esterase components given by the hem&sates axe marked by big arrows, hemoglobin components (Hb) by small arrows. (d) Gel with a narrow pH gradient (7-9). Samples were hemolysates. Enzyme components (l-9) are marked by big arrows on one side, hemoglobin components by small arrows on both sides of the band. Staining with S-lactoylglutathione. Activity applied to the gel was 0.04 unit with S-lactoylglutathione as the substrate.
A. The third band from the alkaline side (PI 8.1) was more intense with S-propionylglutathione and S-acetylglutathione than with S-lactoylglutathione in contrast to the other bands. The location of these bands, was, however, identical with all three substrates. In contrast in the acidic pH range (~15.2) activity bands close to each other were obtained with S-acetylglutathione as the substrate (Fig. 2c) but this area contained no activity for S-lactoylglutathione (Fig. 2a). With S-propionylglutathione the most intense of these bands was visualized (Fig. 2b). A purified S-formylglutathione hydrolase preparation from human liver [6] gave several activity bands in this same area (Fig. 2~). This together with the substrate specificity data of S-formylglutathione hydrolase (Ref. 6 and the results below) shows that this enzyme was represented by the bands at pH 5.2. The best substrate of the latter enzyme, S-formylglutathione, could not be used in the staining because of its incomplete purity and unstability. Partially purified liver glyoxalase II preparations (to step IV according to Ref. 4, spec. act. 10 U/mg) gave several (at least twelve) separate activity bands between pH 6.8 and 8.5 (Fig. 2a and b). Fig. 2d shows the result obtained when the focusing was performed on the gel with a narrow pH gradient (pH 7-9). Nine separate enzyme components were resolved for hemolysates with S-lactoylglutathione. S-Propionylglutathione gave all the same bands and an additional acidic band (S-formylglutathione
283
hydrolase). Partially purified red cell preparations also gave several activity bands (Fig. 4, see below). The site of application of the sample on the gel had no effect on the results obtained. Refocusing experiments were also carried out on gels with the pH gradient 7-9. Individual enzyme bands of the first focusing were cut from the unstained gel part and used as samples for a second focusing on a new similar gel. In every case most of the activity was recovered in the second focusing as a band which corresponded to the original one. Minor additional bands outside the location of the slice of the original gel were observed when bands 6 and 8 of Fig. 2d (i.e. the most central of the ‘unspecific’ thiol esterase bands) were cut. This may indicate partial interconversion of the enzyme components but diffusion during the staining of the first gel was not absolutely excluded as the explanation. The effect of pretreatment of the hemolysates with thiols (30 mM dithiothreitol or 50 mM glutathione, 30 min incubation at 4°C) or neuraminidase was checked. None of these treatments caused any changes in the results of either gel electrophoresis or gel electrofocusing experiments. Isoelectric focusing in liquid column Red cell preparations produced two separate unspecific glutathione thiol esterase components. The more alkaline component (peak A in Fig. 3, pI = 8.40 ? 0.10) reacted best with S-lactoylglutathione. Another smaller component on the acidic side of the former peak usually formed only a shoulder with S-lactoylglutathione but a second peak with S-propionylglutathione and S-acetylglutathione (peak B in Fig. 3, pl= 8.09 + 0.12). Two specific enzymes S-formylglutathione hydrolase (pl = 5.2) and S-succinylglutathione hydrolase
Fig. 3. Column isoelectric focusing of red cell glutathione thiol esterases. Activity assayed with S-fomYlglutathione (a). S-succinylglutathione (A). S-lactoylglutathione (0). S-propionylglutathione (o), and S-acetylglutathione (0). The activity scale is correct for the peak with S-formylglutathione, the activity with S-succinylglutathione has been multiplied by 0.2 and the other activities by 5.0. The sample applied to the column contained 4.9 units of activity with S-lactoylglutathione, 34.0 units with S-fomylglutathione and 96.5 units with S-succinylglutathione as the substrate. and 225 mg protein.
284
(PI = 9.0) were clearly separated from the unspecific glutathione thiol esterases as shown in Fig. 3. Both S-formylglutathione and S-succinylglutathione were hydrolyzed by the unspecific enzyme components shown in Fig. 3 but much slower than by the specific enzymes. Peaks A and B (Fig. 3) were pooled separately and a new similar column electrofocusing experiment conducted for each component. The result suggested that the peaks are separate rather than arise in the experiment since for either of the components activity was only found at the site from where the pooled fractions had been taken. Partial
purification
Column chromatography on DEAE-Sephacel offered a rapid method for partial purification of red cell glyoxalase II. The preparation thus obtained had a specific activity of 4.6 units/mg which represented 230-fold purification over the hemolysate (with 40% yield). Gel chromatography of the hemolysate separated the single sharp peaks of S-formylglutathione hydrolase (apparent M, 56 000) and glyoxalase II (21 000). The partially purified glyoxalase II preparation still gave three separate enzyme bands on electrophoresis (not shown) and a great number of enzyme bands on gel electrofocusing, most of which were between pH values 7.7 and 8.4 (Fig. 4). The number and the location of the bands on gel electrofocusing did not quite correspond to those found for the hemolysates (Fig. 4). For separate hemolysates the number and the location of the enzyme bands has been highly reproducible. Table I presents the relative hydrolytic activities found for a number of glutathione thiol esters in the crude hemolysate and after various fractionation procedures. The values for the partially purified preparations of glyoxalase II (by DEAE-Sephacel chromatography and peak II of Sephadex G-100 chroma-
1
2
3
4
5
J
Fig. 4. Isoelectric focusing of glutathione thiol esterase preparations on polyacrylamide gel (PH 7-9). The samples were: 1, a hemolysate; 2-4. the separate enzyme components I, II and III. respectively, obtained for partially purified red cell glyoxalase II on polyacrylamide gel electrophoresis at pH 5.5 (see Fig. 1); 5. partially purified red cell glyoxalase II (not fractionated by electrophoresis). The hatched bands represent hemoglobin components and the black ones enzyme components. - - - - - -. very weak enzyme components.
285 TABLE
I
RELATIVE Peaks the
ACTIVITIES
FOR
I and II of Sephadex
order
agreeing
of elution, values
when
GLUTATHIONE
G-100
THIOL
chromatography
when
a hemolysate
standard
deviation
is applied is not given.
Preparation
ESTERS
to the column. For further
Activity
*.** * **
(peak (peak
The activities for S-lactoylglutathione This value corresponded to 0.0227
I) II)
100 0 100 100 100
*,*** *+ * * *
100
*
CELL
thiol
PREPARATIONS
rsterasc
peaks
The data are means
details
see text.
obtained.
of two
in
closely
n.d., not determined.
with
S-Lactoylglutathionr Red cell hemolysate (II = 12) Sephadex G-100 chromatography Sephadcx G-100 chromatography DEAE-Sephacel chromatography Isoelectric focusing in column (peak A. Fig. 3) 01 = 6) Isoelectric focusing in column (peak B. Fig. 3) 01 = 6)
IN RED
are the glutathione
S-Propionylglutathione 36.7 0.2 13.1 14.9 52.1 133
S-Acetylglutathionr
S-FlXm,lglutathione
*
4.5
+
7.3
730 100 46.0 n.d. n.d.
t 21.6
+
5.1
f
8.0
28.5 0.5 9.2 11.5 43.6
i 21.4
98.3
and S-formylglutathione have been i 0.0031 unit/ma of protein.
set to 100.
n.d.
respectively
tography) and S-formylglutathione hydrolase (peak I of Sephadex G-100 chromatography) resemble those earlier reported for the corresponding enzymes of human liver [4.6]. The whole activity of the hemolysate for S-propionylglutathione and S-acetylglutathione can apparently not be explained only by glyoxalase II and S-formylglutathione hydrolase. Table I also gives the relative activities for the two unspecific glutathione thiol esterase components of column isoelectric focusing. Peak B (PI= 8.1) gave the highest rate with S-propionylglutathione. Most of the activity of this peak was lost in an attempt for further purification by Sephadex G-100 chromatography. The removal of ampholytes by precipitation of the proteins with ammonium sulfate and washing with saturated ammonium sulfate solution did not change the specificity of this fraction. Correspondence frac tiona tions
of the unspecific
glutathione
thiol ester-use bands of different
When the fractions from column electrofocusing were used as samples in gel electrofocusing, both peak A and B of the column electrofocusing (Fig. 3) gave in the gel electrofocusing all the enzyme bands l-5 as marked in Fig. 2d. The main band given by peak A was band 1 whereas the main band given by peak B was band 4 (Fig. 2d). Other bands were not obtained. The result apparently corresponds to the result of two succeeding gel electrofocusings (see above) when the lower resolution of the column electrofocusing procedure is also taken into account. Both peaks A and B of column electrofocusing (Fig. 3) gave on polyacrylamide gel electrophoresis (acidic pH) the two fastest electrophoretic bands (I and II in Fig. 1) but not the slowest band (III) of the hemolysates. The substrate specificity was apparently the same for the activity recovered in these bands. The three unspecific enzyme components found by the acidic polyacryl-
286
amide gel electrophoresis system (Fig. 1) were cut from the unstained gel and used as samples for gel electrofocusing. Fig. 4 shows the result when a partially purified preparation was used for the electrophoresis. The fastest electrophoretie enzyme band (I, Fig. 1) gave several enzyme bands on gel electrofocusing at pH 7.9-8.5. The middle electrophoretic band (II, Fig. 1) gave several bands at pH 7.9-8.5 and additionally several bands at pH 6.3-7.0. The slowest electrophoretic band (III, Fig. lb) gave several bands but only at pH 6.0-7.0. The result was similar when a hemolysate was used for the electrophoresis preceding the electrofocusing. Discussion The present studies show that human red cells contain at least the three glutathione thiol esterases, glyoxalase II, S-formylglutathione hydrolase and S-succinylglutathione hydrolase which have earlier been found and isolated from human liver [4-71 and that red cell preparations contain substantial glyoxalase II activity, contradictory to some recent claims [ 31. Existence of an additional glutathione thiol esterase which uses well S-propionylglutathione as the substrate is suggested by the comparison of the specificities of purified glyoxalase II and S-formylglutathione hydrolase preparations with those of the hemolysate (Table I). Similar indirect evidence exists from liver [446]. Such a thiol esterase was apparently inactivated during column chromatographies but found by electrofocusing (peak B of Fig. 3). Evidence against artifactual formation of this peak by ampholytes was given in Results. Gel electrophoresis and electrofocusing were used in this work as new fractionation procedures, made possible by specific staining methods. The staining where glutathione formed by the enzyme reacts with 2,6-dichlorophenol indophenol and MTT to produce the formazan bands is the method to be recommended. The other staining described served as a control where the formation of a different product was detected. It required a complex mixture containing S-D-lactoylglutathione, D-lactate dehydrogenase, glutamate-pyruvate transminase and glutamate. The latter two were used to remove constantly the pyruvate formed. Otherwise the unfavourable equilibrium of the D-lactate dehydrogenase reaction would not have allowed significant activity band formation. Charlesworth [ 121 has earlier described a staining method for glyoxalase II and reported that of the 687 separate red cell hemolysates studied by starch electrophoresis every one gave a single enzyme band at the same position. The validity of her staining mixture, containing glyoxalase I, methylglyoxal, oxidized glutathione, lactate dehydrogenase (specific for L-form?), NAD’, phenazine methosulfate and MTT [12] is, however, questionable. Glyoxalase I can only use reduced glutathione and is inactive with the oxidized form [13] and the product of glyoxalase I is D-lactoylglutathione [ 141 which on hydrolysis by glyoxalase II produces D-lactate. This does not react with the usual lactate dehydrogenase. The method [12] does not give any glutathione thiol esterase bands according to the experience of this author. Multiple forms of the unspecific (with respect to the S-acyl group of the substrate) glutathione thiol esterase activity were found to occur in human red cells. Electrophoreses resolved three and gel electrofocusing at least eight com-
287
ponents with similar apparent specificity. Extensive fractionation studies by column chromatographies of human liver preparations have shown that this tissue does riot contain other enzymes which significantly hydrolyze glutathione thiol esters than the glutathione thiol esterases themselves [ 4,5]. A similar finding would be probable in red cells and was supported by the present electrophoresis and electrofocusing results for partially purified red cell and liver preparations. These gave as many ‘unspecific’ bands as the crude solution. Although the best glyoxalase II preparations studied (Table I) need still to be purified, their specificity for various glutathione thiol esters was similar to that of highly purified liver glyoxalase II [4]. This reasoning also excludes association complexes of glyoxalase II with other proteins in the hemolysate as the explanation of the heterogeneity. Multiple forms of glyoxalase II in red cells (and liver) are thus suggested but these might be true or arise by the fractionations. Treatment with thiols or neuraminidase or the alternative use of either persulfate or riboflavin in the gels did not influence the results. Reelectrophoreses showed that the separate electrophoretic forms were not interconvertible and reelectrofocusings gave the original band at least as the major form. Gel electrofocusing of the three electrophoretically separated forms (Fig. 4) gave an individual pattern for every component but they all gave several bands on gel electrofocusing. This might result from the high resolving power of gel electrofocusing but artifactual heterogeneity seems also possible taking into account that, e.g. electrophoretic component II (Fig. 1) gave enzyme bands as far from each other as from pH 6.3 to 8.4 (Fig. 4). Artifactual heterogeneity on electrofocusing has been described for tRNA [ 151, heparin [ 161 and some proteins [17,18] and appears to result from the binding of ampholytes to the macromolecule [ 16,191. The separate forms should then again show heterogeneity on reelectrofocusing and the way and site of sample application should influence the result [ 161. These were not seen in the present case. Thus firm conclusions on the nature of the multiple forms cannot be achieved by the present data. An isolation method which produces relatively high amounts of purified glyoxalase II is required to separate between true multiple forms and irreversible modifications of the enzyme. Acknowledgements These studies have been supported land) and Finnish Cancer Foundation. provided skilful technical assistance.
by the Sigrid Juselius Foundation (FinMrs. Eija Haasanen and Mrs. Silja V2lttila
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Biochimica et Biophysics Acta, 580 (1979) @ Elsevier/North-Holland Biomedical Press
277-288
BBA 38287
GLUTATHIONE
THIOL ESTERASES
FRACTIONATION FOCUSING
LASSE
OF HUMAN RED BLOOD CELLS
BY GEL ELECTROPHORESIS
AND ISOELECTRIC
UOTILA
Department
of Medical Chemistry,
(Received December (Revised manuscript
University of Helsinki, Helsinki (Finland)
28th, 1978) received April 3rd, 1979)
Key words: Glutathione
thiol esterase; Glyoxalasc II; (Human erythrocyte;
Fractionation)
Summary The number and the substrate specificities of glutathione thiol esterases of human red blood cells have been investigated by gel electrophoresis and isoelectric focusing and staining methods devised for the location of these enzymes on gels. Several glutathione thiol esterase forms, both unspecific (with respect to the S-acyl group of the substrate) and specific were found. Electrophoresis on both polyacrylamide and agarose gels resolved three enzyme components with apparently similar substrate specificity. Isoelectric focusing in liquid column separated two unspecific thiol esterase components with S-lactoylglutathione (pZ = 8.4) and S-propionylglutathione (pZ = 8.1) as the best substrates, respectively, and two specific enzymes, S-formylglutathione hydrolase (pZ = 5.2) and S-succinylglutathione hydrolase (pZ = 9.0). Isoelectric focusing on polyacrylamide gel resolved nine unspecific glutathione thiol esterase bands (between pH values 7.0 and 8.4). Partially purified glyoxalase II (S-2EC 3.1.2.6) from erythrocytes or liver hydroxyacylglutathione hydrolase, still gave three components on electrophoresis and several activity bands on gel electrofocusing. These results indicate that human red cells contain at least four separate glutathione thiol esterases. Glyoxalase II, one of these enzymes, apparently occurs in multiple forms. These were neither influenced by pretreatment of the samples with neuraminidase or thiols nor were interconvertible during the fractionations.
Abbreviations: diphenyl
TEMED.
tetrazolium
N,N,N’,N’-tetramethylethylenediamine.
bromide.
MTT,
3-(4,5_dimethylthiazolyl-2)-2.5-
278
Introduction Glyoxalase I (S-lactoyl-glutathione methylglyoxal-lyase (isomerizing), EC 4.4.1.5) and glyoxalase II (S-2-hydroxyacylglutathione hydrolase, EC 3.1.2.6) form the glyoxalase activity ubiquitously found in living cells [ 11. A genetically determined polymorphism of glyoxalase I from human red blood cells has been described [2]. Little is known about red cell glyoxalase II. One recent report has even claimed that glyoxalase II is lacking in red cells [ 31. A highly purified preparation of this enzyme has only been achieved from human liver [ 41. During the studies of liver glyoxalase II two new specific glutathione thiol esterases, S-formylglutathione hydrolase and S-succinylglutathione hydrolase were found [ 51. Both have later been purified to homogeneity [ 6,7]. In the present study the occurrence of glutathione thiol esterases in human red cells has been studied by gel electrophoresis and isoelectric focusing. Staining methods have been devised for the location of these enzymes on gels. Several enzyme components, both specific and unspecific with respect to the S-acyl group of the substrate have been resolved. Materials and Methods Chemicals. 3-(4,5-Dimethyl thiazolyl-2)2,5-diphenyl tetrazolium bromide, 2,6-dichlorophenol indophenol, D-lactate dehydrogenase (EC 1.1.1.28 from Lactobacillus leichmannii) and glutamate-pyruvate transaminase (EC 2.6.1.2) were purchased from Sigma. Acrylamide (specially purified) was obtained from BDH, NJ’-methylenebisacrylamide and N,N,N’,N’-tetramethylethylenediamine (TEMED) from Eastman. Agarose (Indubiose A-37 or A-45) was a product of 1’Industrie biologique Francaise. Ampholytes for isoelectric focusing (40% solutions (w/v) of AmpholineR, pH 3.5-10 and 7-9) and thin-layer electrofocusing gel slabs (Ampholine PAGplates” No. 1804-101, pH 3.5-9.5) were purchased from LKB. The glutathione thiol esters used were prepared by the methods described earlier [ 51. Preparation of red cell hemolysates. Blood samples in 0.31% (w/v) sodium citrate were centrifuged and the plasma and the buffy coat removed. Distilled water (1.5 ~01s.) was added to the washed red cells and after stirring for 45 min at 4” C the hemolysate was centrifuged at 23 000 X g for 10 min. The supernatant was either used immediately or with unchanged results after storage for several weeks at -70°C. Agarose gel electrophoresis. Agarose gels (160 X 65 X 1.5 mm) were run at 4°C. In an acidic system the electrode buffer was 200 mM potassium phosphate, pH 6.2. The gels (l%, w/v) were prepared in l/10 diluted electrode buffer. The samples (about 3 ~1) were applied near the anode into small slots made with a razor blade. The electrophoresis was continued horizontally for 18 h with a potential difference of 8 V/cm between the electrodes. In an alkaline system the electrode buffer was 661 mM Tris/83 mM citric acid pH 9.1. The gel (l%, w/v) was prepared in l/10 diluted electrode buffer. Samples were applied near the cathode. Otherwise the running conditions were similar to those in the acidic system. Polyacrylamide gel electrophoresis. Polyacrylamide slab gels (82 X 82 X 2.7
279
mm) were prepared by the gel slab-casting apparatus of Pharmacia (GSC-8) and run with the Pharmacia gel electrophoresis apparatus (GE 4) at 10°C. In an acidic system the mixture for the gels (7%, w/v) contained seven parts of a 20% (w/v) acrylamide solution (acrylamide and bisacrylamide, 96.5 : 3.5, w/w), two parts 0.4% (v/v) TEMED in 0.135 M acetate buffer, pH 5.5, two parts 0.9% (w/v) ammonium persulfate and nine parts 0.135 M acetate buffer, pH 5.5. The electrode buffer was a 1 : 3 dilution of the acetate buffer mentioned above. The electrophoresis was continued for 20 h at 25 mA/gel. For an alkaline system similar slab gels were prepared according to the small-pore gel system 1 of Maurer [ 81. The electrophoresis was continued for 3 h at 30 mA/gel. Isoelectric focusing on polyacrylamide gel. Some experiments were done on the LKB thin-layer electrofocusing gels, pH 3.5-9.5. Better results were, however, achieved by self-prepared gels (LKB application note No. 306). The gel polymerization mixtures contained 10 ml 29.1% (w/v) acrylamide, 10 ml 0.9% (w/v) bisacrylamide and 37 ml of a solution of 7.5 g sucrose in water. Additionally the mixture for broad pH range contained 3.0 ml Ampholine (pH 3.510) and 0.6 ml 0.004% riboflavin (photopolymerization) and the mixture for narrow pH range 0.03 ml TEMED, 2.4 ml Ampholine (pH 7-9), 0.6 ml Ampholine (pH 3.5-10) and 1.5 ml 2% (w/v) ammonium persulfate. The final ampholyte concentration was 2% (w/v). The anode and cathode solutions were, respectively, 0.2% (v/v) H3POJ and 0.4% (v/v) ethylenediamine. The experiments were continued for 3-4 h on the 2117 Multiphor apparatus with tap water cooling and the following maximum settings on the power supply (LKB, No.2103): power 25 W, voltage 900 V (broad pH range) or 1200 V (narrow pH range) and current 40 mA. Isoelectric focusing in liquid column. The isoelectric focusing column of LKB (model 8101, volume 110 ml) was used. The pH gradient was created with 1% (w/v) Ampholine, pH 7-9, in a sucrose gradient. The gradient solutions contained 1 mM dithiothreitol. The anode (top) solution contained 0.1 ml concentrated sulfuric acid and 20.0 ml H20, and the cathode (bottom) solution 11.0 g sucrose, 14.0 ml HZ0 and 1.0 ml 2 M NaOH. The focusing was continued for 48 h with a voltage of 300 V in the beginning and 600 V in the end. Activities for glutathione thiol esters and pH (4°C) were then measured for each 2 ml fraction. Location of glutathione thiol esteruses on gels. The staining solution used in most cases contained 0.1 M Tris-HCl buffer, pH 8.0, 1 mM glutathione thiol ester, 0.1 mg/ml 2,6-dichlorophenol indophenol and 0.5 mg/ml MTT in 0.7% agar (Bacto-Agar, Difco). The agar solution was kept separately at 50°C before use. In an alternative method the staining solution contained 0.1 M Tris-HCI buffer, pH 8.0, 1 mM S-lactoylglutathione, 3.5 mM NAD’, D-(-)lactate dehydrogenase (8 U/ml), 0.1 mg/ml phenazine methosulfate, 0.5 mg/ml MTT, glutamate-pyruvate transaminase (3.2 U/ml) and 50 mM L-glutamate. The solution was applied on the gel in 0.7% agar. The electrophoresis gels were before staining incubated three times for 5 min in a large volume of 0.1 M Tris-HCl buffer, pH 8.0. Partial purification of glyoxalase II from human red cells. The red cell hemolysate (1 : 5) was dialyzed against 10 mM Tris-HCl buffer, pH 8.0, containing 2 mM 2-mercaptoethanol and then applied to a DEAE-Sephacel column equili-
280
brated with the same solution. The enzyme was slowly eluted by the equilibration buffer whereas most other proteins, among them hemoglobin, were bound. Gel chromatography. A Sephadex G-100 column (2.5 X 70 cm) was equilibrated and eluted with 10 mM Tris-HCl buffer, pH 7.6, containing 200 mM NaCl. The column was calibrated with five standards and the void volume determined by blue dextran. The apparent molecular weights of unknown proteins were determined from a plot of VJV, versus log M, [9], constructed from the results with standards. Treatment with neuraminidase. Hemolysates were incubated for 24 h at 4°C in 20 mM sodium phosphate buffer, pH 6.8, containing 1 mM Ca*’ and 50 U/ml neuraminidase (Calbiochem, from Vibrio cholerue, EC 3.2.1.18). Determination of enzyme activity and protein. Enzyme activity assays were done on a spectrophotometer at 25°C. The disappearance of the thiol esters was measured at 240 nm [4,5] or glutathione formation was recorded in the presence of one of the following agents at 0.2 mM: 5,5’-dithiobis(2nitrobenzoate) (E = 13 600 M-’ - cm-‘, h = 412 nm), 2,2’-dithiodipyridine (E = 7060 M-l * cm-‘, 343 nm) or 4,4’-dithiodipyridine (E = 19 800 M-’ * cm-‘, 324 nm). One enzyme unit catalyzed the hydrolysis of 1 pmol of the substrate with highest velocity under the conditions used. Protein measurements were done by the Lowry method [lo] and for partially purified preparations also fluorometrically [ 111. Results Electrophoresis of glutathione thiol esterases Three separate glutathione thiol esterase components which moved slowly towards the cathode were constantly found in electrophoresis of hemolysates on both agarose and polyacrylamide (Fig. 1) gel in acidic pH. Hemoglobin moved on both gels faster than the enzyme bands. The fastest enzyme component was the strongest one and the slowest the weakest. The highest amount (120) of separate hemolysate samples were analysed by the agarose technique. No variants from that described above were found with any of the techniques used. All bands were visualized with either S-lactoglutathione, S-propidnylglutathione or S-acetylglutathione as the substrate. The reactivity of these compounds decreased in the order mentioned above. The three enzyme components had similar apparent specificity for the glutathione thiol esters studied which indicates that they all represent unspecific glutathione thiol e&erase(s). Reelectrophoresis experiments were also carried out on polyacrylamide gel. Only part of the gel was stained and the separated enzyme components cut from the unstained gel part. After homogenization these were used as samples for new electrophoresis on a similar gel. Both of the two fastest enzyme components gave only the original enzyme form (Fig. 1). The slowest (and weakest) band could not be visualized in reelectrophoresis. In the alkaline electrophoresis systems described three unspecific glutathione thiol esterase components were obtained on both agarose and polyacrylamide gel. The enzyme bands moved towards the anode, again slower than hemoglobin. The strongest enzyme component had now the slowest velocity. The sub-
281
Fig. 1. Polyacrylamide gel electrophoresis of red cell glutathione thiol esterases and reelectrophoresis of the separated enzyme forms (pH 5.5). The sample marked H was a hemolysate (activity applied to the gel 0.18 unit with S-lactoylglutathione as the substrate). The blue enzyme components (I-III) are marked by reman numerals to separate them from the red hemoglobin band (Hb). The other samples were the separated enzyme forms I, II and III, as marked in the figure. which were cut after electrophoresis from unstained gel and then rerun under the same conditions in the gel shown. The faint activity band obtained from form II is marked by an arrow.
&rate specificity of the enzyme components was similar to that found in the acidic systems. No enzyme with the specificity of S-formylglutathione hydrolase which can be visualized with S-acetylglutathione and S-propionylglutathione (see below) was found on electrophoreses. In the acidic systems the latter enzyme did not move towards the cathode and in the alkaline systems it was apparently inactivated. Isoelectric
focusing
on polyacrylamide
gel
Isoelectric focusing on the gel with a broad pH gradient resolved bands between pH values 7.0 and 8.4 with either S-lactoylglutathione S-propionylglutathione (Fig. 2b) or S-acetylglutathione (Fig. 2c) strate. The most intense band was the most alkaline one (~1 8.4). bands were on its acidic side and one further band on both sides of
six enzyme (Fig. Za), as the subThree other hemoglobin
282
Jib
Jib
Fig. 2. Polyacrylamide gel electrofocusing of glutathione thiol esterases. (a-c). Gels with a broad PH gradient (3.5-10) stained with S-lactoylglutathione (a), SJPropionylglutathione (b) and S-acetylglutathione (c). (a and b) both show the staining result for two hemolysates and for a partially purified glyoxalase II preparation from liver [41. (c) for two hemolysatei and for purified S-formylglutathione hydrolase from liver [6]. Unspecific glutathione thiol esterase components given by the hem&sates axe marked by big arrows, hemoglobin components (Hb) by small arrows. (d) Gel with a narrow pH gradient (7-9). Samples were hemolysates. Enzyme components (l-9) are marked by big arrows on one side, hemoglobin components by small arrows on both sides of the band. Staining with S-lactoylglutathione. Activity applied to the gel was 0.04 unit with S-lactoylglutathione as the substrate.
A. The third band from the alkaline side (PI 8.1) was more intense with S-propionylglutathione and S-acetylglutathione than with S-lactoylglutathione in contrast to the other bands. The location of these bands, was, however, identical with all three substrates. In contrast in the acidic pH range (~15.2) activity bands close to each other were obtained with S-acetylglutathione as the substrate (Fig. 2c) but this area contained no activity for S-lactoylglutathione (Fig. 2a). With S-propionylglutathione the most intense of these bands was visualized (Fig. 2b). A purified S-formylglutathione hydrolase preparation from human liver [6] gave several activity bands in this same area (Fig. 2~). This together with the substrate specificity data of S-formylglutathione hydrolase (Ref. 6 and the results below) shows that this enzyme was represented by the bands at pH 5.2. The best substrate of the latter enzyme, S-formylglutathione, could not be used in the staining because of its incomplete purity and unstability. Partially purified liver glyoxalase II preparations (to step IV according to Ref. 4, spec. act. 10 U/mg) gave several (at least twelve) separate activity bands between pH 6.8 and 8.5 (Fig. 2a and b). Fig. 2d shows the result obtained when the focusing was performed on the gel with a narrow pH gradient (pH 7-9). Nine separate enzyme components were resolved for hemolysates with S-lactoylglutathione. S-Propionylglutathione gave all the same bands and an additional acidic band (S-formylglutathione
283
hydrolase). Partially purified red cell preparations also gave several activity bands (Fig. 4, see below). The site of application of the sample on the gel had no effect on the results obtained. Refocusing experiments were also carried out on gels with the pH gradient 7-9. Individual enzyme bands of the first focusing were cut from the unstained gel part and used as samples for a second focusing on a new similar gel. In every case most of the activity was recovered in the second focusing as a band which corresponded to the original one. Minor additional bands outside the location of the slice of the original gel were observed when bands 6 and 8 of Fig. 2d (i.e. the most central of the ‘unspecific’ thiol esterase bands) were cut. This may indicate partial interconversion of the enzyme components but diffusion during the staining of the first gel was not absolutely excluded as the explanation. The effect of pretreatment of the hemolysates with thiols (30 mM dithiothreitol or 50 mM glutathione, 30 min incubation at 4°C) or neuraminidase was checked. None of these treatments caused any changes in the results of either gel electrophoresis or gel electrofocusing experiments. Isoelectric focusing in liquid column Red cell preparations produced two separate unspecific glutathione thiol esterase components. The more alkaline component (peak A in Fig. 3, pI = 8.40 ? 0.10) reacted best with S-lactoylglutathione. Another smaller component on the acidic side of the former peak usually formed only a shoulder with S-lactoylglutathione but a second peak with S-propionylglutathione and S-acetylglutathione (peak B in Fig. 3, pl= 8.09 + 0.12). Two specific enzymes S-formylglutathione hydrolase (pl = 5.2) and S-succinylglutathione hydrolase
Fig. 3. Column isoelectric focusing of red cell glutathione thiol esterases. Activity assayed with S-fomYlglutathione (a). S-succinylglutathione (A). S-lactoylglutathione (0). S-propionylglutathione (o), and S-acetylglutathione (0). The activity scale is correct for the peak with S-formylglutathione, the activity with S-succinylglutathione has been multiplied by 0.2 and the other activities by 5.0. The sample applied to the column contained 4.9 units of activity with S-lactoylglutathione, 34.0 units with S-fomylglutathione and 96.5 units with S-succinylglutathione as the substrate. and 225 mg protein.
284
(PI = 9.0) were clearly separated from the unspecific glutathione thiol esterases as shown in Fig. 3. Both S-formylglutathione and S-succinylglutathione were hydrolyzed by the unspecific enzyme components shown in Fig. 3 but much slower than by the specific enzymes. Peaks A and B (Fig. 3) were pooled separately and a new similar column electrofocusing experiment conducted for each component. The result suggested that the peaks are separate rather than arise in the experiment since for either of the components activity was only found at the site from where the pooled fractions had been taken. Partial
purification
Column chromatography on DEAE-Sephacel offered a rapid method for partial purification of red cell glyoxalase II. The preparation thus obtained had a specific activity of 4.6 units/mg which represented 230-fold purification over the hemolysate (with 40% yield). Gel chromatography of the hemolysate separated the single sharp peaks of S-formylglutathione hydrolase (apparent M, 56 000) and glyoxalase II (21 000). The partially purified glyoxalase II preparation still gave three separate enzyme bands on electrophoresis (not shown) and a great number of enzyme bands on gel electrofocusing, most of which were between pH values 7.7 and 8.4 (Fig. 4). The number and the location of the bands on gel electrofocusing did not quite correspond to those found for the hemolysates (Fig. 4). For separate hemolysates the number and the location of the enzyme bands has been highly reproducible. Table I presents the relative hydrolytic activities found for a number of glutathione thiol esters in the crude hemolysate and after various fractionation procedures. The values for the partially purified preparations of glyoxalase II (by DEAE-Sephacel chromatography and peak II of Sephadex G-100 chroma-
1
2
3
4
5
J
Fig. 4. Isoelectric focusing of glutathione thiol esterase preparations on polyacrylamide gel (PH 7-9). The samples were: 1, a hemolysate; 2-4. the separate enzyme components I, II and III. respectively, obtained for partially purified red cell glyoxalase II on polyacrylamide gel electrophoresis at pH 5.5 (see Fig. 1); 5. partially purified red cell glyoxalase II (not fractionated by electrophoresis). The hatched bands represent hemoglobin components and the black ones enzyme components. - - - - - -. very weak enzyme components.
285 TABLE
I
RELATIVE Peaks the
ACTIVITIES
FOR
I and II of Sephadex
order
agreeing
of elution, values
when
GLUTATHIONE
G-100
THIOL
chromatography
when
a hemolysate
standard
deviation
is applied is not given.
Preparation
ESTERS
to the column. For further
Activity
*.** * **
(peak (peak
The activities for S-lactoylglutathione This value corresponded to 0.0227
I) II)
100 0 100 100 100
*,*** *+ * * *
100
*
CELL
thiol
PREPARATIONS
rsterasc
peaks
The data are means
details
see text.
obtained.
of two
in
closely
n.d., not determined.
with
S-Lactoylglutathionr Red cell hemolysate (II = 12) Sephadex G-100 chromatography Sephadcx G-100 chromatography DEAE-Sephacel chromatography Isoelectric focusing in column (peak A. Fig. 3) 01 = 6) Isoelectric focusing in column (peak B. Fig. 3) 01 = 6)
IN RED
are the glutathione
S-Propionylglutathione 36.7 0.2 13.1 14.9 52.1 133
S-Acetylglutathionr
S-FlXm,lglutathione
*
4.5
+
7.3
730 100 46.0 n.d. n.d.
t 21.6
+
5.1
f
8.0
28.5 0.5 9.2 11.5 43.6
i 21.4
98.3
and S-formylglutathione have been i 0.0031 unit/ma of protein.
set to 100.
n.d.
respectively
tography) and S-formylglutathione hydrolase (peak I of Sephadex G-100 chromatography) resemble those earlier reported for the corresponding enzymes of human liver [4.6]. The whole activity of the hemolysate for S-propionylglutathione and S-acetylglutathione can apparently not be explained only by glyoxalase II and S-formylglutathione hydrolase. Table I also gives the relative activities for the two unspecific glutathione thiol esterase components of column isoelectric focusing. Peak B (PI= 8.1) gave the highest rate with S-propionylglutathione. Most of the activity of this peak was lost in an attempt for further purification by Sephadex G-100 chromatography. The removal of ampholytes by precipitation of the proteins with ammonium sulfate and washing with saturated ammonium sulfate solution did not change the specificity of this fraction. Correspondence frac tiona tions
of the unspecific
glutathione
thiol ester-use bands of different
When the fractions from column electrofocusing were used as samples in gel electrofocusing, both peak A and B of the column electrofocusing (Fig. 3) gave in the gel electrofocusing all the enzyme bands l-5 as marked in Fig. 2d. The main band given by peak A was band 1 whereas the main band given by peak B was band 4 (Fig. 2d). Other bands were not obtained. The result apparently corresponds to the result of two succeeding gel electrofocusings (see above) when the lower resolution of the column electrofocusing procedure is also taken into account. Both peaks A and B of column electrofocusing (Fig. 3) gave on polyacrylamide gel electrophoresis (acidic pH) the two fastest electrophoretic bands (I and II in Fig. 1) but not the slowest band (III) of the hemolysates. The substrate specificity was apparently the same for the activity recovered in these bands. The three unspecific enzyme components found by the acidic polyacryl-
286
amide gel electrophoresis system (Fig. 1) were cut from the unstained gel and used as samples for gel electrofocusing. Fig. 4 shows the result when a partially purified preparation was used for the electrophoresis. The fastest electrophoretie enzyme band (I, Fig. 1) gave several enzyme bands on gel electrofocusing at pH 7.9-8.5. The middle electrophoretic band (II, Fig. 1) gave several bands at pH 7.9-8.5 and additionally several bands at pH 6.3-7.0. The slowest electrophoretic band (III, Fig. lb) gave several bands but only at pH 6.0-7.0. The result was similar when a hemolysate was used for the electrophoresis preceding the electrofocusing. Discussion The present studies show that human red cells contain at least the three glutathione thiol esterases, glyoxalase II, S-formylglutathione hydrolase and S-succinylglutathione hydrolase which have earlier been found and isolated from human liver [4-71 and that red cell preparations contain substantial glyoxalase II activity, contradictory to some recent claims [ 31. Existence of an additional glutathione thiol esterase which uses well S-propionylglutathione as the substrate is suggested by the comparison of the specificities of purified glyoxalase II and S-formylglutathione hydrolase preparations with those of the hemolysate (Table I). Similar indirect evidence exists from liver [446]. Such a thiol esterase was apparently inactivated during column chromatographies but found by electrofocusing (peak B of Fig. 3). Evidence against artifactual formation of this peak by ampholytes was given in Results. Gel electrophoresis and electrofocusing were used in this work as new fractionation procedures, made possible by specific staining methods. The staining where glutathione formed by the enzyme reacts with 2,6-dichlorophenol indophenol and MTT to produce the formazan bands is the method to be recommended. The other staining described served as a control where the formation of a different product was detected. It required a complex mixture containing S-D-lactoylglutathione, D-lactate dehydrogenase, glutamate-pyruvate transminase and glutamate. The latter two were used to remove constantly the pyruvate formed. Otherwise the unfavourable equilibrium of the D-lactate dehydrogenase reaction would not have allowed significant activity band formation. Charlesworth [ 121 has earlier described a staining method for glyoxalase II and reported that of the 687 separate red cell hemolysates studied by starch electrophoresis every one gave a single enzyme band at the same position. The validity of her staining mixture, containing glyoxalase I, methylglyoxal, oxidized glutathione, lactate dehydrogenase (specific for L-form?), NAD’, phenazine methosulfate and MTT [12] is, however, questionable. Glyoxalase I can only use reduced glutathione and is inactive with the oxidized form [13] and the product of glyoxalase I is D-lactoylglutathione [ 141 which on hydrolysis by glyoxalase II produces D-lactate. This does not react with the usual lactate dehydrogenase. The method [12] does not give any glutathione thiol esterase bands according to the experience of this author. Multiple forms of the unspecific (with respect to the S-acyl group of the substrate) glutathione thiol esterase activity were found to occur in human red cells. Electrophoreses resolved three and gel electrofocusing at least eight com-
287
ponents with similar apparent specificity. Extensive fractionation studies by column chromatographies of human liver preparations have shown that this tissue does riot contain other enzymes which significantly hydrolyze glutathione thiol esters than the glutathione thiol esterases themselves [ 4,5]. A similar finding would be probable in red cells and was supported by the present electrophoresis and electrofocusing results for partially purified red cell and liver preparations. These gave as many ‘unspecific’ bands as the crude solution. Although the best glyoxalase II preparations studied (Table I) need still to be purified, their specificity for various glutathione thiol esters was similar to that of highly purified liver glyoxalase II [4]. This reasoning also excludes association complexes of glyoxalase II with other proteins in the hemolysate as the explanation of the heterogeneity. Multiple forms of glyoxalase II in red cells (and liver) are thus suggested but these might be true or arise by the fractionations. Treatment with thiols or neuraminidase or the alternative use of either persulfate or riboflavin in the gels did not influence the results. Reelectrophoreses showed that the separate electrophoretic forms were not interconvertible and reelectrofocusings gave the original band at least as the major form. Gel electrofocusing of the three electrophoretically separated forms (Fig. 4) gave an individual pattern for every component but they all gave several bands on gel electrofocusing. This might result from the high resolving power of gel electrofocusing but artifactual heterogeneity seems also possible taking into account that, e.g. electrophoretic component II (Fig. 1) gave enzyme bands as far from each other as from pH 6.3 to 8.4 (Fig. 4). Artifactual heterogeneity on electrofocusing has been described for tRNA [ 151, heparin [ 161 and some proteins [17,18] and appears to result from the binding of ampholytes to the macromolecule [ 16,191. The separate forms should then again show heterogeneity on reelectrofocusing and the way and site of sample application should influence the result [ 161. These were not seen in the present case. Thus firm conclusions on the nature of the multiple forms cannot be achieved by the present data. An isolation method which produces relatively high amounts of purified glyoxalase II is required to separate between true multiple forms and irreversible modifications of the enzyme. Acknowledgements These studies have been supported land) and Finnish Cancer Foundation. provided skilful technical assistance.
by the Sigrid Juselius Foundation (FinMrs. Eija Haasanen and Mrs. Silja V2lttila
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