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The isolation and immunological properties of two arginase forms from human erythrocytes
BIOCHEMICAL
MEDICINE
AND
METABOLIC
BIOLOGY
39, 247-257 (1988)
The isolation and Immunological Properties of Two Arginase Forms from Human Etythrocytes MARIA
K~DRA-LUBO&KA,*
EL~BIETA
ZAMgCKA,t
*Institute of Cardiology, ul.Alpejska 42, Warsaw-Anin, Institute of Biopharmacy, Academy of Medicine,
Poland,
AND
ZOFIA
P0REMBSKAt.l
and fDepartment
of Biochemistry.
ul. Banacha I, 02-097 Warsaw, Poland
Received March 28, 1985, and in revised form July 16, 1987
In our earlier studies based on DEAE- and CM-cellulose chromatography techniques we postulated the occurrence of only three forms of arginase in human tissues (1). However, after studying the electrophoretic and immunological properties of arginase and in view of its behavior on isoelectric focusing, we have concluded that human tissues contain five forms of arginase, for which altered designations have been proposed: A,, AZ, A3, Ad, AS (2). Data on human erythrocyte arginase are incomplete and divergent. On the basis of the electrophoretic and immunological properties of arginase, the presence of two forms of this enzyme in erythrocytes was postulated (3,4). However Hishibe (5) and Beruter (6) did not contirm the heterogeneity of human erythrocyte arginase, both these authors have detected only a single enzyme form, which they described as identical with arginase present in human liver. Determination of the forms of arginase in human erythrocytes is of considerable clinical significance especially in the case of patients with hyperargininemia which is characterized by selective reduction of arginase activity in some tissues, including erythrocytes (7,8). Moreover, more detailed knowledge of the arginase forms could be of assistance in the elucidation the genetic mechanism of this disease. The present studies involved isolation of two arginases from human erythrocytes, determination of their immunological properties, and comparison of these properties with those of other human arginases. EXPERIMENTAL Reagents. Reagents were purchased as follows: L-arginine (Calbiochem, Los Angeles, CA), Sephadex G-150, Dextran 2000, DEAE-Sephacel (Pharmacia, Uppsala, Sweden), CM-cellulose (CM-2) and DEAE-cellulose (DE-l 1) (Whatman Biochemicals Maidston, Kent, England), Freund’s incomplete and complete adjuvant (Difco Laboratories, Detroit, MI). Marker proteins. Bovine albumin, chicken ovalbumin, bovine globulin and ’ To whom reprint requests should be addressed 0885-4505188 $3.00 Copyright All rights
0 1988 by Academic Press. Inc. of reproduction in any form reserved.
247
248
KEDRA-LUBOIlkSKA,
ZAMBCKA,
AND POREMBSKA
horse myoglobulin (Sigma Chemical Co., St. Louis, MO). All other chemicals were of the purest grades available from standard commercial sources. Materials. Human erythrocytes obtained from a blood bank were washed three times with saline, centrifuged, and stored at - 15°C. Arginase assay. Arginase activity was measured by determining the increase in the amount of the reaction product, ornithine, determined according to Chinard (9) as modified by Porembska and Baranczyk-K&ma (10). One unit of enzymatic activity was defined as 1 pmole of product formed per minute at 37°C. Protein determination. Protein was assayed according to Lowry et al. (11) or spectrometrically by the method of Warburg and Christian (12) with cristalline bovine serum albumin as standard. Molecular weight determination. The molecular weight of human erythrocyte arginase was determined by Sephadex G-150 chromatography, as described by Andrews (13). The column (2 x 42 cm) was equilibrated with 100 mM KC1 in 50 mu Tris-HCl buffer, pH 7.5. Fractions of 2 ml were checked for enzymatic activity and protein content. Horse myoglobulin (17,000 M,), ovalbumin (46,000 M,), bovine serum albumin (69,000 M,), and bovine serum y-globulin (150,000 M,) were used as standards. PuriJication of antigens. Arginase A, from human kidney and arginase AS from human liver were purified according to the procedure described by SkrzypekOsiecka et al. (14) and by Ber and Muszynska (15), respectively. Antiserum. Pure arginase A, from human kidney (sp act 1000 pmole min-’ mg- ’ protein) and pure arginase AS from human liver (sp act 2500 pmole min- ’ mg-’ protein) were raised in female guinea pigs. The animals were immunized every 10 days with 200 pg of protein in 0.5 pmole saline supplemented with 0.5 ml of Freund’s complete adjuvant (first immunization) or 0.5 ml of Freund’s incomplete adjuvant (subsequent immunizations). The animals were bled beginning 6 weeks after the first immunization and blood was centrifuged at 10,OOOg for 15 min in a Sorvall (RC 2-B SS-34 rotor). Serum protein was precipitated by the addition of ammonium sulfate (10 g/20 ml of serum), centrifuged at 15,OOOg for 15 min, dissolved with a small volume of saline, and dialyzed against 5 dm3 of the same solution for 24 hr. Guinea pig serum titer was 32 and 64 for arginase A, and AS, respectively. Double immunodijjfusion. The test was carried out according to Ouchterlony (16) in 1% agar containing 0.01 M Verona1 buffer, pH 8.1. Agar slides were kept at room temperature for 48 hr, whereupon they were washed successively with saline and water, dried, and stained with 0.1% Coomassie blue R-250. Immunoelectrophoresis. The assay was performed at 8°C in agarose gel containing 0.02 M Verona1 buffer, pH 8.6, for 90 min at 9 V/cm. After electrophoresis appropriate antibodies were applied, and following 48 hr incubation at room temperature, the slides were washed successively with saline and water, dried, and stained with 0.1% Coomassie blue R-250.
ARGINASES
FROM HUMAN
249
ERYTHROCYTES
TABLE 1 Purification of Human Erythrocyte Arginases Activity Total protein (mg)
Procedure Extract of erythrocytes 30-60% NH, 2S0, saturation Heated at 55°C I5 min Ethanol precipitation DEAE-cellulose chromatography Arginase I Arginase II DEAE-Sephacel chromatography Arginase I Arginase II CM-cellulose chromatography Arginase 1 n pmole of ornithine
Purijication
Total units
Specific activity”
Purification factor
-
45000
2250
0.05
12000
1800
0.15
3
2000
1200
0.60
12
400
720
1.8
36
30 2
270 1.6
9.0 0.8
180 16
12 0.22
144 0.88
12.0 4.0
240 80
60.0
60.0
1200
I min-’ mg-’ protein.
of Human
RESULTS Erythrocyte Arginase
For the purification, some of the steps previously described by Ber and Muszynska for rat liver arginase (15) and by Baranczyk-Kuima et al. for human blood serum arginase (17) were adopted. Purification by DEAE-Sephacel column chromatography was additionally included. For enzyme stabilization 1 mM MnCl, was used, and 1 mu 2-mercaptoethanol was applied to prevent arginase aggregation (18). All procedures were carried out at 4°C unless otherwise stated. The results of purification are summarized in Table 1. Step 1: Extraction. The erythrocytes were sonicated for 15 to 30 set at 5 A with 3 vol of solution containing 100 mM KCl, 1 mM MnCl,, and 50 mM TrisHCl buffer, pH 7.5. The homogenate was centrifuged at 15,000g for 15 min in a Sorvall (RC 2-B SS-34 rotor) and the pellet was discarded. Step 2: Ammonium Sulfate Fractionation. Solid NH42S04 was added slowly with stirring to the supernatant to 30% saturation (18 g/100 ml). After 60 min, the solution was centrifuged at 10,OOOg for 30 min, the pellet was discarded, and the supernatant brought to 60% saturation (22 g/100 ml) with ammonium sulfate. The precipitate was recovered by centrifugation, dissolved in a small volume of solution containing 1 mM MnCl,, 1 mM 2-mercaptoethanol, 10 mM Tris-HCl
250
KljDRA-LUBOIfiSKA,
ZAMFCKA,
AND
POREMBSKA
buffer, pH 7.5, and then dialyzed against two changes of the same solution for 25 hr. Step 3: Heat treatment. The dialysate was incubated for 15 min in the water bath at 55°C with agitation, cooled, and centrifuged at 15g for 15 min. Step 4: Precipitation with ethanol. Three volumes of ethanol cooled to - 10°C was slowly added to the supematant. The mixture was centrifuged at 10,OOOg at - 10°C for 15 min. The pellet was dissolved with 10 vol of solution containing 1 mM MnCl,, 1 mM 2-mercaptoethanol, 10 mM Tris-HCl buffer, pH 8.3, and dialyzed against the same solution for 24 hr. Step 5: DEAE-cellulose chromatography. The dialysate was centrifuged and placed on a column packed with DEAE-cellulose (24 x 1 cm) stabilized with 800 ml of 10 mM Tris-HCl buffer, pH 8.3. The activity was eluted with 40 ml of the same buffer (fraction I) and with a linear gradient of KC1 from 0.0 to 0.3 M in 300 ml of the same buffer (fraction II) (Fig. la). The active fractions I and II were pooled, concentrated in the presence of Ficoll, and dialyzed as described for Step 4. Each of the two arginase fractions I and II separated by DEAE-cellulose chromatography was then purified separately. Step 6: DEAE-Sephacel chromatography. After dialysis fraction I and fraction II were separately placed on columns packed with DEAE-Sephacel(16 x 1 cm), stabilized with 500 ml of 10 mM Tris-HCl buffer, pH 8.3. Fraction I was eluted with the buffer front (20 ml), fraction II with a linear gradient of KC1 from 0.0 to 0.3 M (Figs. lb and lc). Both active fractions were pooled, concentrated in the presence of Ficoll, and dialyzed 24 hr against a solution of 1 mM MnCl,, 1 mM 2-mercaptoethanol, and 10 mM Tris-HCl buffer, pH 7.5. Step 7: CM-cellulose chromatography. Only fraction I was submitted to CMcellulose chromatography. After dialysis the enzyme was applied on a column (6 x 1 cm), stabilized with 300 ml of 10 mM Tris-HCl buffer, pH 7.5. The enzyme adsorbed on the column was eluted with a KC1 concentration gradient from 0.0 to 0.3 in 100 ml of the same buffer (Fig. Id). The fractions showing arginase activity were pooled, concentrated, and dialyzed as described for Step 6. The arginase preparations obtained after purification represented two forms of enzyme, the first adsorbed on the CM-cellulose column (96% of total activity) and the second adsorbed on DEAE-cellulose as well as DEAE-Sephacel (4% of total activity). Partly purified preparations of the two forms of the enzyme the purification factor being 1200 and 80 for the first and second form, respectively, were used for determination of molecular weight and immunological properties. The two partly purified arginases were free of each other. Molecular
Weight
Both partly purified arginases had a molecular weight of 120,000 ? 5000 as determined by chromatography on Sephadex G-150.
ARGINASES
FROM HUMAN
Elution
ERYTHROCYTES
volume
251
(ml)
FIG. 1. DEAE-cellulose, DEAE-Sephacel. and CM-cellulose chromatography of human erythrocyte arginase. (a) Ethanol precipitate after dialysis (about 400 mg) was applied to DEAE-cellulose column (24 x I cm) equilibrated with IO mM Tris-HCI buffer, pH 8.3. The activity was eluted with 40 ml of the same buffer (fraction I) and with a linear gradient of KCI from 0.0 to 0.3 M in 300 ml of the same buffer (fraction II). The active fractions I and II were pooled, concentrated in the presence of Ficoll and dialyzed 24 hr against the solution containing 1 mM MnCL, 1 mM 2-mercaptoethanol, 10 mM Tris-HCl buffer, pH 8.3. (b, c) Fractions I and II from Step 5 after dialysis (about 30 mg and 2 mg of protein) were placed separately to DEAE-Sephacel column (16 x 1 cm) equilibrated with 10 mM Tris-HCI buffer, pH 8.3. The fractions I and II eluted with the buffer from 20 ml (Fig. lb) and with a linear gradient of KC1 from 0.0 to 0.3 M in 200 ml of the same buffer (Fig. lc), respectively. Both active fractions were pooled, concentrated in the presence of Ficoll, and dialyzed 24 hr against solution containing 1 mM MnCI,, 1 mM 2-mercaptoethanol, and 10 mM Tris-HCI buffer, pH 7.5. (d) Fraction I from Step 6 after dialysis (about 12 mg of protein) was applied to a CMcellulose column (6 x 1 cm) equilibrated with 10 mM Tris-HCI buffer, pH 7.5. The activity was eluted with a KCI concentration gradient from 0.0 to 0.3 M in 100 ml of the same buffer. The fraction showing arginase activity was pooled, concentrated, and dialyzed as described for Step 6. For all four columns fractions of 5 ml were collected at a flow rate of l-l.5 ml/min. Protein and arginase activity were determined as described under Experimental. 0, arginase activity; 0, protein content: -, KC1 gradient.
252
KBDRA-LUBOIfiSKA,
ZAMBCKA,
AND POREMBSKA
FIG. 2. Immunodiffusion of arginases isolated from human erythrocytes in the presence of guinea pig antisera directed against arginase A, from kidney and arginase A, from liver. Center well: (I) antiserum against arginase AI, from liver; (II) antiserum against arginase A, from kidney. (a) Erythrocyte arginase adsorbed on CM-cellulose. (b) Erythrocyte arginase adsorbed on DEAE-cellulose.
Immunological Properties
The arginases isolated from human erythrocytes were investigated by the double immunodiffusion test and by immunoelectrophoresis in the presence of antisera directed against the two parental forms of arginase from human tissues: A, from kidney and AS from liver. It should be noted that total immunological incompatibility of the two parental forms of arginase was reported by Zam,ecka and Porembska (2). Either of the two arginase forms isolated from erythrocytes reacted on immunodiffusion with both antisera, the occurrence of a spur indicating that they were only partially identical (Figs. 21 and 211). Spur formation in the presence of anti-AS serum by the form I adsorbed on CM-cellulose indicates that this form contained more antigenic determinants corresponding to those of form AS from liver (Fig. 21), whereas the second form II, adsorbed on DEAE-cellulose, contained more of the determinants of form A, from kidney (Fig. 211). Such behavior of the two forms of erythrocyte arginase was undoubtedly related to differences in subunit composition of the enzymes. Nonidentity of the two arginase forms was also confirmed by immunoelectrophoresis. When the two forms were electrophoresed simultaneously in the presence arcs differing in their distance of antiserum against arginase A,, two precipitation from the cathode were obtained (Fig. 3). To identify more closely the two erythrocyte arginases, their immunological properties were compared with those of other human arginases. In the presence of the antisera against form AS from liver and form A, from kidney, the first main form of erythrocyte arginase displayed immunological compatibility with arginase A4 from human kidney (Fig. 41), and the second minor form, with arginase A2 from human liver (Fig. 411). On immunoelectrophoresis, the first and second erythrocyte forms showed the same mobility as arginase A., from kidney (Fig. 5a and b) and A2 from liver (Fig. 5c and d), respectively.
ARGINASES
FROM HUMAN
ERYTHROCYTES
253
FIG. 3. Immunoelectroporesis of erythrocyte arginases. Center well: erythrocyte arginases adsorbed on (a) CM- and (b) DEAE-cellulose. Cross-reaction developed with the antiserum against liver arginase A,.
Since the arginase from human erythrocytes was earlier believed (3,lO) to be identical with arginase from human liver, the main form of the erythrocyte enzyme isolated in the present work was compared with the main form from liver, AS. In the double immunodiffusion test (Fig. 6) the main form of erythrocyte arginase displayed only partial identity with form A5 from human liver, whereas on immunoelectrophoresis it showed lower mobility toward the cathode than the latter form (Fig. 7). Moreover, it was found that the main erythrocyte arginase form gives a cross-reaction with both anti-A, and anti-A, antiserum (Fig. 811) whereas the liver A5 cross-reacts only with the homologous, anti-A, antiserum (Fig. 81). DISCUSSION We have demonstrated that two forms of arginase are present in both human liver and kidney A, and A2 and arginases A4 and A,, respectively (2). Arginase A, and arginase A, are parental forms built of one-type subunits; arginase A2 and A4 are hybrids composed of both kinds of subunits (2).
254
KEDRA-LUBOIfiSKA,
1
ZAMECKA,
AND POREMBSKA
2
FIG. 4. The relationship of human erythrocyte arginases with arginase A, from human kidney and arginase Ar from human liver. Center well: (I, 1 and 2) antiserum against liver arginase AS; (II, 1 and 2) antiserum against kidney arginase A,. (la) A, arginase from kidney; (2a) A, arginase from liver; (lb) human erythrocyte arginase adsorbed in CM-cellulose; (2b) human erythrocyte arginase adsorbed on DEAE-cellulose.
Now we have demonstrated that two forms of arginase occur also in human erythrocytes; on the basis of immunological studies they were identified as arginases A, and AZ. Molecular weight of either of them was 120,000 which proves that they are not artifacts formed in the course of purification. The two arginases from erythrocytes have antigenic determinants characteristic of either of the parental arginase forms since they cross-react with both antibodies directed
ARGINASES
FROM HUMAN
ERYTHROCYTES
255
FIG. 5. Immunoelectrophoresis of human erythrocyte arginase when comparing with arginase A, from human kidney and arginase A, from human liver. (a) A, arginase from kidney; (b) erythrocyte arginase adsorbed on CM-cellulose; (c) A, arginase from liver; (d) erythrocyte arginase adsorbed on DEAE-cellulose. Cross-reaction developed in the presence of antiserum against arginase A, from kidney.
against Al and against AS. This confirms also the hybrid nature of the erythrocyte arginases. It should be stressed that, at variance with the earlier suggestions (5,6), the main erythrocyte arginase form A,, was now found to differ from the main enzyme form from liver, AS. The two forms show but partial immunological compatibility due most probably to their different subunit structures. Unlike form
FIG. 6. Cross-reactivity of human liver arginase A5 and erythrocyte arginase adsorbed on CMcellulose. Center well: antiserum against liver arginase AS. (a) A, arginase from human liver; (b) erythrocyte arginase adsorbed on CM-cellulose.
256
KEDRA-LUBOI&SKA, a
ZAMECKA,
AND POREMBSKA b
+ FIG. 7. Immunoelectrophoresis of human liver arginase A, and erythrocyte arginase adsorbed on CM-cellulose. Antiserum against liver arginase A, (a) A5 arginase from human liver; (b) erythrocyte arginase adsorbed on CM-cellulose.
FIG. 8. Cross-reactivity of human liver arginase AS and erythrocyte arginase adsorbed on CMcellulose in the presence of the antisera against liver As and kidney A, arginases. Center well: (I) A, arginase from liver; (II) erythrocyte arginase adsorbed on CM-cellulose. (a) Antiserum against liver arginase AS; (b) antiserum against kidney arginase A,.
ARGINASES
FROM HUMAN
ERYTHROCYTES
257
A5 from liver, which is built of a single type of subunit, arginase & from erythrocytes is a hybrid of two types of subunits corresponding to subunits of the two parental arginase forms. The results obtained could be of significance in studies on the genetic origin of hyperargininemia. It should be taken into account that human arginase is built of two immunologically different subunits which most probably are synthesized by different genes. Determination of the subunit composition of the erythrocyte arginase could be useful for elucidation of the nature of hyperargininemia at the molecular level. SUMMARY 1. Two forms of arginase were isolated from human erythrocytes; the main form adsorbed on CM-cellulose and the second form, occurring in much smaller amount, adsorbed on DEAE-cellulose. 2. The molecular weight of either arginase was 120,000 + 5000. 3. The erythrocyte arginases are similar in immunological properties to arginase A4 from human kidney and AZ from human liver, respectively. 4. Despite the literature data stating that human erythrocyte arginase and human liver arginase are identical, it was found that the main forms of arginase of these tissues A4 from erythrocytes and AS from liver differ in immunological properties. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Porembska, Z., and Kedra, M., Bull. Acad. Polon. Sci. Ser. Biol. 19, 633 (1971). Zampcka, E., and Porembska, Z., Biochem. Med. Metab. Biol. 39, 258 (1988). Bascur, L., Cabello, J. P., Veliz, M., and Gonzales, A., Biochim. Biophys. Actn 128, 149 (1966). Cabello, J., Prajoux, V., and Plaza, M., Biochim. Biophys. Acta 105, 583 (1965). Hishibe, H., Physiol. Chem. Phys. 5, 453 (1973). Beruter, J., Colombo, J., and Bachnamm, C., Biochem. J. 1975, 499 (1978). Cederbaum, S. P., Show, K. N. P., and Valente, M., J. Pediutr. 90, 569 (1977). Michals, V., and Beaudet, A. L., Clin. Genet. 13, 61 (1978). Chinard, F. P., J. Biol. Chem. 199, 91 (1952). Porembska, Z., and Baranczyk-Kuima, A., Diagn. Lab. 3, 239 (1974). Lowry, 0. M., Rosebrough, N. J., Farr, A. L., and Randal, R. J., J. Biol. Chem. 193, 265
12.
Warburg, O., and Christian, W., Biochem. Z. 310, 384 (1941). Andrews, P., Biochem. J. 91, 222 (1964). Skrzypek-Osiecka, I., Robin, Y., and Porembska, Z., Acta Biochim. Polon. 30, 83 (1983). Ber, E., and Muszynska, G., Acta Biochim. Polon. 26, 103 (1979). Ouchterlony, O., in “Handbook of Experimental Immunology” D. M. Weir, Ed., pp. 655-706 Blackwell, Oxford, 1967. Baranczyk-Kuzma, A., Skrzypek-Osiecka, I., and Porembska, Z., Biochem. Med. 26, 174 (1981). Sakai, T., and Murachi, T., Physiol. Chem. Phys. 1, 317 (1969).
(1951).
13. 14. 15. 16. 17. 18.
MEDICINE
AND
METABOLIC
BIOLOGY
39, 247-257 (1988)
The isolation and Immunological Properties of Two Arginase Forms from Human Etythrocytes MARIA
K~DRA-LUBO&KA,*
EL~BIETA
ZAMgCKA,t
*Institute of Cardiology, ul.Alpejska 42, Warsaw-Anin, Institute of Biopharmacy, Academy of Medicine,
Poland,
AND
ZOFIA
P0REMBSKAt.l
and fDepartment
of Biochemistry.
ul. Banacha I, 02-097 Warsaw, Poland
Received March 28, 1985, and in revised form July 16, 1987
In our earlier studies based on DEAE- and CM-cellulose chromatography techniques we postulated the occurrence of only three forms of arginase in human tissues (1). However, after studying the electrophoretic and immunological properties of arginase and in view of its behavior on isoelectric focusing, we have concluded that human tissues contain five forms of arginase, for which altered designations have been proposed: A,, AZ, A3, Ad, AS (2). Data on human erythrocyte arginase are incomplete and divergent. On the basis of the electrophoretic and immunological properties of arginase, the presence of two forms of this enzyme in erythrocytes was postulated (3,4). However Hishibe (5) and Beruter (6) did not contirm the heterogeneity of human erythrocyte arginase, both these authors have detected only a single enzyme form, which they described as identical with arginase present in human liver. Determination of the forms of arginase in human erythrocytes is of considerable clinical significance especially in the case of patients with hyperargininemia which is characterized by selective reduction of arginase activity in some tissues, including erythrocytes (7,8). Moreover, more detailed knowledge of the arginase forms could be of assistance in the elucidation the genetic mechanism of this disease. The present studies involved isolation of two arginases from human erythrocytes, determination of their immunological properties, and comparison of these properties with those of other human arginases. EXPERIMENTAL Reagents. Reagents were purchased as follows: L-arginine (Calbiochem, Los Angeles, CA), Sephadex G-150, Dextran 2000, DEAE-Sephacel (Pharmacia, Uppsala, Sweden), CM-cellulose (CM-2) and DEAE-cellulose (DE-l 1) (Whatman Biochemicals Maidston, Kent, England), Freund’s incomplete and complete adjuvant (Difco Laboratories, Detroit, MI). Marker proteins. Bovine albumin, chicken ovalbumin, bovine globulin and ’ To whom reprint requests should be addressed 0885-4505188 $3.00 Copyright All rights
0 1988 by Academic Press. Inc. of reproduction in any form reserved.
247
248
KEDRA-LUBOIlkSKA,
ZAMBCKA,
AND POREMBSKA
horse myoglobulin (Sigma Chemical Co., St. Louis, MO). All other chemicals were of the purest grades available from standard commercial sources. Materials. Human erythrocytes obtained from a blood bank were washed three times with saline, centrifuged, and stored at - 15°C. Arginase assay. Arginase activity was measured by determining the increase in the amount of the reaction product, ornithine, determined according to Chinard (9) as modified by Porembska and Baranczyk-K&ma (10). One unit of enzymatic activity was defined as 1 pmole of product formed per minute at 37°C. Protein determination. Protein was assayed according to Lowry et al. (11) or spectrometrically by the method of Warburg and Christian (12) with cristalline bovine serum albumin as standard. Molecular weight determination. The molecular weight of human erythrocyte arginase was determined by Sephadex G-150 chromatography, as described by Andrews (13). The column (2 x 42 cm) was equilibrated with 100 mM KC1 in 50 mu Tris-HCl buffer, pH 7.5. Fractions of 2 ml were checked for enzymatic activity and protein content. Horse myoglobulin (17,000 M,), ovalbumin (46,000 M,), bovine serum albumin (69,000 M,), and bovine serum y-globulin (150,000 M,) were used as standards. PuriJication of antigens. Arginase A, from human kidney and arginase AS from human liver were purified according to the procedure described by SkrzypekOsiecka et al. (14) and by Ber and Muszynska (15), respectively. Antiserum. Pure arginase A, from human kidney (sp act 1000 pmole min-’ mg- ’ protein) and pure arginase AS from human liver (sp act 2500 pmole min- ’ mg-’ protein) were raised in female guinea pigs. The animals were immunized every 10 days with 200 pg of protein in 0.5 pmole saline supplemented with 0.5 ml of Freund’s complete adjuvant (first immunization) or 0.5 ml of Freund’s incomplete adjuvant (subsequent immunizations). The animals were bled beginning 6 weeks after the first immunization and blood was centrifuged at 10,OOOg for 15 min in a Sorvall (RC 2-B SS-34 rotor). Serum protein was precipitated by the addition of ammonium sulfate (10 g/20 ml of serum), centrifuged at 15,OOOg for 15 min, dissolved with a small volume of saline, and dialyzed against 5 dm3 of the same solution for 24 hr. Guinea pig serum titer was 32 and 64 for arginase A, and AS, respectively. Double immunodijjfusion. The test was carried out according to Ouchterlony (16) in 1% agar containing 0.01 M Verona1 buffer, pH 8.1. Agar slides were kept at room temperature for 48 hr, whereupon they were washed successively with saline and water, dried, and stained with 0.1% Coomassie blue R-250. Immunoelectrophoresis. The assay was performed at 8°C in agarose gel containing 0.02 M Verona1 buffer, pH 8.6, for 90 min at 9 V/cm. After electrophoresis appropriate antibodies were applied, and following 48 hr incubation at room temperature, the slides were washed successively with saline and water, dried, and stained with 0.1% Coomassie blue R-250.
ARGINASES
FROM HUMAN
249
ERYTHROCYTES
TABLE 1 Purification of Human Erythrocyte Arginases Activity Total protein (mg)
Procedure Extract of erythrocytes 30-60% NH, 2S0, saturation Heated at 55°C I5 min Ethanol precipitation DEAE-cellulose chromatography Arginase I Arginase II DEAE-Sephacel chromatography Arginase I Arginase II CM-cellulose chromatography Arginase 1 n pmole of ornithine
Purijication
Total units
Specific activity”
Purification factor
-
45000
2250
0.05
12000
1800
0.15
3
2000
1200
0.60
12
400
720
1.8
36
30 2
270 1.6
9.0 0.8
180 16
12 0.22
144 0.88
12.0 4.0
240 80
60.0
60.0
1200
I min-’ mg-’ protein.
of Human
RESULTS Erythrocyte Arginase
For the purification, some of the steps previously described by Ber and Muszynska for rat liver arginase (15) and by Baranczyk-Kuima et al. for human blood serum arginase (17) were adopted. Purification by DEAE-Sephacel column chromatography was additionally included. For enzyme stabilization 1 mM MnCl, was used, and 1 mu 2-mercaptoethanol was applied to prevent arginase aggregation (18). All procedures were carried out at 4°C unless otherwise stated. The results of purification are summarized in Table 1. Step 1: Extraction. The erythrocytes were sonicated for 15 to 30 set at 5 A with 3 vol of solution containing 100 mM KCl, 1 mM MnCl,, and 50 mM TrisHCl buffer, pH 7.5. The homogenate was centrifuged at 15,000g for 15 min in a Sorvall (RC 2-B SS-34 rotor) and the pellet was discarded. Step 2: Ammonium Sulfate Fractionation. Solid NH42S04 was added slowly with stirring to the supernatant to 30% saturation (18 g/100 ml). After 60 min, the solution was centrifuged at 10,OOOg for 30 min, the pellet was discarded, and the supernatant brought to 60% saturation (22 g/100 ml) with ammonium sulfate. The precipitate was recovered by centrifugation, dissolved in a small volume of solution containing 1 mM MnCl,, 1 mM 2-mercaptoethanol, 10 mM Tris-HCl
250
KljDRA-LUBOIfiSKA,
ZAMFCKA,
AND
POREMBSKA
buffer, pH 7.5, and then dialyzed against two changes of the same solution for 25 hr. Step 3: Heat treatment. The dialysate was incubated for 15 min in the water bath at 55°C with agitation, cooled, and centrifuged at 15g for 15 min. Step 4: Precipitation with ethanol. Three volumes of ethanol cooled to - 10°C was slowly added to the supematant. The mixture was centrifuged at 10,OOOg at - 10°C for 15 min. The pellet was dissolved with 10 vol of solution containing 1 mM MnCl,, 1 mM 2-mercaptoethanol, 10 mM Tris-HCl buffer, pH 8.3, and dialyzed against the same solution for 24 hr. Step 5: DEAE-cellulose chromatography. The dialysate was centrifuged and placed on a column packed with DEAE-cellulose (24 x 1 cm) stabilized with 800 ml of 10 mM Tris-HCl buffer, pH 8.3. The activity was eluted with 40 ml of the same buffer (fraction I) and with a linear gradient of KC1 from 0.0 to 0.3 M in 300 ml of the same buffer (fraction II) (Fig. la). The active fractions I and II were pooled, concentrated in the presence of Ficoll, and dialyzed as described for Step 4. Each of the two arginase fractions I and II separated by DEAE-cellulose chromatography was then purified separately. Step 6: DEAE-Sephacel chromatography. After dialysis fraction I and fraction II were separately placed on columns packed with DEAE-Sephacel(16 x 1 cm), stabilized with 500 ml of 10 mM Tris-HCl buffer, pH 8.3. Fraction I was eluted with the buffer front (20 ml), fraction II with a linear gradient of KC1 from 0.0 to 0.3 M (Figs. lb and lc). Both active fractions were pooled, concentrated in the presence of Ficoll, and dialyzed 24 hr against a solution of 1 mM MnCl,, 1 mM 2-mercaptoethanol, and 10 mM Tris-HCl buffer, pH 7.5. Step 7: CM-cellulose chromatography. Only fraction I was submitted to CMcellulose chromatography. After dialysis the enzyme was applied on a column (6 x 1 cm), stabilized with 300 ml of 10 mM Tris-HCl buffer, pH 7.5. The enzyme adsorbed on the column was eluted with a KC1 concentration gradient from 0.0 to 0.3 in 100 ml of the same buffer (Fig. Id). The fractions showing arginase activity were pooled, concentrated, and dialyzed as described for Step 6. The arginase preparations obtained after purification represented two forms of enzyme, the first adsorbed on the CM-cellulose column (96% of total activity) and the second adsorbed on DEAE-cellulose as well as DEAE-Sephacel (4% of total activity). Partly purified preparations of the two forms of the enzyme the purification factor being 1200 and 80 for the first and second form, respectively, were used for determination of molecular weight and immunological properties. The two partly purified arginases were free of each other. Molecular
Weight
Both partly purified arginases had a molecular weight of 120,000 ? 5000 as determined by chromatography on Sephadex G-150.
ARGINASES
FROM HUMAN
Elution
ERYTHROCYTES
volume
251
(ml)
FIG. 1. DEAE-cellulose, DEAE-Sephacel. and CM-cellulose chromatography of human erythrocyte arginase. (a) Ethanol precipitate after dialysis (about 400 mg) was applied to DEAE-cellulose column (24 x I cm) equilibrated with IO mM Tris-HCI buffer, pH 8.3. The activity was eluted with 40 ml of the same buffer (fraction I) and with a linear gradient of KCI from 0.0 to 0.3 M in 300 ml of the same buffer (fraction II). The active fractions I and II were pooled, concentrated in the presence of Ficoll and dialyzed 24 hr against the solution containing 1 mM MnCL, 1 mM 2-mercaptoethanol, 10 mM Tris-HCl buffer, pH 8.3. (b, c) Fractions I and II from Step 5 after dialysis (about 30 mg and 2 mg of protein) were placed separately to DEAE-Sephacel column (16 x 1 cm) equilibrated with 10 mM Tris-HCI buffer, pH 8.3. The fractions I and II eluted with the buffer from 20 ml (Fig. lb) and with a linear gradient of KC1 from 0.0 to 0.3 M in 200 ml of the same buffer (Fig. lc), respectively. Both active fractions were pooled, concentrated in the presence of Ficoll, and dialyzed 24 hr against solution containing 1 mM MnCI,, 1 mM 2-mercaptoethanol, and 10 mM Tris-HCI buffer, pH 7.5. (d) Fraction I from Step 6 after dialysis (about 12 mg of protein) was applied to a CMcellulose column (6 x 1 cm) equilibrated with 10 mM Tris-HCI buffer, pH 7.5. The activity was eluted with a KCI concentration gradient from 0.0 to 0.3 M in 100 ml of the same buffer. The fraction showing arginase activity was pooled, concentrated, and dialyzed as described for Step 6. For all four columns fractions of 5 ml were collected at a flow rate of l-l.5 ml/min. Protein and arginase activity were determined as described under Experimental. 0, arginase activity; 0, protein content: -, KC1 gradient.
252
KBDRA-LUBOIfiSKA,
ZAMBCKA,
AND POREMBSKA
FIG. 2. Immunodiffusion of arginases isolated from human erythrocytes in the presence of guinea pig antisera directed against arginase A, from kidney and arginase A, from liver. Center well: (I) antiserum against arginase AI, from liver; (II) antiserum against arginase A, from kidney. (a) Erythrocyte arginase adsorbed on CM-cellulose. (b) Erythrocyte arginase adsorbed on DEAE-cellulose.
Immunological Properties
The arginases isolated from human erythrocytes were investigated by the double immunodiffusion test and by immunoelectrophoresis in the presence of antisera directed against the two parental forms of arginase from human tissues: A, from kidney and AS from liver. It should be noted that total immunological incompatibility of the two parental forms of arginase was reported by Zam,ecka and Porembska (2). Either of the two arginase forms isolated from erythrocytes reacted on immunodiffusion with both antisera, the occurrence of a spur indicating that they were only partially identical (Figs. 21 and 211). Spur formation in the presence of anti-AS serum by the form I adsorbed on CM-cellulose indicates that this form contained more antigenic determinants corresponding to those of form AS from liver (Fig. 21), whereas the second form II, adsorbed on DEAE-cellulose, contained more of the determinants of form A, from kidney (Fig. 211). Such behavior of the two forms of erythrocyte arginase was undoubtedly related to differences in subunit composition of the enzymes. Nonidentity of the two arginase forms was also confirmed by immunoelectrophoresis. When the two forms were electrophoresed simultaneously in the presence arcs differing in their distance of antiserum against arginase A,, two precipitation from the cathode were obtained (Fig. 3). To identify more closely the two erythrocyte arginases, their immunological properties were compared with those of other human arginases. In the presence of the antisera against form AS from liver and form A, from kidney, the first main form of erythrocyte arginase displayed immunological compatibility with arginase A4 from human kidney (Fig. 41), and the second minor form, with arginase A2 from human liver (Fig. 411). On immunoelectrophoresis, the first and second erythrocyte forms showed the same mobility as arginase A., from kidney (Fig. 5a and b) and A2 from liver (Fig. 5c and d), respectively.
ARGINASES
FROM HUMAN
ERYTHROCYTES
253
FIG. 3. Immunoelectroporesis of erythrocyte arginases. Center well: erythrocyte arginases adsorbed on (a) CM- and (b) DEAE-cellulose. Cross-reaction developed with the antiserum against liver arginase A,.
Since the arginase from human erythrocytes was earlier believed (3,lO) to be identical with arginase from human liver, the main form of the erythrocyte enzyme isolated in the present work was compared with the main form from liver, AS. In the double immunodiffusion test (Fig. 6) the main form of erythrocyte arginase displayed only partial identity with form A5 from human liver, whereas on immunoelectrophoresis it showed lower mobility toward the cathode than the latter form (Fig. 7). Moreover, it was found that the main erythrocyte arginase form gives a cross-reaction with both anti-A, and anti-A, antiserum (Fig. 811) whereas the liver A5 cross-reacts only with the homologous, anti-A, antiserum (Fig. 81). DISCUSSION We have demonstrated that two forms of arginase are present in both human liver and kidney A, and A2 and arginases A4 and A,, respectively (2). Arginase A, and arginase A, are parental forms built of one-type subunits; arginase A2 and A4 are hybrids composed of both kinds of subunits (2).
254
KEDRA-LUBOIfiSKA,
1
ZAMECKA,
AND POREMBSKA
2
FIG. 4. The relationship of human erythrocyte arginases with arginase A, from human kidney and arginase Ar from human liver. Center well: (I, 1 and 2) antiserum against liver arginase AS; (II, 1 and 2) antiserum against kidney arginase A,. (la) A, arginase from kidney; (2a) A, arginase from liver; (lb) human erythrocyte arginase adsorbed in CM-cellulose; (2b) human erythrocyte arginase adsorbed on DEAE-cellulose.
Now we have demonstrated that two forms of arginase occur also in human erythrocytes; on the basis of immunological studies they were identified as arginases A, and AZ. Molecular weight of either of them was 120,000 which proves that they are not artifacts formed in the course of purification. The two arginases from erythrocytes have antigenic determinants characteristic of either of the parental arginase forms since they cross-react with both antibodies directed
ARGINASES
FROM HUMAN
ERYTHROCYTES
255
FIG. 5. Immunoelectrophoresis of human erythrocyte arginase when comparing with arginase A, from human kidney and arginase A, from human liver. (a) A, arginase from kidney; (b) erythrocyte arginase adsorbed on CM-cellulose; (c) A, arginase from liver; (d) erythrocyte arginase adsorbed on DEAE-cellulose. Cross-reaction developed in the presence of antiserum against arginase A, from kidney.
against Al and against AS. This confirms also the hybrid nature of the erythrocyte arginases. It should be stressed that, at variance with the earlier suggestions (5,6), the main erythrocyte arginase form A,, was now found to differ from the main enzyme form from liver, AS. The two forms show but partial immunological compatibility due most probably to their different subunit structures. Unlike form
FIG. 6. Cross-reactivity of human liver arginase A5 and erythrocyte arginase adsorbed on CMcellulose. Center well: antiserum against liver arginase AS. (a) A, arginase from human liver; (b) erythrocyte arginase adsorbed on CM-cellulose.
256
KEDRA-LUBOI&SKA, a
ZAMECKA,
AND POREMBSKA b
+ FIG. 7. Immunoelectrophoresis of human liver arginase A, and erythrocyte arginase adsorbed on CM-cellulose. Antiserum against liver arginase A, (a) A5 arginase from human liver; (b) erythrocyte arginase adsorbed on CM-cellulose.
FIG. 8. Cross-reactivity of human liver arginase AS and erythrocyte arginase adsorbed on CMcellulose in the presence of the antisera against liver As and kidney A, arginases. Center well: (I) A, arginase from liver; (II) erythrocyte arginase adsorbed on CM-cellulose. (a) Antiserum against liver arginase AS; (b) antiserum against kidney arginase A,.
ARGINASES
FROM HUMAN
ERYTHROCYTES
257
A5 from liver, which is built of a single type of subunit, arginase & from erythrocytes is a hybrid of two types of subunits corresponding to subunits of the two parental arginase forms. The results obtained could be of significance in studies on the genetic origin of hyperargininemia. It should be taken into account that human arginase is built of two immunologically different subunits which most probably are synthesized by different genes. Determination of the subunit composition of the erythrocyte arginase could be useful for elucidation of the nature of hyperargininemia at the molecular level. SUMMARY 1. Two forms of arginase were isolated from human erythrocytes; the main form adsorbed on CM-cellulose and the second form, occurring in much smaller amount, adsorbed on DEAE-cellulose. 2. The molecular weight of either arginase was 120,000 + 5000. 3. The erythrocyte arginases are similar in immunological properties to arginase A4 from human kidney and AZ from human liver, respectively. 4. Despite the literature data stating that human erythrocyte arginase and human liver arginase are identical, it was found that the main forms of arginase of these tissues A4 from erythrocytes and AS from liver differ in immunological properties. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Porembska, Z., and Kedra, M., Bull. Acad. Polon. Sci. Ser. Biol. 19, 633 (1971). Zampcka, E., and Porembska, Z., Biochem. Med. Metab. Biol. 39, 258 (1988). Bascur, L., Cabello, J. P., Veliz, M., and Gonzales, A., Biochim. Biophys. Actn 128, 149 (1966). Cabello, J., Prajoux, V., and Plaza, M., Biochim. Biophys. Acta 105, 583 (1965). Hishibe, H., Physiol. Chem. Phys. 5, 453 (1973). Beruter, J., Colombo, J., and Bachnamm, C., Biochem. J. 1975, 499 (1978). Cederbaum, S. P., Show, K. N. P., and Valente, M., J. Pediutr. 90, 569 (1977). Michals, V., and Beaudet, A. L., Clin. Genet. 13, 61 (1978). Chinard, F. P., J. Biol. Chem. 199, 91 (1952). Porembska, Z., and Baranczyk-Kuima, A., Diagn. Lab. 3, 239 (1974). Lowry, 0. M., Rosebrough, N. J., Farr, A. L., and Randal, R. J., J. Biol. Chem. 193, 265
12.
Warburg, O., and Christian, W., Biochem. Z. 310, 384 (1941). Andrews, P., Biochem. J. 91, 222 (1964). Skrzypek-Osiecka, I., Robin, Y., and Porembska, Z., Acta Biochim. Polon. 30, 83 (1983). Ber, E., and Muszynska, G., Acta Biochim. Polon. 26, 103 (1979). Ouchterlony, O., in “Handbook of Experimental Immunology” D. M. Weir, Ed., pp. 655-706 Blackwell, Oxford, 1967. Baranczyk-Kuzma, A., Skrzypek-Osiecka, I., and Porembska, Z., Biochem. Med. 26, 174 (1981). Sakai, T., and Murachi, T., Physiol. Chem. Phys. 1, 317 (1969).
(1951).
13. 14. 15. 16. 17. 18.