Five forms of arginase in human tissues

BIOCHEMICAL

MEDICINE

AND

METABOLIC

BIOLOGY

39, 258-266 (1988)

Five Forms of Arginase in Human Tissues EL~BIETA Department of Biochemistry,

ZAMFCKA

AND ZOFIA POREMBSKA’

Institute of Biopharmacy, Academy of Medicine, ul.Banacha 02-097 Warsaw, Poland

I,

ReceivedAugust 20, 1986,andin revisedform July 16, 1987

Heterogeneity of arginase in human tissues has been postulated since 1965 (I), the data, however, on the number of the enzyme forms and their properties are fragmentary and inconsistent. The occurrence of three arginase forms in human tissues has been demonstrated by CM- and DEAE-cellulose chromatography (2), starch-gel electrophoresis (3), and the technique of anodic and cathodic electrophoresis on polyacrylamide gel (4). Two molecular forms of arginase have been reported to be present in human liver and erythrocytes (5). On the other hand, Nishibe (6) and Beruter et al. (7) have questioned the occurrence of various molecular forms of arginase in the liver and erythrocytes of man; they postulated that various forms of the enzyme isolated and described by other workers are artifacts arising during the purification procedure. So far, it has proved impossible to definitely determine the number of arginase forms in human tissues and to examine their properties, because of the unavailability of sufficiently well-purified preparations of the enzyme. On the other hand, the properties of various arginase forms occurring in rat tissues have been rather extensively studied. Recently, five forms of rat arginase have been isolated in our laboratory (8). They were designated, beginning from the most anodic form, as follows: A, kidney, A2 liver, A3 salivary gland, A4 kidney, and A5 liver. All forms have the same molecular weight, 120,000, and dissociate in the presence of EDTA into subunits of M, 30,000 (8). Arginase A, from kidney and arginase A5 from liver are built each of a single type of subunits, the respective subunits differ, however, in the electric charge and immunological properties. Both of these types of subunits and the native forms of arginases A, and A5 show complete immunological incompatibility, whereas arginases AZ, A3, and A4 display partial immunological similarity both with respect to each other and to arginases Al and A5 (8). These results indicate that in rat tissues arginase A, from kidney and A5 from liver are parental forms, and that their subunits combining in varying proportions produce the remaining three forms: AZ, A3, and Ad. ’ To whomreprint requestsshouldbe addressed. 0885-4505188 $3.00 Copyright All rights

0 1988 by Academic Press, Inc. of reproduction in any form reserved.

258

ARGINASE

IN HUMAN

TISSUES

259

Previously we have postulated the presence of only three arginase forms in human tissues (2). The results obtained for rat tissues prompted us to reexamine this question by studying the arginase forms occurring in human liver, kidney, and submaxillary gland. METHODS Reagents. Reagents were purchased as follows: L-arginine (Calbiochem, Los Angeles, CA), Sephadex G-100, Sephadex G-150, and Dextran 2000 (Pharmacia, UppsaIa, Sweden), CM-cellulose (CM-52) 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 horse myoglobulin were from Sigma Chemical Co., St. Louis, Missouri. All other chemicals were of the purest available grades from standard commercial sources. Human tissues. Liver, kidney, and submaxillary gland were taken within 2030 hr after death of 20- to 30-year-old persons, killed in traffic accidents, with no pathological changes observed at autopsy. Isolation of different forms of arginase. The nomenclature used to designate the isolated arginase was that proposed by Porembska and Zam,ecka (8). Pure arginase AS from human liver (sp. act 2500 pmole*min-’ mg-’ protein) was obtained according to Ber and Muszynska (9), arginase A’ from human kidney (sp. act 1000 pmole*min~’ mg-’ protein) and partly purified arginase A4 from kidney (sp. act 30 pmole-min-’ mg-’ protein) were prepared as raported by Skrzypek-Osiecka et al. (10). To obtain partly purified arginase A3 from salivary glands (sp. act 25 pmole.min-’ rng-’ protein) and arginase A2 from liver (sp. act 20 pmole*min~’ mg-’ protein) ammonium sulfate precipitation, acetone fractionation, heat treatment, as well as CM- and DEAE-cellulose chromatography were applied according to Skrzypek-Osiecka ef al. (IO). Each preparation represented only one form of arginase. Arginase assay. The enzymatic activity was determined according to Chinard (II), as modified by Porembska and Baranczyk-Kuima (12). Protein determination. Protein was assayed according to Lowry et al. (13) or spectrophotometrically by the method of Warburg and Christian (14), with crystalline serum albumin as standard. Molecular weight determination. The molecular weight of human arginase was determined by Sephadex G-100 and G-150 chromatography, as described by Andrews (15). The column (2 x 40 cm) was previously equilibrated with 100 mM KC1 in 50 mM Tris-HCl buffer, pH 7.5. Horse myoglobulin (MW 17,000) ovalbumin (M, 46,000), bovine serum albumin (MW 69,000), and bovine y-globulin (M, 150,000) were used as standards (5 mg in 1 ml). Fractions of 2 ml were checked for enzymatic activity and protein content. Isoelectric focusing. Isoelectric focusing was performed according to the method of Yesterberg and Svensson (16). Preparations of the isolated arginases were fractionated in linear sucrose density gradients O-50% solution with 1% Ampholin, pH 3.5-10, at 4°C for 48 hr. Fractions of 1 ml were collected, and pH was

260

ZAMFCKA

AND

PROEMBSKA

determined with the use of Raiometer pH-meter provided with a microelectrode. Protein content was determined by the spectrophotometric method. Double immunodiffusion. The test was carried out according to Ouchterlony (17) 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. Zmmunoelectrophoresis. 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. Antiserum. Pure arginase A, from human kidney and pure arginase A, from human liver were raised in female guinea pigs. The animals were immunized every tenth day with 200 pg of protein in 0.5 ml 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; blood was centrifuged at 10,OOOg for 15 min in a Servall (RC 2-B SS-34 rotor). Serum protein was precipitated by addition of ammonium sulfate (10 g/20 ml) of serum, centrifuged at 15,OOOg for 15 mitt, 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 arginase Aj, respectively. RESULTS Behavior of Different Chromatography

Forms

of Arginase

on CM- and DEAE-Cellulose

Two distinct forms of the enzyme were isolated from human liver A, and AZ and two forms from kidney A, and A4, whereas single arginase A3 was isolated from the submaxillary salivary gland. Only forms A4 and A5 were adsorbed on the CM-cellulose column and they could be eluted with a KC1 concentration gradient at 0.12 and 0.16 M, respectively (Fig. la). When the mixture of these two forms was cochromatographed, only highly purified preparations gave two distinctly separated peaks. On chromatography of crude extracts from liver and kidney or of slightly purified preparations, no distinct separation was observed, and the two forms of the enzyme were eluted as a single broad peak with 0.12-0.16 M KCl. Unlike forms A4 and As, arginase forms A,, AZ, and A3 were adsorbed only on DEAE-cellulose, and were eluted from the column with 0.18, 0.09, and 0.05 M KC1 (Fig. lb), respectively. When form Al from kidney and AZ from liver were cochromatographed, even crude extracts gave two distinctly separated active peaks, whereas separation of forms A2 and A3 was possible only with the use of highly purified preparations. The similarity in the behavior of forms AS from liver and & from kidney on CM-cellulose led us previously to the conclusion that they represented the same form, referred to as form A, in the previously used nomenclature (18).

ARGINASE

20

IN HUMAN

40

60

Elution

volume

TISSUES

60

261

100

(ml)

FIG. 1. CM-cellulose and DEAE-cellulose chromatography of arginase from human tissues. (a) The partly purified arginase A, from liver (sp act 1500 Units) and arginase A, from kidney (sp act 25 Units) were applied to about 10 mg and 10 mg of protein, respectively, to a CM-cellulose column (1 x 15 cm) equilibrated with 10 mM Tris-HCI buffer, pH 7.5, and eluted with a HCI concentration from 0.0 to 0.3 M in 100 ml of the same solution. Fractions of 3 ml were collected at a flow rate of l-l.5 ml/min. (b) The partly purified arginase A, from kidney (sp act 500 Units) A, from liver (sp act 10 Units) and arginase A, from salivary gland (sp act 15 Units) were placed in about 10, 15, and 20 mg of protein on a column packed with DEAE-cellulose (1 x 15 cm) equilibrated with 10 mM Tris-HCI buffer, pH 8.3. Elution conditions were as described for Fig. la. 0, Activity; 0; protein content; -, KC1 gradient.

Molecular

Weight

All arginases isolated from human tissues were found to have the same molecular weight: 120,000 ? 5000 (Fig. 2). Form A, from kidney and AS from liver retained the M, of 120,000 even on prolonged storage at - 2O”C, whereas the other forms were much less stable, especially arginase AZ which dissociated into subunits. Zsoelectric Focusing Each of the isolated arginase forms, subjected separately to isoelectric focusing, gave a single peak. The values of the isoelectric point were 7.1, 7.7, 8.4, 8.9, When and 9.3, for the arginase forms A,, AZ, A3, Ad, and AS, respectively. isoelectric focusing was performed for a mixture of the arginase forms (Fig. 3a and b) the number of the peaks corresponded to the number of forms present in the mixture; the pH values corresponded to those obtained previously for distinct arginase forms.

262

ZAMl$CKA

AND POREMBSKA

molecular

weight

(x 1 04)

FIG. 2. Molecular weight determination of arginase from human tissues by Sephadex G-150 filtration. Partly purified arginases A, from kidney, Az from liver, A, from salivary glands, & from kidney, and A, from liver (sp act 500, 10, 15, 25, 1500 Units, respectively), or marker proteins (2 mg) were applied. (1) Horse myoglobulin (17,000 M,); (2) chicken egg albumin (45,000 MJ; (3) bovine serum albumin (69,000 Mr); (4) bovine serum y globulin (150,000 M,); (5) arginase.

Immunological

Properties

In a double-immunodiffusion test arginase A, from kidney gave a cross-reaction exclusively with its own antiserum (Fig. 4 Ia), similarly, A5 from liver crossreacted only with anti-A5 antiserum (Fig. 4 IIb). Arginase A, gave no crossreaction with antiserum against A5 (Fig. 4 IIa) and arginase A5 did not precipitate with antiserum against form A, (Fig. 4 IB).

0.4

-

A08 /--I-* /c-hihi 1i _lC

0.2 *

7

.

r’

A4

1 20

40 Elution

60 volume

80

100

Ll12-t50 100

FIG. 3. Isoelectric focusing of arginase. Partially purified arginase A*, Aa, and A4 /lo, 5 mg of protein, respectively, were focused 60 hr at 500 V in pH gradient 3.5-10. Fractions of 1.5 ml were collected. 0, Activity; 0, protein; -, pH.

ARGINASE

I FIG. A, and against A,, (Ib

IN HUMAN

263

TISSUES

II

4. Ouchterlony double-immunodiffusion showing the precipitin reaction of purified arginase arginase A5 with the antiserum against both forms of enzyme. Center well: (I) antiserum arginase A, from kidney: (II) antiserum against arginase A, from liver, (Ia and IIa) arginase and Ilb) arginase A,.

When arginases A, and A5 (center well) were subjected simultaneously to the double-immunodiffusion test in the presence of the respective two types of antiserum, the precipitin arcs crossed each other, indicating that the two arginase forms showed complete immunological incompatibility (Fig. 5a and b). This behavior proves that arginase A, from kidney and arginase A5 from liver have completely different antigenic determinants. Arginase AZ, A3, and A4 reacted with both kinds of antiserum against A, and A5 forms (Fig. 6A and B). The presence of a spur in each case pointed to their partial immunological identity with forms A, and AS, as well as to partial immunological identity of forms AZ, A3, and A4 among themselves. Comparison of the immunological properties of the various arginases in the presence of anti-A, and anti-A, serum showed that arginase A2 from liver, A3 from salivery gland, and A, from kidney have two types of antigenic determinants: those of arginase Al from kidney and those of arginase A5 from liver. The isolated arginase forms differed in immunoelectrophoretic mobility (Fig. 7). Arginase A, migrated most rapidly to the anode (Fig. 7 Ia), and A5 migrated most rapidly to the cathode (Fig. 7 He). The former gave a precipitin arc only with antiserum against arginase A, (Fig. 7 I) and the latter gave a precipitin arc only with anti-A, serum (Fig. 7 II). Arginases AZ, Ax, and A, gave three precipitin arcs between those of forms A, and A5 at the same sites with both kinds of antiserum (Fig. 7 I and II, b, c, d). OtSCUSSlON Five forms of arginase, isolated from human tissues in the present work, were designated similarly as those isolated from rat tissues (8), beginning from the most anodic form, as arginase A, (main form from kidney), A, (minor form from

264

ZAMFCKA

AND POREMBSKA

a

b

FIG. 5. Cross-reactivity of antiserum against arginase A, from kidney and arginase A, from liver treated by the mixture of pure arginases A, and AS. Center well: pure arginase A, and pure arginase AS; (a) antiserum against arginase A,; (b) antiserum against arginase AS.

liver), A3 (single form from salivary gland), A4 (minor form from kidney), and A5 (main form from liver). Arginases A, and A5 were purified to homogeneity; the other three forms were obtained in a partly purified state. The preparations of all five forms of arginase showed the same molecular weight: 120,000, proving that they were not degradation products of native forms. The isolated arginases differed in their behavior on CM- and DEAE-cellulose chromatography as well as in electrophoretic mobility. They were characterized by different values of isoelectric points. Immunological properties of the arginases adduced strong evidence for the presence of five distinct forms of the enzyme. Like the analogous arginases from rat tissues (8), arginase A, from rat kidney showed complete immunological incompatibility with arginase A5 from liver. Since arginase AZ, A3, and A4 from human tissues gave a cross-reaction with both anti-A, and -A5 serum it can be concluded that they contain both types of determinants, being built of the corresponding two kinds of subunits. The quantitative subunit composition of these hybrids is now the subject of our work. SUMMARY

Five forms of arginase, A,, AZ, A3, Aq, and AS, were found to be present in human tissues. The molecular weight of all these forms is the same, 120,000, but they differ in the behavior on DEAE- and CM-cellulose, electrophoretic mobility, isoelectric point, and immunochemical properties. Forms A, from kidney and A5 from liver show complete immunological incompatibility, whereas forms

IN IIUMAN

ARGINASE

a

265

‘I‘ISSUES

a

b

b

A

B

C

II

I

FIG. 6. Ouchterlony double-immunodiffusion showing the precipitin reaction of arginases isolated from human tissues with the antiserum against arginase A, and arginase A,. Center well: (I A,B,C) antiserum against arginase A,; (II A,B,C) antiserum against arginase A,. (I) Aa, kidney A,; Ab, liver AZ; Ba, liver A,; Bb. salivary glands A,; Ca, salivary glands A,, Cb, kidney A,. (II) Aa, liver A,, Ab, kidney A,; Ba, kidney A,, Bb, salivary glands A,; Ca, salivary glands, A,, Cb, liver A,.

a

FIG. 7. Immunoelectrophoresis A2 from liver; (c) arginase A3 from liver. Cross-reaction developed in and antiserum against arginase As

b

c

d

e

of human arginase forms. (a) arginase A, from kidney; (b) arginase salivary glands; (d) arginase A, from kidney; (e) arginase A, from the presence of antiserum against arginase A, from kidney (I), from liver (II).

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A2 from liver, A3 from salivary gland, and A4 from kidney exhibit partial incompatibility with respect to each other and to forms A, and AS. ACKNOWLEDGMENT The authors thank Ms. Katarzyna Dunin-Szpotanska

for her skillful technical assistance.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Cabello, J., Prajoux, V., and Plaza, M., Biochim. Biophys. Acta 105, 583 (1965). Porembska, Z., and Kedra, M., Bull. Acad. Polon. Sci. Ser. Sci. Biol. 19, 633 (1971). Borcic, O., and Straus, N., J. C/in. Chem. Cl&z. Biochem. 14, 533 (1976). Penchala, F., Reddi, W., Knox, E., and Herzfeld, A., Enzyme 20, 305 (1975). Bascur, L., Cabello, J. P., Veliz, M., and Gonzeles, A., Biochim. Biophys. Acta 128, 149 (1966). Nishibe, H., Physiol. Chem. Phys. 5, 453 (1973). Beruter, J., Colombo, J. P., and Bachmann, C., Biochem. J. 175, 499 (1978). Porembska, Z., and Zamecka, E., Acta Biochim. Pal. 31, 223 (1984). Ber, E., and Muszynska, G., Acta Biochim. Pol. 26, 103 (1979). Skrzypek-Osiecka, I., Robin, Y., and Porembska, Z., Acta Biochim. Pal. 30, 83 (1983). Chinard, F. P., .I. Biol. Chem. 199, 91 (1952). Porembska, Z., and Baranczyk-Kuima. A., Diagn. Lab. 3, 239 (1974). Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem. 193, 265 (1951). Warburg, O., and Christian, W., Biochem. Z. 310, 384 (1941). Andrews, P., Biochem. J. 91, 222 (1964). Yesterberg, O., and Svensson, H., Acta Chem. &and. 20, 820 (1966). Ouchterlony, C., in “Handbook of Experimental Immunology” (D. M. Weir, Ed.), pp. 655706. Blackwell, Oxford, 1967. Gasiorowska, I., Porembska, Z., Jachinowicz, J., and Mochnacka, I., Acta Biochim. PO/. 17, 19 (1970).