Comparative physical-chemical studies of mammalian arginases

Comp. Biochem. Physiol., 1970, Vol. 37, pp. 345 to 359. Pergamon Press. Printed in Great Britain

COMPARATIVE P H Y S I C A L - C H E M I C A L S T U D I E S OF MAMMALIAN ARGINASES* H E L G A H I R S C H - K O L B , J O H N P. H E I N E , H E L M U T D A V I D M. G R E E N B E R G

J. K O L B and

Cancer Research Institute, University of California School of Medicine, San Francisco, California 94122 (Received 18 M a y 1970)

A b s t r a c t - - 1 . Liver arginase (E.C. 3.5.3.1) of various mammalian species (rat, mouse, dog, rabbit, pork, monkey) were partly purified and their isoelectric properties determined by carboxymethyl cellulose column chromatography and isoelectric focusing. 2. The ureotelic arginase can be divided into mainly two groups; (i) basic and (ii) slightly acidic or neutral proteins. 3. The two groups of arginases showed marked differences in the binding of Mn ~+. 4. Only beef liver arginase could be activated by Co *+ and Ni 2+, all other mammalian arginases were inhibited by these metal ions. 5. The molecular weights were found to range from 120,000-160,000 daltons. 6. High Michaelis constants (6-20 mM) were obtained for all arginases studied. 7. Similarities were also found in the pH optima (pH 9"3-10'5) as well as the optimal Mn 2+ concentration (40 mM) required to obtain maximal catalytic activity.

INTRODUCTION IT HAS been reported by several research groups (Mora et al., 1965, 1966; Soru, 1965 ; Brown, 1966 ; Soberon et al., 1967; Reddy & Campbell, 1968 ; Rossi & Grazi, 1969; Middlehoven, 1969) that significant differences in ureotelic, uricotelic and ammonotelic liver arginases have been observed with respect to antigenic properties, molecular weights, Michaelis constants, substrate inhibition and stability during dialysis. Besides this, there are n u m e r o u s publications reporting different properties a m o n g m a m m a l i a n arginases (Greenberg, 1955; Olomucki, 1955; G r e e n b e r g et al., 1956; G r a s s m a n et al., 1958; Bach & Killip, 1958, 1961; Bach et al., 1963; Schimke, 1964; Cabello et al., 1965; Rossman et al., 1966), e.g. there seems to be a wide discrepancy in the isolation procedures described, the affinity for M n binding, metal ion activation as well as molecular weights. * Aided by research grants from the National Cancer Institute (CA 02915 and CA 03175) National Institutes of Health. 345

346

H. HIRSCH-KOLB,J. P. HEINE,H. J. KOLB AND D. M. GREENBERG

I t was t h e p u r p o s e of this i n v e s t i g a t i o n to c o m p a r e arginases f r o m v a r i o u s m a m m a l i a n species u n d e r i d e n t i c a l c o n d i t i o n s as to t h e i r kinetic, isoelectric an d molecular properties. MATERIALS AND METHODS L-Arginine (free base) and Tris (hydroxymethyl) aminomethane (A grade) were obtained from Calbiochem. T h e chlorides of M n 2+, Co 2+ and Ni 2+, analytical grade, were purchased from Mallinckrodt, New York. Carboxymethylcellulose, purchased from BioRad Laboratories, was prepared according to Sober & Peterson, 1956. Frozen livers of monkey, rabbit and mouse were obtained from Pel-Freez. For the purification of rat, beef, pork and dog liver arginase, fresh livers were used.

Enzyme purification Arginase from rat liver was prepared as described elsewhere (Schimke, 1964; Hirsch-Kolb & Greenberg, 1968). Beef liver arginase was isolated according to a procedure given by Greenberg, 1955. Arginases from the other species were partially purified according to the following procedure. It not stated otherwise, the purification steps were carried out at 4°C. Liver homogenates were prepared by homogenizing 50 g of livers in a Waring blendor at low speed for 3 rain in 150 ml 0'01 M T r i s - H C l buffer, pH 7"5, containing 0"05 M MnC12 and 0'1 M KC1. T h e homogenate was centrifuged at 20,000 g for 60 min. Th e precipitate was discarded. After cooling the supernatant in an ice-salt bath, 1"5 vol. of acetone (reagent grade) per volume of supernatant were added slowly, while stirring the mixture vigorously. During this purification step the temperature was kept at - 1 0 ° C . After the addition of acetone the solution was centrifuged in an International refrigerated vacuum centrifuge at - 10°C for 10 min at 5,000 g. T h e supernatant liquor was poured off and discarded. T h e precipitates were collected and dissolved in 75 ml of 0"01 M Tris-HC1 buffer, pH 7"5, containing 0-05 M MnC12. To facilitate solution, the precipitate and the buffer solution were homogenized in a blendor for about 1 rain at low speed. Th e undissolved material was then removed by centrifugation. In order to remove traces of acetone and small molecular weight components the protein solution was dialyzed over night against the same buffer (0-01 M Tris-HC1, 0-05 M MnC12, p H 7-5). Heat treatment was carried out by incubation of the protein solution (adjusted to pH 7'7 with very dilute NaOH) in a 60°C water-bath for 12 rain. Th e denatured proteins were removed by centrifugation (20,000 g, 15 rain). Th e supernatant was dialyzed against 2 liters of 5 x 10 -4 M Tris-HC1 buffer, p H 7-5, for 24 hr, with two changes of the dialysis bath. T h e dialyzed solution was then lyophilyzed to a small volume (about 15 ml) and dialyzed again for one day against the same buffer. Precipitates were removed by centrifugation. Carboxymethyl cellulose (CMC) column chromatography (column size: 10 x 2"5 cm), using a peristaltic pump, was carried out at a flow rate of 30 ml per hr. Fractions of 2'5 ml were collected. If not stated otherwise, the column was equilibrated with 5 x 10 -4 M T r i s HC1 buffer. After the dialyzed protein solution was applied on the column, the latter was washed with 100 ml of the same buffer and then eluted with 50 ml of 0"1 M arginine solution (adjusted to p H 7'5 with HC1). T h e collected fractions were analyzed for protein and arginase activity. Th e most active fractions were pooled, concentrated to about 5 rot, dialyzed against 0"01 M Tris-HC1 buffer, p H 7"5, and stored by freezing at - 20°C. Protein determinations were carried out with the Folin-Ciocalteu reagent (Layne, 1957), using bovine serum albumin (crystallized, purchased from Pentex) as a standard. Arginase activity was determined with isonitrosopropiophenone (Greenberg, 1966), obtained from Eastman Kodak Company.

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One enzyme unit is defined as the amount of enzyme that produces one micromole urea per rain at 25°C (Van Slyke & Archibald, 1946). Specific activity is expressed in enzyme units per mg protein nitrogen. Metal ion activation of previously dialyzed arginase preparations was carried out by adding MnC12, CoC1, or NiC1, solutions (in 0"05 M Tris-HC1 buffer, adjusted to pH 7'5) to a final concentration of 0"05 M. The enzyme together with the divalent metal were then incubated in a 40°C water-bath for 1 hr. Isoelectric focusing was performed according to a method described by Vesterberg & Svensson (1966) and Ahlgren et al. (1967), using carrier ampholytes by LKB-Produkter, Stockholm, to establish a pH-gradient of 3-10. One ml arginase solution, concentrated in a collodion bag (Sartorius-Membranfilter), containing 1200-9000 enzyme units, was applied on the column. The experiments were carried out for a period of 64 hr. The voltage was increased stepwise from 200-500 V, and kept constant at the upper figure. The column temperature was 8°C. Fractions of 2 ml were collected from the bottom of the column and enzyme activity and pH were determined for each fraction, pH-measurements were carried out at 4°C with a Beckman Zeromatic II pH-meter, equipped with a combination electrode and an automatic temperature compensator. Molecular weight determinations were performed by Sephadex column chromatography (Whitaker, 1963 ; Determann & Michel, 1966). A Sephadex G 200 column (2 × 65 cm) was prepared, equilibrated with 0'01 M Tris-HC1 buffer, pH 7"5, and eluted with the same buffer at a flow rate of 20 ml/hr. The temperature of the column was 4°C. After concentration in collodion bags, arginase samples of 1 ml (containing 900-4000 enzyme units) were applied on the column. Fractions of 1"5 ml were collected using a Gilson fraction collector, equipped with a drop counting device. For the calibration of the column, bovine serum albumin, crystallized (Pentex), chymotrypsinogen (Calbiochem) and glyceraldehydephosphate dehydrogenase from rabbit muscle (Boehringer, Mannheim) were used as standards. The void volume V0 of the column was determined with blue dextran. The maximum of the eluted arginase peak was determined by activity measurements. RESULTS

Purification of arginases W i t h the exception of arginase f r o m rat liver, w h i c h was purified to h o m o geneity (S.A. ~ 20,000), and beef liver, w h i c h had a final specific activity of 4500, the arginases f r o m the other species were purified b y acetone precipitation, heat t r e a t m e n t and C M C c o l u m n c h r o m a t o g r a p h y . T h e specific activity of these arginases was increased 80-1,500 fold (see T a b l e 1). After purification the e n z y m e s were concentrated in collodion bags and stored frozen. N o noticeable decrease in activity could be observed after several weeks of storage. T h e adsorption behavior of the arginase preparations on C M C , equilibrated with 5 x 10 -4 M T r i s - H C l buffer at p H 7.5, was used as a criterion as to w h e t h e r the arginase f r o m a certain species was a m o r e neutral or basic protein. Figures la and l b show two typical elution patterns for a slightly acidic arginase (from rabbit liver, Fig. la) and a basic arginase (from dog liver, Fig. lb). T h e first t y p e of arginase is not a d s o r b e d on the c o l u m n at the p H and ionic strength applied and the e n z y m e is eluted f r o m the c o l u m n with the void volume. I n case of the basic arginases the e n z y m e s were b o u n d tightly to C M C w h e n the c o l u m n was washed with buffer solution (5 x 10 -4 M T r i s - H C 1 , p H 7.5) and the active protein was then eluted with 0.1 M arginine (adjusted to p H 7.5 with HC1).

348

H. HIRSCH-KOLB, J. P. HEINE, H. J. KOLBANDD. M . GREENBERG

TABLE i--SPECIFIC ACTIVITIES OBTAINED FOR UREOTELIC LIVER ARGINASES FROM VARIOUS SPECIES AND CHARACTERIZATION OF THEIR ISOELECTRIC PROPERTIES ACCORDING TO THEIR ADSORPTION BY CMC IN 0"01 M T r i s - H C l BUFFER, pH 7'5

Isoelectric characteristic derived from C M C

Species Rat Beef Pork Dog t Mouse Rabbit Monkey

Sp. act.

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FIG. 1. a. Elution pattern of rabbit liver arginase from C M C . Prior to chromatography the enzyme solution was dialyzed against 5 × 10 -4 M T r i s - H C 1 buffer, p H 7"5. T h e c o l u m n was equilibrated and washed with the same buffer. Solid line, protein m e a s u r e d at 280 m/z; dashed line, arginase activity determined with isonitrosopropiophenone, b. Elution pattern of dog liver arginase u n d e r the same conditions as described above.

PHYSICAL-CHEMICAL

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MAMMALIAN

349

ARGINASES

Exceptions from the above typical elution patterns were observed with the arginases from pork, and monkey liver. Under the conditions described, pork arginase was separated into two peaks by CMC column chromatography; one peak of arginase activity appeared within the void volume, suggesting a comparatively neutral (uncharged) protein. The second peak was eluted by 0" 1 M arginine solution, which is typical for a more basic protein. Also in the case of monkey liver arginase a separation into two enzyme fractions could be observed. From an isoelectric focusing experiment--as described in a later section--the pI of these two fractions was determined to be at pH 6.8 and 7-5. When a mixture of the two peaks (previously dialyzed against 10 -~ M TrisHC1 buffer, at pH 6.8 or 7.5, respectively) was applied under identical conditions on two CMC columns, equilibrated with either pH 6.8 or 7.5 buffer (10 -3 M TrisHCI) the elution patterns shown in Figs. 2a and 2b were obtained. At pH 7.5 one arginase peak was eluted in the void volume (monkey I), the second one was u

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FRACTION NO. F l a . 2. E l u t i o n p a t t e r n s of m o n k e y liver arginase f r o m C M C c o l u m n c h r o m a t o graphy, a. P r i o r to c h r o m a t o g r a p h y t h e e n z y m e was dialyzed against 5 x 10 -4 M T r i s - H C 1 buffer, p H 7.5. T h e c o l u m n was e q u i l i b r a t e d a n d w a s h e d w i t h t h e same buffer; t h e n t h e a d s o r b e d p r o t e i n s were eluted w i t h 0"1 M a r g i n i n e solution (adjusted to p H 7"5 w i t h HC1). Solid line, p r o t e i n m e a s u r e d at 280 m/x; d a s h e d line, arginase activity, b. Similar e x p e r i m e n t as in (a), b u t all p r o c e d u r e s were carried o u t at p H 6"8.

350

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HIRSCH-KOLB,J. P. HEINE,H. J. KOLBANDD. M. GREENBERG

washed off the column with 10-3 M buffer (monkey II). From the column equilibrated with p H 6"8 buffer the first peak emerged at the buffer front (monkey I), while the second one could only be eluted with 0.1 M arginine solution (monkey II).

Isoelectricfocusing Our experiments with pH-gradients from 3-10 as described under "Materials and M e t h o d s " showed that the pI values of arginases from various mammalian vertebrates differ remarkably. Accordingly, arginases can be divided into two groups: (1) basic arginases with a pI around p H 9.4 (Figs. 3a and 3b) and (2) neutral or slightly acidic arginases (Figs. 3c and 3d). F r o m monkey liver two arginases with pI values of p H 7-5 and 6.8 could be isolated by column chromatography (Figs. 2a and 2b) and isoelectric focusing (Fig. 3d). A mixture of the pork liver enzyme yielded a distinct separation into two major peaks with maxima at p H 6.9 and 8.8 (Fig. 3e). It is not clear, whether the small peak repeatedly obtained at p H 6.0 represents a third isozyme or is due to an artefact in the experimental procedure. Interesting is the difference in M n activation for the two major peaks. While the peak at p H 8.8 was activated about two-fold as usual, the arginase activity for the peak at p H 6-9 could be increased five-fold by M n 2+ activation. In Table 2 the isoelectric points of several arginases are presented. In the legend to Fig. 3 the percentage of recovery of catalytic activity after isoelectric TABLE 2--KINETIC AND MOLECULAR CONSTANTS FOR UREOTELIC LIVER ARGINASES

KBb Species

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20 7 (4)

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9"4 --

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10-10'5" 10-10.5 10-10" 5 10 9' 5-10' 5 * 10'2

6'9 and 8'8 9.4 -6'5 6' 8 and 7' 5 --

118,000 (13) Dissociation into subunits of 30,000 140,000 ? 137,000 160,000 140,000 138,000

* A mixture of the peaks separated on CMC was used. focusing is given. A noticeable difference in stability of the two groups of arginases was observed: basic arginases were far more stable to isoelectric focusing than neutral or slightly acidic arginases. This could also be observed during certain purification steps, e.g. acetone precipitation, dialysis and column chromatography. T h e higher activity loss of neutral or slightly acidic arginases is probably a consequence of an increased instability due to the easier dissociation of M n ~+ from the e n z y m e - M n complex.

PHYSICAL-CHEMICAL

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FIG. 3. Isoelectric focusing of arginases f r o m rat, dog, rabbit, m o n k e y a n d p o r k liver. Solid line, n o n - a c t i v a t e d e n z y m e ; d a s h e d line, e n z y m e activated i n 0"05 M M n C I ~ at 40°C for 1 hr. Electrofocusing e x p e r i m e n t s were carried o u t in a p H 3 - 1 0 g r a d i e n t at 8°C. a. R a t liver arginase. A b o u t 9,000 e n z y m e u n i t s of p u r e arginase were applied o n t h e c o l u m n . R e c o v e r y of e n z y m a t i c activity 55 p e r cent. A similar r u n in a p H 7 - 1 0 g r a d i e n t also yielded a p I of 9'4. b. D o g liver arginase: 2500 e n z y m e u n i t s of a 560-fold purified arginase were p u t o n t h e c o l u m n . Recovery was a b o u t 45 p e r cent. p I = 9"4. c. R a b b i t liver arginase. T h e e x p e r i m e n t was s t a r t e d w i t h 3800 e n z y m e u n i t s of a 130-fold purified r a b b i t liver arginase. L o w r e c o v e r y of 7 p e r c e n t ; p I = 6"4. d. M o n k e y liver arginase. ( M i x t u r e of b o t h isozymes.) A p p r o x i m a t e l y 220-fold p u r i f i e d ; 4500 e n z y m e units. L o w r e c o v e r y of a b o u t 5 p e r cent. T h e n o n - a c t i v a t e d as well as t h e activated e n z y m e curves s h o w clearly two peaks w i t h p I = 6"8 a n d 7"5. e. P o r k liver arginase. R e c o v e r y 9 p e r cent. S e p a r a t i o n into two distinctly m a j o r peaks of arginase activity w i t h p I at 6"9 a n d 8"8 occurs. A smaller peak at p H 6"0 was o b s e r v e d i n two identical e x p e r i m e n t s . I n t e r e s t i n g is t h e different activation b y M n z+.

352

H. HIRSCH-KOLB,J. P. HEINE,H. J. KOLBANDD. M. GREENBERG

All arginases were completely soluble at their isoelectric point in the presence of ampholytes. On activation in 0.05 M MnC] 2 solution for 1 hr at 40°C and pH 7.5, the activity was in most cases increased approximately twofold.

Manganese binding Several publications report (Rogers & Moore, 1963 ; Brown, 1966) that according to the results obtained from dialysis experiments, the binding of M n 2 '~ to the protein, which is important for the catalytic activity, seems to vary for arginases isolated from different species. In our experiments dialysis of dilute arginase solutions (about 0.1 mg/ml) was carried out for 1-4 days against 0.01 M Tris-HC1 buffer, pH 7-5, with daily changes of the dialysis bath. The decrease in catalytic activity of the dialyzed samples of several arginases is given in Fig. 4. These experiments show clearly that basic and neutral or slightly acidic arginases have a different behavior on dialysis. The catalytic activity of basic arginases (rat, mouse and dog) is reduced to about 40-50 per cent of the original (maximal) value after two days dialysis. This value then remains fairly constant within two more days of dialysis. There were only minor traces of precipitate found in the dialysis bag after 4 days. When activated with M n 2+, 90-100 per cent of the original activity could be regained in all dialyzed samples. 10{

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FIG. 4. Loss of enzymatic activity of various arginases by continued dialysis for 1-4 days. The fully Mna+-activated original sample was taken as 100 per cent. Dialysis of the 2 ml samples, containing approximately 0'1 mg arginase/ml, was performed against 0.01 M Tris-HC1 buffer, pH 7'5, with daily changes of the dialysis bath. ©--©, Mouse arginase; @--@, rat arginase; 0 - - 0 , dog arginase ; 0 - - 0 monkey I arginase ; 0 - - 0 , monkey I I, arginase; O---O, beef liver arginase.

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Neutral or slightly acidic arginases (beef, monkey I and II, and rabbit) lost considerably more catalytic activity under the same conditions. It should be pointed out that after a short dialysis time (1 day) 85-100 per cent of the original catalytic activity could be recovered in these samples. On continued dialysis, however, a permanent loss of activity occurred. This agrees with the observation that in these samples a much heavier protein precipitate was formed, which could not be reactivated by Mn 2+. Since the catalytic activity is directly correlated to Mn 2+ binding, as could be shown by magnetic resonance studies of rat liver arginase (Hirsch-Kolb et aL, 1970) it can be concluded from the above results that neutral or slightly acidic arginases bind the Mn 2+ less tightly and, therefore, lose it to a greater extent in the above experiment. The loss of Mn obviously renders the enzyme more unstable, which results in a higher denaturation of the enzyme on prolonged dialysis.

Metal ion activation Previously dialyzed beef liver arginase, which had lost part of its Mn 2+, could be reactivated at pH 7.0 with 0.05 M Co 2+ or Ni 2 L solutions on incubation in a 40°C water bath for one hour. The catalytic activity increased markedly when the activity assay was carried out at pH 7.5. Beef liver arginase can bind Co 2+ and Ni 2+ instead of Mn 2+ to form an enzymatically active complex. The same observations are reported for horse liver arginase (Mohamed & Greenberg, 1945). These Co s+ and Ni 2+ activated arginases show two pH-optima at pH 7"5 and 9-5, compared to only one pH optimum at pH 9-5 for the Mn-activated enzyme. When dialyzed arginase preparations from rat, dog, mouse, monkey (I and II) and rabbit liver were incubated with 0"05 M Co 2÷ or Ni ~+ solutions at pH 7.0 under the same conditions as described above for beef liver arginase, and activity tests of these samples were carried out at pH 7.5 and 9.5, no increase in activity could be observed at either pH. On the contrary, there was a 10-50~o inhibition compared to the initial activity. In another experiment it could be shown that this inhibitory effect is dependent upon the Co s+ and Ni 2~ concentration. These results suggest that Co s+ and Ni 2+ probably are bound by the enzymes, but render them less active. Kinetic studies K,,, values for several mammalian arginases were determined according to the method of Lineweaver-Burk (1934). Activity tests were carried out at substrate concentrations of 5-100 mM arginine at pH 9.5 and 25°C. In the case of isozymes a mixture of both elution peaks from CMC was used for the kinetic determinations. From Table 2 it can be seen that the Michaelis constants determined for a number of arginases lie between 6 and 20 mM, a comparatively high value. The pH dependence of the arginase activity was determined for several arginases at a substrate concentration of 100 mM and 25°C. The pH of the arginine solutions was adjusted with HC1. As shown in Table 2, the pH optima for the Mn 2+ activated arginases are in the range from 9-3 to 10-5. In most cases the 13

354

H. HIRSCH-KOLB,J. P. HEINE, H. J. KOLBAND D. M. GREENBERG

optimum is spread over half a pH unit. For dialyzed, non-activated arginases the same values were obtained. Mn2+-activation studies of dialyzed arginase preparations (rat, mouse, dog and monkey) were carried out in 5-100 mM MnCI~ solutions, adjusted to pH 7.5 with 0.05 M Tris-HC1 buffer. For metal ion activation the samples were incubated in a 40°C water bath for one hour. With increasing Mn ~+ concentrations increasing catalytic activities were obtained. The maximum activity was reached at 40 mM MnC12. At higher Mn 2+ concentrations (100 raM) the catalytic activity remained constant. In contrast to human erythrocyte arginase (Kalab & Pellikan, 1964) no inhibition due to protein denaturation occurred at these high Mn 2+ concentrations.

Determination of molecular weights The molecular weights of rat and horse liver arginase have been determined previously by ultracentrifugation (Greenberg et al., 1956; Hirsch-Kolb & Greenberg, 1968). As shown in Table 2 the molecular weights obtained by gel filtration lie generally between 120,000 and 160,000 for the mammalian arginases investigated, (Fig. 5), and thus differ remarkably from those of urieotelic arginase (1).

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FIC. 5. Determination of molecular weights by Sephadex G 200 gel filtration. Among the results obtained by Sephadex chromatography, beef liver arginase represents an exception. From the elution pattern of the beef liver enzyme a molecular weight of only 30,000 was calculated. Since sedimentation and diffusion studies carried out in our laboratory with highly purified beef liver arginase (sp. act. 4,500) suggested a molecular weight between 120,000-140,000, the low value for the molecular weight obtained using Sephadex gel filtration could represent that of a subunit. This also would be in good agreement with the subunit molecular weight determined for rat liver arginase (Hirsch-Kolb & Greenberg, 1968).

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The fact that a high percentage of the catalytic activity (about 80%) of the beef liver enzyme was lost by column chromatography also agrees well with the assumption that this arginase easily dissociates on the Sephadex column into subunits. From these results no conclusions can be made whether the subunit of the beef liver enzyme is catalytically active or whether an association of the subunit to an active enzyme molecule occurs. A somewhat similar result was obtained repeatedly for dog liver arginase. The enzyme was eluted in a wide peak, whose maximum gave a molecular weight of 60,000. However, since the elution peak was wide-spread, we believe that the above molecular weight represents an average value of a dissociation-association equilibrium rather than the intrinsic molecular weight of the enzyme. In the case of monkey, and pork liver two arginase peaks could be separated on CMC. However, when the two fractions from monkey liver were applied separately on Sephadex the same molecular weight of 140,000 was obtained for each. Pork liver arginase was resolved on Sephadex G 200 into two peaks with arginase activity (ratio ~ 1 : 3). For the major peak the usual molecular weight of 140,000 was calculated, whereas, a smaller peak, probably due to aggregation of the enzyme, appeared right after the void volume. DISCUSSION The comparative physical-chemical properties of a number of mammalian arginases have been investigated. It could be concluded from the behavior on CMC that ureotelic arginases generally divide into two groups: (a) basic and (b) neutral or slightly acidic proteins. Consequently, it follows that purification procedures, which try to attain homogeneity of the enzyme preparation, will differ in principal for both types. It could be shown that basic arginases such as from rat, dog and mouse, can be purified to a high degree by the procedure given above, using CMC column chromatography as a final purification step. For neutral arginases, however, a procedure applying DEAE column chromatography might be useful. The results obtained from CMC chromatography which strongly suggest a differentiation of arginases into basic and neutral proteins, were confirmed by isoelectric focusing experiments. Generally, a pI of pH 8.8-9.4 was obtained for basic arginases, and pH 6.4-7.5 for neutral arginases. In all experiments carried out, it was observed that basic arginases were much more stable to the procedure of isoelectric focusing than the neutral proteins. This is probably due to the fact that the neutral arginases lose their Mn easier--as shown by dialysis experiments-which renders the enzyme more unstable. The catalytic activity of the enzyme fractions obtained after electrofocusing was usually increased twofold by Mn activation. An exception was one fraction of pork liver arginase (pI = 6"9), where the enzyme activity could be increased fivefold by incubation with Mn 2+. It was interesting to observe for pork liver arginase--as suggested by the CMC elution pattern--a separation into two major peaks (Fig. 3e) with pI values of 6.9 and 8-8.

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In the case of monkey liver arginase, which seem to have some similarity with human liver arginase (Cabello et al., 1965), two isozymes with pI = 6"8 and 7.5 were obtained by isoelectric focusing (Fig. 3d), again confirming the result obtained from CMC chromatography. Unlike human liver arginase, where the ratio of the two peaks eluted from CMC is as high as 10 : 1, a ratio of approximately 2 : 1 was obtained for the two isozymes in monkey liver. The requirement for Mn 2~ for full catalytic activity seems to be common to all mammalian arginases. In dialysis experiments basic and neutral arginases showed different characteristics. Upon 2 days of dialysis basic arginases lost about 50 per cent of their catalytic activity due to dissociation of the Mn. The enzymatic activity then remained constant for several days of continued dialysis. By Mn activation 90-100 per cent of the original activity could be recovered in all samples. There was only little formation of a precipitate due to denaturation of the enzyme. When neutral arginases were dialyzed for 1-2 days a much larger amount of catalytic activity (75-90 per cent) was lost. Most of the original activity could be recovered by incubation with Mn. However, on continued dialysis a heavy precipitate formed in the dialysis bag and tile loss of catalytic activity became irreversible. The precipitated arginase remained insoluble and could no longer be activated by Mn2+. A comparison of the results obtained for basic and neutral arginases suggests, that there might be a difference in the binding affinity for Mn. However, the fact appears to be common for both types of arginases that once more than 50 per cent of the Mn is lost, the enzyme becomes more and more unstable. It has been reported that previously dialyzed beef and horse liver arginase, which have lost part of their Mn by dissociation, can be activated by Co 2+ and Ni 2+ (Mohamed & Greenberg, 1945 ; Greenberg et al., 1956). These Co and Niactivated enzymes have a pH-optimum curve with two optima (around 7.5 and 9.5), compared to only one optimum at pH 9-5 for Mn-activated arginase. When we tried to activate several mammalian arginases (rat, dog, mouse, rabbit and monkey) by incubation in 0.05 M Co 2t and Ni 2+ solutions at pH 7.0, and then carried out the activity assays at pH 7.5 as well as at 9"5, no increase at either pH was observed. On the contrary, an inhibition depending on the metal ion concentration occurred. For comparison an experiment with beef liver arginase was carried out under identical conditions, which yielded a considerable increase in activity after Co 2+ and Ni 2 ~ incubation. The results suggest that divalent Co and Ni are probably bound by the enzyme, but whereas beef and horse liver arginase are activated by these metals, the other arginases show a reduced enzymatic activity. The Michaelis constants for the ureotelic arginases investigated were found to be in the range from 6-20 mM (Table 2). This agrees well with the results obtained by other research groups for a number of ureotelic arginases (Mora et al., 1965). Mora et al. have pointed out the striking difference between the K m values of ureotelic and uricotelic arginases (10--20 mM, and 100-200 mM, respectively).

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The pH-activity curves for various mammalian arginases do not differ greatly with respect to shape and maximum activity. The pH-optima lie between pH 9-3 and 10.5 and are usually spread over half a pH unit. Also, the optimal Mn concentration for metal ion activation seems to be rather similar for ureotelic arginases. A Mn concentration of 40 mM was found to be optimal. This value is much higher than the 0.6 mM required for optimal Mnactivation of human erythrocyte arginase (Kalab & Pelikan, 1904) or 0.5 mM for avian liver arginases (Brown, 1966). In contrast to the latter arginases is the finding that the above liver enzymes were not inhibited by higher concentrations of Mn 2+ (0.1 M). The molecular weights for a number of mammalian arginases obtained from Sephadex G 200 column chromatography and analytical ultra-centrifugation are listed in Table 2 and range between 120,000 and 160,000. A higher molecular weight was only found for pork liver arginase, where a small peak (1 : 3) with arginase activity was eluted right after the void volume of the Sephadex column. It is likely that this peak is due to aggregation of the enzyme. Exceptions from the range given above are beef and dog liver arginase according to their elution patterns from Sephadex. However, since analytical ultracentrifugation of a rather pure preparation of beef liver arginase (sp. act. ~ 4500) yielded a molecular weight between 120,000-140,000, it can be assumed that this arginase is split into its subunits on the Sephadex column. The molecular weight of 30,000 found for beef liver arginase by Sephadex G 200 gel filtration agrees well with the subunit molecular weight obtained for rat liver arginase (Hirsch-Kolb & Greenberg, 1968). Dissociation into subunits and formation of a dissociation-association equilibrium is probably also the reason for the low molecular weight found for dog liver arginase. Molecular weights in the range from 120,000-160,000 generally obtained for ureotelic liver arginases differ remarkably from the molecular weights found for uricotelic and fungal arginases (lizard liver arginase, mol. wt. = 276,000; arginase from Neurospora crassa, mol. wt. = 278,000) (Mora et al., 1965, 1966) which seem to be twice as high. In this connection, it should be pointed out that Reddi and Campbell (1968) found a molecular weight of 27,000 for an arginase isolated from earth worm gut, which seems to represent the subunit molecular weight of ureotelic arginases. REFERENCES AHLGREN E., ERIKSSON K. E. & VESTERBERG O. (1967) Characterization of celluloses and related enzymes by isoelectric focusing, gel filtration and zone electrophoresis. I. Studies on Aspergillus enzymes. Acta chem. scand. 21, 937-944.

ANDREWSP. (1964) Estimation of molecular weights of proteins by Sephadex by gel filtration. Biochem. ff . 91,222-233. BACH S. J., HAWKINSR. A. & SWAINED. (1963) A short method for the purification of arginase from ox liver. Biochem.ff. 89, 263-265. BACH S. J. & KILLIP J. D. (1958) Purification and crystallization of arginase. Biochnn. biophys. Xcta 29, 273-280.

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BACH S. J. & KILLIP J. D. (1961) Studies on the purification and the kinetic properties of arginase from beef, sheep and horse liver. Biochim. biophys. Acta 47, 336-343. BROWN G. W. (1966) Studies in comparative biochemistry and evolution. I. Avian liver arginase. A r chs Biochem. Biophys. 114, 184-194. CABELLO J., PRAJOUXV. & PLAZAM. (1965) Immunodiffusion studies on human liver and erythrocyte arginases. Biochim. biophys. Acta 105, 583-593. DETERMANN H. & MICHEL W. (1966) The correlation between molecular weight and elution behaviour in the gel chromatography of proteins. -7. Chromat. 25, 303-313. GRASSMANN W., HORMANN H. & JANOWSKY O. (1958) Arginase--I. Elektrophoretische reinigung des enzymes. Z. phys. Chem. 312, 273-285. GREENBERG D. M. (1955) Arginase. In Methods in Enzymology Vol. II, pp. 368-374. Academic Press, N.Y. GREENBERG D. M. (1966) The Amidases. In Hoppe-Neyler/Thierfelder, Handbuch der physiologisch-andpathologisch-chemischen Analyse, 10th edn., Vol. 7, pp. 354-368. GREENBERG D. M., BAGOTA. E. & ROHOLT O. A., JR. (1956) Liver arginases--III. Properties of highly purified arginase. Archs Biochem. Biophys. 62, 446453. HIRScH-KOLB H. & GREENBERGD. M. (1968) Molecular characteristics of rat liver arginase. -7. Biol. Chem. 243, 6123-6129. HIRScH-KOLB H., KOLB H. J. & GREENBERG D. M. (1970)-7. Biol. Chem. (Publication pending.) KALAB H. & PELIKAN V. (1964) On arginase activity--X. Effect of manganese and high temperature on erythrocyte arginase. Acta Univ. palaekianaeolomuceusis36, 131-134. KOSSMAN K. T., HINTZER., LANGEK. & MENNE F. (1966) Reindarstellung yon Arginase aus Rattenleber. Z. phys. Chem. 346, 163-170. LAYNE E. (1957) Spectrophotometric and turbidometric methods for measuring proteins. In IVIethods in Enzymology, (Edited by COLOWICKS. P. & KAPLAN N. O.). Vol. 3, pp. 447-454. Academic Press, New York. LINEWEAW.R H. & BURR D. (1934) The determination of enzyme dissociation constants. -7. Am. Chem. Soc. 56, 658-666. MIDDLEHOVEN W. J. (1969) The ferrous ion as the cofactor of arginase in vivo--I. Properties of yeast arginase metallocomplexes of known composition and of native arginase. Biochim. biophys. Acta 191, 110-121. MOHAMED M. S. & GREENBERGD. M. (1945) Liver Arginase--I. Preparation of extracts of high potency, chemical properties, activation-inhibition, and pH activity. Archs Biochem. 8, 349-364. MORA J., MARTUSCELLIJ., ORTIz-PINEBAJ. & SOBERON G. (1965) The regulation of ureabiosynthesis enzymes in vertebrates. Biochem. -7. 96, 28-35. MORA J., TARV~B R. & BOJAIL L. F. (1966) On the structure and function of different arginases. Biochim. biophys. Acta 118, 206-209. MORA J., TARRAB R., MARTUSCELLIJ. & SOBERSON G. (1965) Characteristics of arginases from ureotelic and non-ureotelic animals. Biochem.-7. 96, 588-594. OLOMUCKI O. ~ VERRIERJ. M. (1955) Purification de l'arginase hepatique. Bull. Soc. Chim. Biol. 37, 1353-1361. REDDY S. R. R. ~; CAMPBELLJ. W. (1968) A low molecular weight arginase in the earthworm. Biochim. biophys. Acta 159, 557-560. ROGERS S. & MOORE M. (1963) Studies of the mechanism of action of the Shope rabbit papilloma virus--I. Concerning the nature of the induction of arginase in the infected cells..7, expl. Med. 117, 521-542. RossI N. & GRAZI G. (1969) Characterization of a new type of arginase from chicken liver. Eur.-7. Biochem. 7, 348-352. SCHIMKE R. T. (1964) The importance of both synthesis and degradation in the control of arginase levels in rat liver. -7. Biol. Chem. 239, 3808-3817.

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SOBER H. A. & PETERSON E. A. (1956) Chromatography of p r o t e i n s - - I . Cellulose ionexchange adsorbents, ft. Am. Chem. Soc. 78, 751-755. SOBERON G., ORTIz-PINEDA J. • TARRAB R. (1967) Characteristics of the ureotelic arginase and its role in the advent of ureotelism during the metamorphosis of the Mexican axolotl. U.S. Nat. Cancer Inst. Monograph No. 27, pp. 283-295. SoRv E. (1965) Purification of bacterial arginase, ft. Chromatography 20, 325-333. VAN SLYKE D. D. & ARCHIBALD R. M . (1946) Gasometric and photometric measurement of arginase activity, ft. biol. Chem. 165, 293-309. VESTERBERG O. & SVENSSONH. (1966) Isoelectric fractionation analysis, and characterization of ampholytes in natural p H gradients--IV. Further studies on the resolving power in connection with separation of myoglobin. Acta chem. scand. 20, 820-834. WHITAKER J. R. (1963) Determination of the molecular weight of proteins by gel filtration and elution behaviour in the gel chromatography of proteins. Analyt. Chem. 35, 19501953. Key Word Index--Arginase; liver arginase; mammalian liver arginases; comparison; molecular weights ; kinetic constants ; manganous ion binding; isoelectric values ; comparative properties; comparative stability; Michaelis constants; adsorption on carboxymethyl cellulose; isoetectric focusing. Latin Names of species--Mouse, Mus musculus ; dog, canis vulgaris ; cat, Felix domestica ; horse, Equus caballus; beef, Bos taurus; pig, sus scrofa; monkey, Macca mulatta; guinea-pig, Cavia cobaya ; rabbit, Lepus europaeus ; rat, Mus rattus.