catalytically active monomer forms of immobilized arginase

Eur J Biochem 130, 373-381 (1983) 7, FEBS 1983

Catalytically Active Monomer Forms of Immobilized Arginase

Raul AGUIRRE and Volker KASCHE

Fachbereich Biologie, N W 11, Universitit Bremen

(Received July 12/August 18, 1982) - EJB 5767

Native rat liver arginase was covalently coupled to Sepharose beads and the resulting matrix-bound tetramer was subsequently dissociated by acid or EDTA treatment. The immobilized derivatives that remain in the matrix were renatured in the presence of the cofactor Mn2+ and analyzed in terms of recoverable activity before and after incubation with soluble enzyme. The activity of the immobilized enzyme was determined under conditions where it is directly proportional to the enzyme content. Both the acid-treated and EDTA-treated and renatured immobilized rat liver arginase have about one-fourth the activity of the untreated immobilized enzyme. The activity of the renatured immobilized enzyme after reassociation with free enzyme was 85 ”/,(acid-dissociated oligomer) and 60 :;< (EDTA-dissociated oligomer) of the activity of the initially immobilized oligomer. The acid-dissociated and renatured immobilized arginase had a Kn, value (18 mM) that is approximately four times the value of the immobilized tetrameric enzyme (3.4 mM). After renaturation and reassociation with free enzyme the K,,, value of the immobilized enzyme was found to be equal to the value of the immobilized oligomer before dissociation. This indicates that the dissociated and renatured immobilized enzyme is monomeric. The Mn2+ ions normally associated with oligomeric arginase are easily removed from this dissociated form by washing with Mn2+-free buffer. The immobilized tetramer, on the other hand, does not lose Mn2+ after this procedure, and this ion seems to be bound even more tightly than in the case of the free tetramer. The reconstituted oligomer had a K,,, value approximating the value of the initially bound oligomer.

From these results and binding curves we conclude that rat liver arginase oligomer can be dissociated into monomers, which are catalytically active after renaturation with cofactor (Mn”) at neutral pH. Thus, for arginase the tertiary structure is sufficient for the expression of the function of this enzyme.

The immobilized monomeric arginase has been used to purify crude arginase by subunit exchange (affinity) chromatography.

Whether the quarternary structure of oligomeric enzymes with identical subunits, or the tertiary structure of a single subunit alone is sufficient for the expression of catalytic properties has been extensively studied recently. Two different experimental approaches have been used here. The oligomer dissociation/reassociation reaction and the catalytic properties of the intermediates are studied either in heterogeneous systems, after initial immobilization of one subunit in the oligomer [I - 1 I ] or in homogeneous solution [I1 - 181. The processes studied in these systems can be represented by the following scheme

oligomer monomer oligomer dissociation renaturation reconstitution

(reassociation) (M), ___+ MI- M”- (M)n ( 1 )

t free monomers f

where (M), represents the oligomer, M’ an inactive monomer (without cofactors and/or partly unfolded) and M ” the renatured monomer (after adding cofactors and removal of the denaturing solvent). In several studies, where chaotropic agents were used to dissociate the oligomer, the renatured monomer M” had been found to possess a low activity compared to the activity per monomer in the oligomeric enzyme.

Lniymes Arginase (EC 3.5.3.1); urease (EC 3.5.1.5)

This applies to studies either on oligorners in solution or on the immobilized monomer M‘, which was renatured by a removal of the chaotropic solvent in one step [3,6,9,12]. The latter can still bind free monomers so that the reconstituted oligomer has an activity similar to the original oligomer. The conclusion drawn from these studies was that the monomer M” is inactive. However, this may also indicate that this monomer form was not formed under these experimental conditions. For example this has been observed for immobilized lactate dehydrogenase [3], phosphoglucose iso- merase [ 5 ] , pyruvate kinase [9], free and immobilized alcohol dehydrogenase [6,16], free malate dehydrogenase [I21 and fumarase [14].

When milder dissociation conditions, such as a pH change or cofactor (for example, metal ions) removal, are used and the renaturation is performed under mild conditions, the monomers M ” were found to be fully active [4,10,18]. This also applies to monomers produced by chaotropic solvents, if the renaturation is performed by gradual removal of the chaotrophic agent in the presence of disulfide-exchange re- agents (dithioerithrol). This has been observed for immobilized ~-glyceraldehyde-3-phosphate dehydrogenase [7,8] and free and immobilized creatine kinase [2,17].

That in some cases the activity of the monomer cannot be restored may be attributed to the experimental conditions, which did not allow the process M’+M” (renaturation) before the reconstitution of the oligomer. In solution the

3 74

I TURMWER MICHAEUS BWND 1

W 8 E R -MENTEN ACTIVE I CaVSTANT O U W M R ’

CONTENT I

I c

TFTRAMER

DIMER

MONOMER r BEFORE DISSOCIATION

TURNWER MICHAELIS FRLCTION NUMBER -MENTEN Gf OLTWMERS

CONSl.4M INITIALLY IMMlBIuzED AND ACTIVE AFTER RENATURATION

AFTER DISSOCIATION AND RENATURATION

Fig. 1, Scheme qfpo.ssihle .species ohfairzedafter dissociation and renaturation of’ immobilized tetrameric arginase. The dimeric species obtained after these processes can also result from reassociation between adjacent bound monomers

renatured monomer may be formed after the M’ has formed a complex with other monomers. In this case an isolated active monomer, M”, cannot be directly analyzed. This problem may be circumvented using immobilized monomers where the renaturation process M’-+M” can be performed in the absence of interfering monomer association. The data in [ 1 - IS] indicate that for some oligomeric enzymes the quarternary structure is necessary for the expression of catalytic activity. For other oligomeric enzymes the tertiary structure is sufficient for the expression of catalytic activity. To exclude the possibility that the experimental procedure influences these conclusions and to determine why such structural differences, necessary for the expression of catalytic activity, have been evolved, further studies on different enzymes are necessary. In the immobilization method several experimental problems can, as indicated above, be avoided. However, the method may introduce experimental errors of its own that must be considered.

In the case of immobilized oligomers and monomers it must be verified that the observed enzyme activity is directly proportional to the active-site content. This is hardly ever done. It is known, however, that over a certain density of immobilized enzyme the stationary effectiveness factor de- creases with increasing enzyme content [I91 that is, the activity is then not proportional to the enzyme content. Another problem is that some protein (up to 30%) may be determined as immobilized oligomer but can be removed by electrophoretic desorption [20] or by chaotropic solvents. Other methods, such as titration with specific ligands, e.g. free monomers, must then be used to quantify the amount of M” in Eqn (1). Then also crude oligomer samples can be used to study the activity of the monomers.

In this study on rat liver arginase by the immobilization method the factors influencing the kinetics of immobilized enzymes, when compared to the enzyme in solution, have been considered in the quantitative evaluation of the experimental data. The applicability of this method when studying crude protein preparations is also demonstrated. For human liver arginase, studied by the same method, the results indicate that the renatured monomer is active [4]. However, in this study the observed activity was not demonstrated to be directly proportional to the enzyme content. Rat liver arginase has been reported to be a tetramer of

molecular weight 120000 composed of subunits of identical molecular weight [21]. Gel filtration of crude preparations reveals small but significant amounts of arginase activity corresponding to a species of about 30000 molecular weight [22]. This indicates that active subunits and tetrainers exist simultaneously and raises questions concerning the role of both forms. We have, therefore, studied the catalytic properties of the monomers produced by dissociation of tetramers of rat liver arginase. Two relative mild procedure producing dissociation of rat liver arginase have been reported: the removal of the activator cation Mn2+ by EDTA [23] and the incubation of the oligomer at pH 2.7 [24]. The monomers produced by these methods may be renatured by addition of Mi?+ or by restoration of neutral pH [23,24].

Preliminary data on the use of the renatured subunits for purification of free arginase by subunit-exchange chromatography have been reported earlier [29].

Quunritutive RelationsJor the Enzymic Cizaructerizution of Monomers of Immohili-.ed Oligomcric Ennrjwics

The properties, (a) enzyme activity and (b) amount of bound protein and active enzyme, of an immobilized oligomeric enzyme determined before and after dissociation, renaturation and reassociation can be used to investigate whether the monomeric form retains thc catalytic activity. With the definitions in Fig. 1, the following relations for these properties from which CI, /I and can be determined, can be derived.

a) The observed activity of an immobilized oligomeric enzyme is generally not (as for the free enzyme in solution) directly proportional to the immobilized enzymc content [I 91. For an oligomeric enzyme following simple Michaelis-Menten kinetics the observed activity is given by

where the subscript o denotes the properties of the oligomeric enzyme and yo is the steady-state effectiveness factor that is a function of Vi. K&,, the bulk substrate concentration [S], and other inicroenvironmental and macroenvironmental prop-

375

erties of the system [19]. The ratio of the activities after and before dissociation/renaturation, is for [S] 9 all KA values

1:" YO kca t , ,

where generally the effectiveness factor qd for the dissociated and renatured enzyme + 11" and kLa,,o + k&,d # klat.,. After dissociation and renaturation the immobilized enzyme may bind monomers. The ratio between the total activity of free enzyme bound at saturation to the total activity of the dissociated and renatured immobilized enzyme used in this binding experiment is (when [S] 9 all K , values)

where the unprimed quantities denote properties of the free enzyme. From Eqns (3) and (4) we obtain

where B and ( c d / r 0 ) can be directly determined from activity measurements.

b) The ratio of immobilized active protein after and before dissociation and renaturation (cd/cO), where cd is the immobilized active protein content after the dissociation and renaturation, can be obtained from protein determinations only when all protein subunits are active. Some experimental problems limit the use of such determinations here. Is has been shown that some 'immobilized' protein can be removed from the support by electrophoretic desot-ption [20]. Similar processes may occur when an immobilized oligomeric protein is dissociated by chaotropic solvents or milder treatments. However, the ratio cd/cu can always be determined from reassociation experiments using free monomers as specific ligands. Then the same enzyme preparation must be used for the immobilization and the binding experiment to dissociated and renatured immobilized enzyme. The ratio is given by

B Y co A i m m 2 4 ( 6 )

where A b and A, are the activities of the free oligomerlml gel before and after incubation with the dissociated and renatured enzyme, and Aim, is the activity of the initially immobilized enzyme/ml gel determined from the activity balance in the solution before and after immobilization.

The quantities, 2 , /j and 7 , which give information on the relative amount of renatured moiiomers c;) and dimers (B) , can be determined from the relations in Eqns ( 5 ) , (6) and

= a + + ('d - Aim, - (Ah - Aa) - . .. _ ~ . ~ ~ .. ~ - ~ .~

properties and can also give information on whether com- plexes other than the oligonier are active [IC)]. When all subunits are equal in their substrate-binding property and in the absence of cooperative effects Kk,o = l/n KL,, , for an n-meric enzyme [25]. Such differences in K, have been observed for malate dehydrogenase [lo] and creatine kinase [2]. However, in these studies it was not shown whether y = 1 .

The experiments necessary to determine ( B ud/f',) in Eqn (5), (cd/co) in Eqn (6) and KA d o not require the use of pure oligonieric protein. Thus, in this case immobilization is a useful tool to study the enzymic properties of crude oligomeric protein preparations.

MATERIALS AND METHODS

Rat liver arginase was purified according to Schimke [23] up to the ethanol precipitation step without modifications. The animals used were adult albino male rats (strain Sprague- Dawley) weighing about 200g. Sepharose CL-2B, 4B and BrCN-activated Sepharose 4B were supplied by Pharmacia Fine Chemicals (Uppsala, Sweden), BrCN by Merck, L-arginine hydrochloride by Sigma. Urease was obtained from P-L Biochemicals Inc. (78 unitsjmg). All other chemicals were of analytjcal grade.

Coupling of Arginasc to Srphurose

Arginase was bound to Sepharose as described previously [19,26] with the sole modification that washing at low pH was carried out at pH 4.5. The amount of coupled protein was estimated from absorption measurements by determining the difference between the added amount of protein and the amount that was washed out of the gel after the coupling procedure. The immobilized enzyme was stored at 4' in 0.1 M Tris/HCl buffer (pH 7.5) containing 0.05 M MnCI2. No activity loss was observed over a period of 4months. A fraction of BrCN-activated Sepharose was incubated in the coupling buffer but in absence of enzyme, and further submitted to the same washing treatments of the enzyme- containing gel. This fraction was used as control gel in some experiments.

Dctcwninuiions of' Eiizjww Activitj>

Activity measurements of immobilized arginase were carried out by potentiometric determination ofNH,' generated in the presence of an excess of urease. The potential ( E ) was registered with a NHZ-selectivc electrode (Philips IS 561- NH:) connected to a pH meter in conjunction with a chart recorder. A saturated calomel electrode (Philips Type R 44,'2- SDj1) with a built-in salt bridge (filled with 0.3 M Tris,'HCl, pH 7.5) was used as reference. Unless otherwise stated, the reaction mixture (20 ml) contained the following: 0.05 M L-arginine, 0.05 M MnCI2, 0.1 mM NH4CI, 0.05 mgiml urease and 0.1 M Tris/HCI buffer, pH 7.5. Usually 5 inin after the addition of these components to the reaction vessel a constant background potential was obtained. The sample of Sepharose]

activity after reconstitution activity before dissociation

arginlse suspension was then added, and the potential variation was recorded. All measurements were carried out in a thermostated vessel at 25 C under constant stirring at a speed where the reaction rate was independent of the stirring speed.

( 7 )

when all measurements are performed in systems where (kcal.ojk~a,.o) is known and ti,, = 1. The ratio kCdl.,,/k&o in Eqn ( 5 ) can be determined from the activity yield for the immobilization or reassociation process, when all immobi-

the K , values determined by usual procedures are molecular d t "Hi+] dr

, y + [ j + - , = ~.~~~~~ ~- .

From the time differential of the Nernst equation

dE 1 d " G I ~ ~ = 0.05y. -~ .~ . .- ~ ~- ~

lized oligomers retain their enzyme function. For 4, and q d = 1

3 76

it follows that when the experimental conditions are such that the reciprocal of ammonium ion concentration changes much more slowly with time than the time differential of [NH: 1, the determination of AE/At gives a direct measure- ment of the NH2 production rate. Owing to an excess of urease this rate represents the activity of arginase. The slope AElAt, determined 5 min after the addition or arginase, was found to be linear for arginase amounts between 0.5 unit and 5.0 units.

EDTA Treatment of Immobilized Enzyme

MnZ + was removed from immobilized arginase by washing samples of gel (2-10 i d ) with 0.03 M or 0.1 M EDTA at pH 8.0. The EDTA solution was pumped through a small jacketed column containing the enzyme suspension in a cyclic system. Both the EDTA reservoir (250 ml) and the column were maintained a t 37 'C. At given times, aliquots of the gel suspension were withdrawn and washed extensively with 0.1 M TrisIHC1 buffer, at pH 7.5 at room temperature and the enzyme activity was determined. Reactivation tests were performed by incubation of EDTA-inactivated immobilized preparations in 0.1 M Tris/HCl buffer, pH 7.5, which contained 0.05 M MnCIz, for 1 h at 37°C. EDTA treatment of soluble enzyme was carried out as described by Carvajal et al. ~41.

Low p H Treatment

Low pH dissociation was performed by washing a given volume of Sepharose/arginase with glycinejHCl buffer, I = 0.05 M, pH 2.7, on a glass filter at room temperature. The procedure was repeated seven times with two volumes of this buffer, and subsequently the gel was extensively washed with 0.1 M Tris/HCl buffer at pH 7.5, containing 0.05 M MnC12. The gel was suspended in this buffer and the activity was determined of this suspension. The immobilized enzyme submitted to such procedure will be referred to as acid-treated arginase in the text.

Treatment with GuanidinrlHCI

A given volume of Sepharosejarginase was washed with two volumes of 6 M guanidine hydrochloride solution containing 10 mM 2-mercaptoethanol in a glass filter six times. The slurry (1 : 1 gel/guanidine hydrochloride solution) was subsequently transferred to a dialysis tube and dialysed in 200 volumes of 0.1 M Tris/HCl buRer, containing 0.05 M MnC12, at room temperature during 3 h with two changes of buffer. The dialyzed suspension was allowed to settle to determine the gel volume. Then the activity of the resuspended gel was determined.

Kffhc. t iveness Estimation q f thc Immobilized Enzynze

The stationary effectiveness factor 4 was estimated according to the method described in (191. The Thiele modulus (R2V/DiKm)1/2 , where R is the particle radius (z 50 pm), V the maximum activity of the immobilized oligomer, 0: the diffusion coefficient of the substrate in the gel and K , the Michaelis-Menten constant for the free enzyme (I the value for the immobilized enzyme), was calculated from experimental data using the value Dh = cm2/s.

Then the substrate concentration range where y~ = 1, can be determined from the graphs in [19] using a Sherwood number zz 10, which is normally attained in these systems (it is a measure of the external mass transfer limitation). Alter- natively an analogous method described by Engasser [27] can be used to determine 4. For all preparations used here, q was found to be about 1 even at substrate concentrations nearly equal to K,, for the free enzyme.

RESULTS Immohilizution Yields

A semipurified preparation (990 units/mg; 1 unit is the amount of enzyme that produces 1 pmol urea in I min at 37 T) was used for the immobilization to Sepharose CL-2B and 4B, which were BrCN-activated in our laboratory, and to Sepharose 4 8 supplied by Pharmacia (Uppsala) as BrCN- activated Sepharose. 20mg BrCN/ml gel was used for the activation. The coupling reaction was carried out in all cases at p H 7.0 and 25 "C for 15 h. Table 1 shows the yield for different matrices. The stationary effectiveness Factor 17 was found to be about 1 for all these preparations. Thus the activity is directly proportional to the active enzyme content. A check of the immobilized activity and that washed out of the gel, indicated that no activity was lost after the immobilization procedure and that k:,,,, = k,,,., within the experimental error ( & 10 %).

Dissociation and Renaturation of the Immobilized Enzyme

Immobilized rat liver arginase was considerably more stable than its soluble form towards inactivation by EDTA. A 1 -h treatment with 0.03 M EDTA at 37 'C does not produce a noticeable inactivation of Sepharose-4B-immobilized enzyme, whereas the soluble form completely loses its bound Mn2+ after this treatment ([23] and personal observations). Even after 1 h of treatment with 0.1 M EDTA at 37°C the immobilized enzyme loses only about 10% of its activity. Fig. 2 shows the inactivation kinetics of the immobilized enzyme. Total inactivation is obtained only after 50 h of EDTA treatment. When Mn2+ is added to completely inactivated enzyme (50 h), the recovered activity is about 25 ".', of the original value.

The activities of the dissociated and renatured immobilized enzyme are given in Table 2 from which cd/z., in Eqn (3) and Eqn ( 5 ) may be determined.

When the immobilized oligomer was dissociated with guanidine hydrochloride the immobilized subunit M' (Eqn 1) could not be renatured upon dialysis against renaturation buffer.

K, Determinations

Native immobilized arginase, EDTA-treated and acid- treated immobilized species were further characterized with respect to the K,,, for L-arginine. Experiments with EDTA- treated immobilized arginase were performed with gel samples first completely inactivated by EDTA and subsequently re- activated by incubation in Tris buffer pH 7.5 with 0.05 M MnClz at 37 "C for 1 h. The results are presented in Fig. 3 as Eadie-Hofstee plots. The K , values obtained from these plots were 3.4 mM, 5.0 mM, and 18 mM for the native, EDTA- treated and acid-treated immobilized form respectively. The

377

corresponding value for the free oligomer was found to be 3.9 mM. When acid-treated immobilized enzyme was incubated in the presence of an excess of free enzyme, the K, value of the reassociated oligonier was found to be equal to that of the immobilized oligomer before dissociation.

The linearity of the curves in Fig. 3 indicates that the active enzyme forms in each sample are homogeneous with respect to their K , values. The linearity for substrate concentrations below the K, values shows that diffusion limitations for these immobilized enzymes are negligible; i.e. q = 1 also at low

Table 1. Jmnmbilization yirld.s of' rut liver arginasr

Sepharose type Binding yield Protein bound Activity bound

"/ / o mg/ml unitslml

4B 40 0.17 180 4B" 72 0.75 700 CL-2B 50 0.35 300

a Pharmacia activated Sepharose.

0 40 80 Cycling time (hi

Fig. 2. Kinetics of inactivation by EDTA of' immobilized arginasr (Se- phuro.se 4 B , 180 unitsiml gel) . The incubation was performed in 0.1 M EDTA at 37 'C and the cycling flow was 1.5 nilimin. The graph indicates the activity measured without Mn2+ (0) and after incubation in 0.05 m Mn2" (1 h, at 37" and pH 7.5) (0)

0 LO 80 120

substrate content. The K , and k,,, values for the dissociated enzyme indiciate that they are values for immobilized monomeric arginase.

Mn2+ Dissociation of AcidTveuted Arginase

IHirsch-Kolb et al. [25] have found that the tetrameric form of rat liver argiiiase has two weakly ( K d = 2 x M) and two strongly (Kd = 3 x lo-' M) bound Mn2+ ions. These two kinds of MnZ+ sites can be easily differentiated by dialysis against Mn2+-free buffer at pH 7.5.

To compare the affinity of the immobilized tetramer with that of the acid-produced immobilized subunits to Mn2+, matrices containing these forms were washed with neutral buffer. The tests were done by eluting gels with Tris buffer pH 7.5 in small columns at 4'C. Gel samples were withdrawn to assay activity Initially and after 1 h incubation in 0.05 M MnClz at 37°C. The results are presented in Fig.4. Im- mobilized subunits lose a considerable fraction of their Mn2+ very easily, whereas the tetrameric form retains most of its Mn2+ even after prolonged elution. I t is interesting to note that the activity decay in this form is considerably less than the 50 y i observed by dialysis of soluble tetramers [28]. This observation is consistent with the results presented above where a stabilization of bound tetramer was observed during EDTA inactivation.

Reassociation o f tlzr Oligomevic Enzyrrze

The ability of the immobilized renatured subunits to bind free monomers was studied as follows : samples of immobilized enzyme, dissociated with EDTA or by incubation at pH 2.7. were mixed with an excess (10 times the recoverable activity in the gel) of soluble enzyme previously dissociated with EDTA or a t pH 2.7. The mixture was dialyzed overnight against 0.1 M Tris/HCl buffer, pH 7.5, containing 0.05 M MnC12, at room temperature.

The activities before dissociation, after renaturation and reassociation are summarized in Table 2.

The binding of specific ligands, here free subunits, to the immobilized subunits was studied with acid-dissociated enzyme.

0 10 20

lo7 v (M s-' ml gel-')

Fig.3. ~ f l d f e - I ~ ~ f ~ ~ i e r , p l o t . s f o r f ) . r r ~ and immohiliirdrat h e r arginasc. The untreated immobilized derivative was arginase-Sepharose 4B with 180 unitslml gel. (A) The results obtained with free (0) (0.25 mgiassay) and immobilired untreated tetrameric enzyme (0). (B) Results with immobilized subunits: (0) dissociated by EDTA; (0) dissociated by acid treatment

37H

3

Woshing volume 11)

Fig.4. MnZ+ elution of ( A ) untrruted tetnrmeric and ( B ) acid-treutd, monomeric, immohiiizrd rut urgii~use. The immobilized derivatives were arginase-Sepharose 4B containing 280 and 48 units/ml gel respectively. Small columns (2 ml gcl) were washed at 4 C with 0.01 M Tris/HCI (pH 7.5) at a tlow rate of 25 niljh. The graph shows the gel activity measured in absence of Mn" (0, 0) and after incubation in 0.05 M MnZ + (0, .)

Table 2. h~rrnohili~edru~ fivrr urKitww: crc'tivifj. h(.li,re urzduficr cliwsoriution and reconstitution with .solirhfc. rtizj'nic.

Activity assays were performed iit pH 7.5 (0.1 M 'TrisiHCI, 0.05 M MnCIz, 0.1 tnM NH4CI and 0.05 M L-arginine) and 25 C

1 mmo bilized Treatment preparation

Activity in the gel

Sepharose 48 None (180 unitsiml gel) dissociation with EDTA

(pH = 7 . 5 ) renaturation with MnZ ' reconstitution in the presence of excess of soluble enLymc

dissociation at pH = 2.5 renaturation with MnZ +

reconstitution in the presence of excesb or solu ble cnzynie

dissociation at pH = 2.5 renaturation with MnL+ reconstitulion in thc presence of cxcess of soluble enzyinc dissociation with 6 M guanidine;HCl and subsequent renaturation with MnZ+

Sepharose CL-2B none (300units!ml gel)

Sepharose 48 none (700 unitshnlgel)

of untreated iinmobilized enzyme

100

0 25

60 I00

0 27

85 100

0 21

8 5

0

0

Variable amounts of soluble enzyme were mixed at pH 2.7 with a constant amount of acid-treated iminobilized enzyme and neutralized by dialysis in small dialysis chambers. After attainment of the equilibrium, an aliquot of the mixture soluble enzyme + immobilized enzyme was withdrawn to measure its activity. The rest of the mixture was allowed to settle, and a sample of soluble enzyme was taken to determine the activity remaining in the soluble phase. The activity hound by the inimobilized preparation is estimated by the difference between soluble activity found in a parallel series of experi-

0

log (Supernatant act iv i tyJ

Fig. 5. The assoriuiion of Jiee urginusr to immohilirrcl ucirt-trcuied untl rmaturrd urginaw. Variable amounts of free enzyme, dissolved in glyclne bullfer ( I = 0.05 M and pH 2.7), were mixed with a constant amount of immobilized arginase and subsequently dialyzed in 0.1 M Tris HCI buffer, pH 7.5. containing 0.05 M MnCIZ, for 3 h at room temperature. The binding numbcr is the activity of free enzyme associated,activity of immobilized enzyine (unitsiunil). The abscissa parameter is the enzyme activity found in the supernatant after the incubation. The immobilized enLyine preparation was arginase-Sepharose 4 8 with 700 units ml gel in the untreated form

ments using control gel without enzyme and the soluble activity found in the experiments with immobilized enzyme. The binding number is defined as the ratio between the activity of the associated soluble enzyme and the dissociated and renatured immobilized enzyme (or 'acid-treated' arginase) and equals B in Eqn (4) at saturation. The results are presented in Fig.5, which resembles a titration curve with at least two affinity constants. It is interesting to note that an initial plateau is obtained for a binding number nearly equal to 1 (dimer stoichiometry), whereas at higher free enzyme concentrations the curve tends to a binding number equal to 3 , thus three enzyme units are associated per unit of bound ligand (tetramer stoichiometry). From the data on the maximum amount of free enzyme bound to the immobilized subunits and the initially bound enzyme content Eqn (6) the ratio cd/co was found to be x 0.25. This also indicates that the dissociated and renatured enzyme is predominantly an active monomer.

Determinution of' the Fruction of Active Tetrumers, Diniers mid Monoiners it? Dissociuted und Rcnaturrd Immohilix~d En-y 7 me

The K , values and the Mn2+ elution experiment (Fig.4) show that the dissociated and renatured enzyme contains negligible amounts of tetramers. Consequently, x in Fig. 1 equals zero. Then p and y can be calculated from Eqns(S)and(6) [orEqns(6)and(7)J, when thequantitics c d ' r o , rd/(.n. l ~ ~ ~ , . ~ / k : ~ ~ . ~ ~ B and q0 are known. The quantity rd. ro is given in Table 2; cd / ro is given above; l<cat,c,/k:t,,.,l was found to he = 1 as determined from the activity yield during the immobilization or in the binding of free monomers to immobilized subunits; qU has been shown to be equal to unity: B in Eqns (4) and ( 5 ) is the maximal value of the binding number and was determined from Fig. 5 (= 2.8).

The results of these calculation for the acid-treated enzyme are :

5 0.3, y 2 0.7,

379

I\ I ‘ 25

0 0 0 10 40 60 80 100

Elution volume (mil

Fig. 6. Subunit-rxchungr cliromrrtogruphy of rut liver rrrginux (A) 10 ml arginase-Sepharose CL-2B were washed with neutral buffer and subsequently with acid buffer (arrow). (B) This material was suspended in acid buffer and 40 mg of a freeze-dried semipurified (990 unitsling protein) preparation of free arginase %as added. The suspension was dialyzed in neutral buffer containing 0.05 M MnC12. The gel was poured onto a column and cluted successively with Tris;HC:l pH 7.5. acetate buffer ( I = 0.05 M, pH 4.5) and glycine buffer ( I = 0.05 M, pH 2.7) at 4 ° C

whcre y is a low estimate since the value used for B is lower than the value at saturation. They show that immobilized renatured arginase monomers are catalytically active.

Suhunit-E.xchange Chronqatography

The ability of immobilized subunits to pick up free subunits, by displacing the equilibrium towards reassociation conditions, offers the possibility of applying this system to enzyme purification. The application scheme can be outlined as follows.

a) Immobilized subunits are mixed with an impure prcp- aration under dissociating conditions (low pH).

b) The association conditions are established in the mixture (neutralization).

c) The subunits are eluted by washing at low pH. d) The immobilized subunits may be used again to purify

more enzyme by repetition of the steps (a), (b) and (c). Results of subunit-exchange chromatography to purify

arginase are shown in Fig.6. The elution of the originally bound enzyme at low pH gives a peak with a specific activity three times higher than the material coupled to the matrix. The elution at low pH, after reassociation with free enzyme [step (c) of the purification outline], gives a peak (Fig.6B) with a specific activity 4.5-fold higher than the original material. This approximately corrcsponds to the specific activity of a pure arginase preparation [21].

DISCUSSION

Immobilized rat liver arginase could be dissociated by treatment with EDTA or at low pH. The subunits still immobilized to the support, could be renatured by incubation with the cofactor MnZ+. This indicates that the inactive subunits, M’ from Eqn ( I ) are only slightly unfolded. They could not be renatured when the dissociation was performed with

guanidine hydrochloride. The renatured subunits M” could bind free subunits so that practically the original activity of the oligomer was obtained. The difference (l0-20:;, in Table 2) can be partly due to the fact that either desorption of non-covalently bound oligomer occurs during the dissociation process or that during the reassociation process some subunits M” are so sterically hindered that they have not bound free monomer at all. The K,,, values and MilZf elution properties of the enzyme before and after dissociation and renaturation showed that virtually no enzyme was present as a tetramer after the dissociation. The enzyme content before and after renaturation was determined by titration with the specific ligand monomeric arginase. This and activity determinations before and after dissociation/renaturation and reassociation (under conditions where the activity is directly proportional to the enzyme content) were used to determine the quantities a, f l and y in Fig. 1 from Eqns (3, (6) and (7). The results show that more than 70 %of the subunits obtained after dissociation and renaturation are active monomers (M” in Eqn 1). Thus in the oligomeric enzyme rat liver arginase the tertiary structure of the monomer alone is required for the expression of the catalytic activity. The results and methods used in this study demonstrate the applicability of the immobilization method to study possible monomer activity when using an impure oligomer preparation. Similar results for human liver arginase have been reported in [4], but the authors did not show whether the condition q = 1 is fulfilled.

The monomers produced by either dissociation mcthod were found to have turnover numbers (kLAt.,) that were approximately a quarter of the value (kb , , , ) for the oligomeric enzyme. However, one should not generally assume, as is frequently done, that k La,,, = k idI.Jn, where n is the number of monomers in the oligomer. The K L values were, as expected, increased for thc monomers when compared with the immobilized oligomer.

The acid-produced immobilized subunit was found to have a K, value significantly higher than that for the EDTA-produced monomer. This suggests that acid-produced and EDTA- produced immobilized subunits have different conformational states (conformers [30]). The question of whether these as- sumedly different conformational states are interconvertible remains unanswered. Prolonged incubation of the acid-produced form in Mn2+-containing buffer at 37‘C did not produce changes in K,.

A striking difference between free and immobilized tetramer has been observed with respect to its affinity for Mn2+. The soluble form easily loses two of its four Mn2+ ions by dialysis [28]. The immobilized tetramers lose only a small fraction of their activity after washing with large amounts of buffer (Fig.4). Since activity is directly related to the amount of Mn2+ bound to the enzyme [28], the observed MnZ+ binding indicates that even the weakly bound Mn2+ (Kd = 0.2 mM [28]) in free oligomers is more firmly bound in immobilized oligomcrs. EDTA treatment also indicates that the immobilized oligomer is more stable towards dissociation than the frec oligomer. While the free enzyme is completely inactivated after only 15 min of incubation in 0.01 moljl EDTA at 37 ’C, this treatment does not reduce the activity of the immobilized tetramer. This has also been observed in [31]. The importance of the quarternary structure to stabilization is demonstrated by the fact that washing immobilized subunits with Mn2+-free buffer produced a considerable degree of irreversible inactivation (Fig. 3 B). In this context this observation is used to prove that the fraction of tetrameric arginase in the dissociated and renatured enzyme is negligible. The M n 2 +

380

binding results indicate that the conformation of the immobilized tetramer may differ from the conformation of the soluble tetramer. This gives no activity changes at the high MnZf concentrations used in this study. Differences in the affinity for Mn2 + between free enzyme and membrane-bound arginase in natural systems have also been observed. The MnZ ’ concentration necessary to obtain the maximal activity (0.05 M) of the soluble enzyme is considerably higher than the Mn2 + concentration found in the cytoplasm of hepatocytes (0.01 mM [32]). Recently published results indicate that arginase associated with mitochondria1 membranes has a higher affinity for Mn2+ than the soluble enzyme and that solubilization of the bound enzyme lowers its affinity for Mn” 1331.

The results obtained in this and other similar studies on oligomeric enzymes [2, 7, 8, 10, 15, 17, 181 show that the renatured M” has enzyme activity provided that the dissociation is performed under mild conditions or the renaturation of M‘, produced by chaotropic agents, is carried out under conditions where monomer renaturation, i.e. the transition M‘+M” in Eqn (l), can be analyzed. Thus the tertiary structure of a single monomer seems to be sufficient for the expression of the enzyme activity of these oligomeric enzymes with identical monomers. The quaternary structures seems, at least for arginase, to be of importance for the regulation of the enzyme activity and its stability with respect to cofactor binding. The results for arginase and other enzymes, where the monomers have been shown to be active, have been mainly obtained with immobilized monomers. Several observations support the conclusion that these results may be extrapolated to soluble monomers. Monomeric enzymes that have been immobilized in polysaccharide supports always retain their enzymic function although K,, and kot may be perturbed [35,36]. Thus the tertiary structure is not considerably perturbed upon immobilization. Such immobilized monomers can be reversibly denatured when the BrCN content, used in the activation of the support, is similar to the BrCN concentration used here [37]. This shows that the immobilization does not change the spontaneously folded confirmation so that the enzyme function is lost. This should also apply to denatured monomers of oligomeric enzymes that refold in the absence of an inter- action with other monomers either in solution or when they are immobilized and have active monomers as observed for arginase here.

Several authors have shown that the monomer forms M’ and M” (in Eqn 1) for other oligomeric enzymes with identical subunits have no catalytic properties [3,6,9,12- 141. This may in some cases be due to the experimental conditions when the process M‘+M” cannot be followed prior to reconstitution. In other cases it indicates that for some oligomeric enzymes the quaternary structure is required for the expression of catalytic properties. Further studies are, therefore, necessary to estab- lish the differences between these two groups of oligomeric enzymes with identical subunits.

The immobilization method, as shown here, is also suitable for this purpose when crude enzyme preparations are used. In such studies it must be shown that the observed activity is directly proportional to the enzyme content, i.e. 4 = 1. When this applies also for substrate contents lower than the K, value, the latter can be determined for the immobilized oligomer and monomer. This value is then a molecular property independent of diffusion limitations in the gel. The active enzyme content before and after dissociation, renaturation and reassociation can be determined either by protein determination or, as here, by titration with the specific ligand-free

monomer. In the latter case it is not necessary to use pure oligomer samples for the immobilization. The activity determinations before and after dissociation, renaturation and reassociation can then be used to determine the relative amount of winers (LX, B and y using Eqns 5, 6 and 7) (for a tetramer). From this it can be established whether the renatured enzyme M” is an active monomer.

The reversible dissociation produced by the acid treatment of the immobilized oligomer was used to purify soluble arginase by subunit-exchange affinity chromatography (Fig. 6) [29,34]. It was observed that the elution of the oligomer- containing gel at pH 4.5 only produces a small desorption of reactivable enzyme (Fig. 6B). This suggests some stabilization of the immobilized oligomer towards acid dissociation, since it has been observed that the soluble tetramers quantitatively dissociate into dimers at this pH [24].

This work has been supported by Deutscher Akudemischrr Ausiuusch- dienst (R. A,) , Deutsche Forschun~.s~emrin.~c/zujt and Fonds der chemi.schen Induustrie (V. K . ) .

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R. Aguirre, Laboratoire de Chimie Bacterienne du Centre National dc la Recherche Scientifique. 31 Chemin Joseph-Aiguier, F-I 3274 Marseille-Cedex 2, France

V. Kasche, Fachbei-eich Biologie-Chemie, Universitat Brenien, N W 2, Loebener StraBe, D-2800 Bremen 33, Federal Republic of Germany

catalytically active monomer forms of immobilized arginase

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