Purification and properties of D -3-hydroxybutyrate dehydrogenase from Paracoccus denitrificans

300

Biochimica et Biophysica Acta 839 (1985) 300-307 Elsevier

BBA22027

Purification and properties of D-3-hydroxybutyrate

dehydrogenase

from

Paracoccus denitrificans

I. M a t y s k o v / ~ , J. K o v / ~ * a n d P. R a c e k Department of Biochemistry, Facul(vof Science, J.E. PurlqFnbUniversio', Kotlizkskgt 2. 611 37 Brno (Czechoslovakia)

(Received September 24th. 1984)

Key words: Hydroxybutyratedehydrogenase; Malate dehydrogenase: (Paracoccus) D-3-Hydroxybutyrate dehydrogenase from Paracoccus denitrificans has been purified to near homogeneity. The enzyme was prepared using DEAE-cellu[ose chromatography, affinity chromatography on immobilized Cibacron blue (Matrex Gel Blue A) and gel permeation chromatography. The pure enzyme was obtained by chromatofocusing as the final isolation step. The purification procedure yielded the enzyme with a specific activity of about 100 u n i t s / m g protein. The enzyme is specific for D-3-hydroxybutyrate and NAD and it exhibits anomalous kinetics (hysteresis) at low enzyme and coenzyme concentrations, it is relatively stable in the presence of EDTA at pH 7 - 8 and higher salt concentrations. D-3-Hydroxybutyrate dehydrogenase is a tetramer with a molecular weight of 130000 + 10000, its isoelectric point equals 5.10 + 0.05. The enzyme is applicable to the determination of acetoacetate and D-3-hydroxybutyrate concentrations.

Introduction D-3-Hydroxybutyrate dehydrogenase (D-3-hydroxybutyrate:NAD + oxidoreductase, EC 1.1.1.30) is an enzyme which is widely distributed in both eucaryotic and procaryotic organisms [1,2]. It catalyzes the reversible oxidation of D-3-hydroxybutyrate to acetoacetate. The enzyme has been purified from various microorganisms and characterized [1,3-5]. This enzyme is important especially for the bacterial species which are able to accumulate poly-3-hydroxybutyrate as an intracellular reserve material and depolymerize it under nutrient-limited conditions [6-8]. As the enzyme from these microorganisms (e.g., Pseudomonas, Rhodopseudomonas or Zooglea) is not tightly bound to membrane structures (at variance with the mammalian enzyme [9]) it can be obtained from the bacterial cytosol using current

* To whom correspondence should be addressed.

separation techniques [1,3-5]. D-3-Hydroxybutyrate dehydrogenase can be employed for the determination of acetoacetate and D-3-hydroxybutyrate concentrations in biological materials [10,111. The present paper reports a method of purification and several properties of this enzyme from Paracoccus denitrificans (which belongs to the microorganisms using poly-3-hydroxybutyrate as a reserve material [12]).

Materials and Methods Culture of organism P. denitrificans N C I B 8944 was grown at 30°C on a shaker in the medium consisting of 15 mM Na2HPO4, 30 m M K H 2 P O 4, 50 mM NH4C1, 1 m M MgSO 4, 30 ~M ferric citrate and 50 mM succinic acid (pH 7.3). The bacterium was harvested in the late logarithmic growth phase by centrifugation at 3000 × g for 60 min at 4°C. The washed cells were aerated by shaking in the same

0304-4165/85/$03.30 ~) 1985 Elsevier Science Publishers B.V. (Biomedical Division)

301

medium as given above, except for the succinic acid concentration (0.1 mM), for several hours, washed and separated using the same centrifugation as described above. The bacterial cells were stored at - 60°C.

En,~vme and protein assay The assay mixture for enzyme activity (1 ml) consisted of 1.8 mM N A D and 25 mM sodium D,L-3-hydroxybutyrate in 0.15 M Tris-HC1 buffer (pH 8.5; containing 1 mM EDTA). The rate of N A D reduction was followed at 340 nm at 25°C in a Cary 118 spectrophotometer. The activity of malate dehydrogenase was measured spectrophotometrically according to Ref. 13. Protein concentration was determined with the biuret reagent or by measuring the absorbance at 280 and 260 nm [14]. Purification of the enzyme (all manipulations were carried out at 0 - 8 ° C unless otherwise stated) Step 1. Preparation of the crude extract. Frozen cells of P. denitrificans were thawed in 0.2 M potassium phosphate buffer (pH 7.5). The cells were incubated with lysozyme (5 m g / g wet weight) for 30 min at 30°C and then disintegrated mechanically using a DynoMill disintegrator. The cytosol was separated by a centrifugation at 20 000 × g for 20 min and stored at - 2 0 or - 6 0 ° C . Step 2. Chromatography on D E A E cellulose. The pH and conductivity values of the thawed cytosol were brought to those of the starting buffer (50 mM sodium phosphate (pH 7.4) with 1 mM EDTA). Protein concentration in the cytosol applied to a 5 0 × 350 mm column packed with DEAE-cellulose was about 50 mg/ml. The column was washed with the starting buffer until the value of Az80 dropped below 0.1. The enzyme was eluted by a discontinuous gradient of pH and ionic strength (0.4 M sodium phosphate buffer (pH 6) with 1 mM EDTA). The fractions containing high activities of D-3-hydrobutyrate dehydrogenase were pooled, the pH was brought to 7.4, and the solution was concentrated using an Amicon cell with a XM-50 membrane. Step 3. Affinity chromatography. A column (26 × 300 mm) containing Matrex Gel Blue A was equilibrated with 0.1 M sodium phosphate buffer (pH 7.4) with 0.4 M NaC1 and 1 mM EDTA

(buffer A). The partially purified enzyme obtained in the previous step was loaded onto the column and washed until A280 decreased below 0.1. Thereafter a linear gradient of ionic strength was used; the final buffer was 0.1 M sodium phosphate (pH 7.4) with 1.2 M NaC1 and 1 mM EDTA (buffer B). The fractions with high enzyme activities were pooled and concentrated by ultrafiltration as described above. Step 4. Gel permeation chromatography. The sample obtained in the previous step was applied to a Sephadex G-200 column (16 x 700 mm). The active fractions (eluted with 0.3 M sodium phosphate buffer (pH 7.4) containing 1 mM EDTA) were collected and concentrated as given above. The chromatography on a 7.5 × 600 mm UltroPac TSK 3000SW column (LKB, Bromma) was carried out at room temperature under analogous conditions as the separation on the Sephadex column (with approx. 5 mg protein). Step 5. Chromatofocusing. The material obtained by gel chromatography was concentrated and diafiltrated using a TCF2 apparatus (Amicon) against the starting buffer (buffer A: 25 mM imidazole + HC1 (pH 7.4)) and loaded on a MonoP ( H R 5 / 2 0 ) column (Pharmacia, Uppsala). 10% Polybuffer 74 + HC1 (pH 4) [15] was used as the eluent (buffer B). The separations proceeded at room temperature. The fractions containing the highest specific activity of the enzyme were pooled, concentrated and diafiltrated against 0.2 M sodium phosphate buffer (pH 7.4) containing 1 mM EDTA. Chromatofocusing was carried out either semi-preparatively (total protein amount of about 20 mg) or analytically (with 1-2 mg protein).

Chromatographic analysis The enzyme purity was checked by the chromatography on a Polyanion SI ( H R 5 / 5 ) column (Pharmacia/Uppsala). 10 mM sodium phosphate buffer (pH 7.5) (buffer A) and 1 M sodium phosphate buffer (pH 5.8) (buffer B) were used for the gradient elution (carried out at room temperature). The chromatographic equipment consisted of two P-500 pumps, a GP-250 gradient programmer, a V-7 valve, a UV-1 optical unit (~ = 280 nm), a REC-482 recorder and a FRAC-100 collector (Pharmacia).

302

Electromigration methods The agarose electrophoresis was carried out in 1% agarose gels in 30 mM Tris-HCl buffer (pH 9.1) as described in Ref. 16. The separation proceeded in a LKB Multiphor apparatus (for 40 min at 4°C at 16 V/cm). The enzyme activity in the gels was detected with the reaction mixture containing 2.5 mM D,L-hydroxybutyrate, 0.2 mM NAD, 0.1 mM phenazine methosulphate and 0.5 mM 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride in 30 mM Tris-HCl buffer (pH 8.5:3 h at 27°C). Coomassie Blue R-250 [17] was used for staining the protein fractions. The optimized SDS-polyacrylamide gel electrophoresis [18,19] using a gradient of N,N'-methylenebisacrylamide (0.5-3%) was carried out in a Protean double slab electrophoresis cell (Bio-Rad). The protein zones were stained as described above The electropherograms were analyzed quantitatively in a Vitatron densitometer.

Chemicals D,L-3-Hydroxybutyrate was the product of BDH Chemicals (U.K.), L-(+)-3-hydroxybutyrate was obtained by resolution of the racemate by repeated crystallization of the quinine salts from acetone [20]. Lithium acetoacetate was supplied from Serva, Heidelberg (F.R.G.). The coenzymes (NAD, NADH, N A D P and N A D P H ) were purchased from Boehringer, Mannheim (F.R.G.). Lysozyme was the product of Reanal (Hungary), Sephadex G-200 as well as Polybuffer 74 were from Pharmacia, (Sweden, Matrex Gel Blue A was obtained

from Amicon (U.S.A.), DEAE-cellulose (DE52) was the product of Whatman (U.K.). A lowmolecular-weight calibration kit for gel filtration of Pharmacia was completed with horse liver alcohol dehydrogenase and glutamate dehydrogenase from Boehringer, and lactate dehydrogenase from Reanal (Hungary). A low-molecularweight calibration kit for SDS-polyacrylamide gel electrophoresis of Pharmacia was used. The materials for electrophoretic separations were mostly from Serva. Results and Discussion

Purification of the enzyme It is known that fractionation with ammonium sulphate and heat denaturation are useful methods in the preparation of partially purified D-3-hydroxybutyrate dehydrogenases from several microorganisms [1,3-5]. However, these methods failed in the case of the enzyme from P. denitrificans (negligible increases in specific activity were attained, and poorly sedimenting precipitates were formed). On the other hand, chromatography on DEAE-cellulose proved to be a very convenient method which could be carried out as the first separation step. The enzyme activity was eluted with 0.4 M sodium phosphate buffer (pH 6) as a relatively narrow zone nearly in the void volume of the column. The yield of D-3-hydroxybutyrate dehydrogenase activity amounted to about 80-85%, and the specific activity increased essen-

TABLE I P U R I F I C A T I O N OF D-3-HYDROXYBUTYRATE D E H Y D R O G E N A S E F R O M P A R A C O C C U S D E N I T R I F I C A N S The initial preparation conained about 60 g (wet weight) of bacterial cells. Values are the averages from four preparations; 4b and 5b refer to micro-preparative variants of the last two steps. Step

Protein (rag)

Total act. (U)

Spec. act. (U/mg)

Yield (%)

1. 2. 3. 4a. 4b. 5a. 5b.

4300 770 60 19

650 535 385 300

0.15 0.70 6.4 16 29 81 97

(100) 82 59 46 30 -

Crude extract DEAE-cellulose Matrex Gel Blue A Sephadex G-200 UltroPac TSK 3000SW Chromatofocusing (after 4a) Chromatofocusing (after 4b)

2.4 -

195

303

tially (nearly 5-times, see Table I). The partially purified enzyme was subjected to affinity chromatography on Matrex Gel Blue A (containing Cibacron blue 3GA as the affinant) suitable for biospecific separations of NAD-dependent dehydrogenases [21]. The enzyme exhibited a sufficient affinity to this material, since the total activity present in the sample retained at the column in spite of a relatively high ionic strength of the buffer. The enzyme was eluted by tile described NaC1 gradient (see Materials and Methods and Fig. 1). Using this affinity step the enzyme was purified nearly 10-times, the recovery of the activity being higher than 70% (cf. Table I). Gel permeation chromatography was the third chromatographic step in the proposed purification procedure. The elution profiles obtained using Sephadex G-200 (preparations at larger scales) and UltroPac TSK 3000SW (semi-preparative purification) are shown in Fig. 2. Both methods gave similar results, the separation on the UltroPac TSK column being, as expected, more efficient and essentially quicker. The recovery of the enzyme activity was about 80% in both cases and the specific activity increase was 2-3-fold and 4-5fold, respectively (cf., Table I). A relatively pure enzyme which was quite suitable for most of the F A280 ; i 20 k

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Fig. 1. Affinity chromatography of D-3-hydroxybutyrate dehydrogenase from P. denitrificans on Matrex Gel Blue A (step 3). V~, elution volume; - - , absorbance at 280 n m (A280); . . . . . , enzyme activity; . . . . . . , NaC1 gradient (buffer A, 0.1 M sodium phosphate (pH 7.4) with 1 m M E D T A and 0.4 M NaCI; buffer B, the same as buffer A but with 1.2 M NaCI); flow rate, approx. 0.8 m l / m i n .

......

6 A (U/m[)

A280 20

1.0

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50

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100

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- 2

'¢~ tmL!

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% Imp) Fig. 2. Gel permeation chromatography of D-3-hydroxybutyrate dehydrogenase (step 4). (A) Separation on Sephadex G-200; flow rate, o.5 m l / m i n ; - - . , D-3-hydroxybutyrate dehydrogenase activity; . . . . . . , malate dehydrogenase activity. (B) Separation on a UltroPac TSK 3000SW column; flow rate, 1.2 m l / m i n ; 1, D-3-hydroxybutyrate dehydrogenase activity; 2, malate dehydrogenase activity; the other symbols are the same as those in Fig. h

applications (e.g., for the analytical purposes, see below) was obtained at this stage. Moreover, gel permeation chromatography yielded a relatively pure malate dehydrogenase (see Fig. 2) which was the only significant NAD-dependent dehydrogenase activity accompanying D-3-hydroxybutyrate dehydrogenase in the described purification procedure. A semi-preparative chromatofocusing on a Mono P column was the final step in the proposed method of D-3-hydroxybutyrate dehydrogenase isolation. The results of an analytical variant of this method (indicating that the isoelectric point of the enzyme equals 5.10 + 0.05) are shown in Fig. 3. The increase in specific activity was 5-6-fold for the less pure samples obtained by the Sephadex G-200 chromatography and about 3-fold for those passed through the UltroPac column prior to chromatofocusing (Table I). An almost pure enzyme was obtained using this efficient and rapid method (see below); the recovery was about 70%. The results of the whole purification procedure (summarized in Table I) showed that the increase in specific activity was nearly 700-fold, and that

304 gradient. On the other hand, the purified enzyme (after chromatofocusing) yielded only one significant protein peak which coincided with the activity peak (not shown). A semi-preparative variant of this c h r o m a t o g r a p h y can also be used as a slightly less effective (but less expensive) substitution for chromatofocusing (the final step of the isolation procedure). The purified enzyme was shown homogenous on agarose gel electrophoresis; the band of the enzyme activity coincided with that of protein (not shown). The mobility of D-3-hydroxybutyrate dehydrogenase from P. denitrificans was 3.3 - 10 ~ c m 2 / V per min under the conditions of the experiment (see Materials and Methods).

iFA780j / jjj~

pH

04 5O

~"~ j~/l z~

02

5E

!1!1 x 63

~.J

,,

20

30

4O

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Fig. 3. Analytical chromatofocusing of D-3-hydroxybut~rate dehydrogenase (step 5). - - , A2s0; . . . . . . , pH value of the eluate; flow rate, 0.8 ml/min; 2 mg of protein were loaded onto the column, the protein peak with D-3-hydroxybutyrate dehydrogenase activity is indicated by an arrow.

Characterization of the enzyme The results of the c h r o m a t o g r a p h y on UltroPac T S K 3000SW (Fig. 2B) were c o m p a r e d with the elution profiles of molecular weight standards. The relative molecular weight of the enzyme under study was assessed as 132000_+ 10000 (cf. Fig. 5A). In order to ascertain the subunit composition and the subunit molecular weight, electrophoresis on a polyacrylamide gel in the presence of sodium dodecylsulphate was carried out. The purified enzyme yielded one significant protein peak with a molecular weight of 32000_+ 2000 (cf. Fig. 4B). Two minor peaks (with molecular weights of about 20000 and 40000) which were observed in the electropherogram (not shown) could correspond to a slight contamination of the purified enzyme with traces of other proteins, since their relative amounts were several times lower than that of the main

the specific activity of the final product obtained in four chromatographic steps (nearly 100 U / m g ) was comparable to that of the enzyme prepared from Zooglea ramigera in eight steps [1] (the initial specific activities in the cytosol being nearly the same). The enzyme purity was checked during preparation by the analytical anion exchange chrom a t o g r a p h y on a Polyanion SI col.umn (Fig. 4). The elution profile of the crude extract was similar to that shown in Fig. 4, except for a greater n u m b e r of high peaks which were eluted either with the starting buffer or at the beginning of the

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A280

B

A280

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.

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A280

20

.

.

c

// 0.1

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7e (mL)

.

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0

2O

Fig. 4. Analytical chromatography of D-3-hydroxybutyrate dehydrogenase on Polyanion S1. Buffer A, 10 mM sodium phosphate (pH 7.5); buffer B, 1 M sodium phosphate (pH 5.8); , A2s0; . . . . . . , elution gradient; ,L, peaks with enzyme activity; flow rate, 1.5 ml/min. (A) Sample after DEAEcellulose chromatography (step 2); (B) sample after affinity chromatography (step 3); (C) sample after chromatography on Sephadex G-200 (step 4).

305 [09 M

(ml)

/..8

18

16

4.6

X

\

14 4.4

~

12 Log M

mr

Fig. 5. Determination of the relative molecular weight (M) of D-3-hydroxybutyrate dehydrogenase (e). (A) Chromatography on an UltroPac TSK 3000SW column (see Fig. 2b), Ve stands for the elution volume. Standards ((3): (1) chymotrypsinogen (25000); (2) bovine serum albumin (67000); (3) liver alcohol dehydrogenase (80000); (4) lactate dehydrogenase (142000); (5) aldolase (147000); (6) catalase (240000); (7) glutamate dehydrogenase (324000). (B) SDS-polyacrylamide gel electrophoresis, m, denotes the relative mobility. Standards ((3): (1) bovine serum albumin (67000); (2) ovoalburnin (43000); (3) carbonate anhydrase (30000); (4) trypsin inhibitor (20100).

protein zone. These results are compatible with the assumption that D-3-hydroxybutyrate dehydrogenase from P. denitrificans is a tetramer (composed of identical subunits), with a relative molecular weight of about 130 000, similar to that of the enzyme from Z. ramigera, whose molecular weight was assessed as 112 000 [1]. The enzyme from P. denitrificans is specific for D-3-hydroxybutyrate and N A P , since no activity was detected if L-( + )-3-hydroxybutyrate or N A D P were used. N A D H cannot be replaced by N A D P H in the reverse reaction (acetoacetate reduction). The substrate and coenzyme specificity of the enzyme prepared from P. denitrificans is identical with that of the enzymes stemming from other microorganisms [1,4]. Preliminary experiments revealed that D-3-hydroxybutyrate dehydrogenase from P. denitrificans exhibited anomalous kinetic behaviour (hysteretic effects detectable as lag phases in the progress curves) when the reactions were initiated by small additions of low-activity enzyme solutions. The initial rates amounted to about one half of those attained after the lag phases had been terminated.

The half-time of the lag phases depended essentially on enzyme concentration, they were negligible (below 2 s) if the final enzyme activities were above 0.01 U / m l and acquired the value of about 10 s at 0.002 U / m l . The observed effects were induced by N A P , since the half-time of the lag phase was reduced at high N A P concentrations, and a preincubation (5 min) of the enzyme with N A P (0.2 raM) resulted in a normal initial velocity response. These kinetic data suggest that the binding of the coenzyme to the enzyme triggers the formation of enzyme aggregates with an increased enzyme activity. This assumption was confirmed by the chromatography on the UltroPac TSK 3000SW column. If 0.2 m M N A P was present in the mobile phase (and the other conditions were the same as those given in Fig. 2B) a new peak (containing an essential fraction of the loaded enzyme activity) with an elution volume corresponding to a relative molecular weight higher than 250000 was detected. The described hysteretic effects (which are of interest from the viewpoint of theoretical enzymology and might be important for the regulation of the enzyme activity in

306

the living cells of P. denitrificans) will be analyzed more thoroughly elsewhere.

Stability of the enzyme D-3-Hydroxybutyrate dehydrogenases from other bacteria (e.g., from Rhodopseudomonas spheroides, Rhodospirilum rubrum or Z. ramigera ) have been reported not to be very stable enzymes [1,4]. o-3-Hydroxybutyrate dehydrogenase from P. denitrificans is in this respect similar; it is rather unstable at low protein and salt concentrations. This property of the enzyme was taken into account in the purification procedure (and other manipulations with the enzyme samples). As no change of the medium was necessary in the described procedure (except prior to chromatofocusing; of. Table I) the activity losses were considerably reduced. The stability of the enzyme was checked in media of various pH, salt and protein concentrations and in the presence of several potential stabilizers. The samples kept at - 2 0 ° C (or at lower temperatures) were stable for several months, the lyophilized enzyme retained the activity for more than 2 months at 4°C. The pH values between 7 and 8 (sodium phosphate buffers with the salt concentration higher than 0.1 M) were found optimal for the enzyme kept in the solution at 4°C. Under these conditions, the enzyme activity decreased to one half in about 5 days (if the total protein concentration was above 5 m g / m l ) or in about 2 days (in the case of samples with protein concentration below 2 m g / m l ) . The stability increase in the presence of 0.5% mercaptoethanol or 10% glycerol was rather slight (at variance with the protective effect of these compounds in the case of the enzyme from Z. ramigera [1]). As the stability of the enzyme in solutions was increased essentially in the presence of 1-10 mM E D T A this metal-chelating compound was added into most of the buffers used in the purification procedure and into the reaction mixture for activity measurements.

Analytical applications As the coenzyme and substrate specificities of D-3-hydroxybutyrate dehydrogenase from P. denitrificans are the same as those of other bacterial o-3-hydroxybutyrate dehydrogenases [10,11], the possibility of its analytical use should be analo-

gous. We determined D-3-hydroxybutyrate and acetoacetate concentrations in model mixtures of known actual concentrations, using the methods described for other bacterial D-3-hydroxybutyrate dehydrogenases [10,11]. The enzyme activities, the incubation times and the other conditions during the tests were the same as those given in the original methods. The acetoacetate and 3-hydroxybutyrate concentrations in the model samples were in the ranges which are relevant from the viewpoint of clinical biochemistry [11] (i.e., 20-150/~M and 10-300/~M, respectively). The concentrations calculated from the observed changes in absorbance at 340 nm were nearly identical with the actual concentrations of these compounds (the errors being in the range of +5%). These results confirm that D-3-hydroxybutyrate dehydrogenase from P. denitrificans could have the same application scope in analytical biochemistry as the analogous enzymes from other bacteria. References 1 Nakada, T., Fukui, T., Saito, T., Miki, K., Oji, C., Matsuda, S., Ushijima, A. and Tomita, K. (1981) J. Biochem. 89, 625-635 2 Preuveneers, M.J., Peacock, D., Crook, E.M., Clark, J.B. and Blocklehurst, K. (1973) Biochem. J. 133, 133-157 3 Shuster, C.W. and Doudoroff, M. (1962) J. Biol. Chem. 237, 603-607 4 Bergmeyer, H.U., Gawehn, K. and Klotzsch, H. (1967) Biochem. J. 102, 423-431 5 Williamson, D.H., Mellanby, J. and Krebs, H.A. (1962) Biochem. J. 82, 90 96 6 Dawes, E.A. and Ribbson, D.W. (1964) Bacteriol. Rev. 28, 125 149 7 Stevenson, L.H. and Socolofsky, M.D. (1966) J. Bacteriol. 91, 304-310 8 Crabtree, K., McCoy, E., Boyle, W.C. and Rohlich, G.A. (1965) Appl. Microbiol. 13, 218-226 9 Lehninger, A.L., Sudduth, H.C. and Wise, J.B. (1960) J. Biol. Chem. 235, 2450-2455 10 Young, D.A.B. and Renold, A.E. (1965) Enzymol. Biol. Clin. 5, 65-69 11 Mellanby, J. and Williamson, D.H. (1970) in Methoden der Enzymatischen Analyse (Bergmeyer, H.U., ed.), Vol. 3., pp. 1772 1779, Academic Verlag, Berlin 12 Stanier, R.Y., Doudoroff, M. and Adelberg, E.A. (1972) General Microbiology, Maxmilian Press, London 13 Biochemica Information I (1973) Boehringer, Mannheim 14 Krebs, H.A., Mellanby, J. and Williamson, D.H. (1962) Biochem. J. 82, 96-98 15 Chromatofocusing (1982) Pharmacia Fine Chemicals, Uppsala

307 16 Kov~ff, J., Racek, P. and V16kov~, V. (1983) Comp. Biochem. Physiol. 76B, 161-165 17 Webster, R.G. and Datyner, A. (1963) Biochim. Biophys. Acta 71, 377-379

18 19 20 21

Racek. P. (1983) Bull. Cs. Sp. Biochem. 11, 5-43 Laemli, U.K. (1970) Nature 227, 680 683 Clarke, H.T. (1959) J. Org. Chem. 24, 1610-1611 Matrex Gels (1980) Amicon Corporation