Eur. J. Biochem. 61, 589-596 (1976)

Uronic Acid Dehydrogenase from Pseudomonas syringae Purification and Properties Gunter WAGNER and Siegfried HOLLMANN Institut fur Physiologische Chemie I, Universitat Diisseldorf (Received April 30iOctober 29, 1975)

1. Uronic acid dehydrogenase was purified to homogeneity. After a 338-fold purification a yield of 16% was achieved with a specific activity of 81 pmol NADH formed min-' mg protein-'. 2. The purity of the enzyme was controlled by disc electrophoresis, sodium dodecylsulfate electrophoresis and ultracentrifugation. 3. A molecular weight of 60000 was determined by gel chromatography and by ultracentrifugation. 4. The native enzyme is composed of two subunits, their molecular weight being 30000 as estimated by sodium dodecylsulfate electrophoresis. The subunits as such are inactive. 5. The absorption spectrum with a maximum at 278 nm shows no evidence for a prosthetic group. 6. For catalytic activity no SH groups and no metals seem to be necessary. 7. The Michaelis constants determined with the pure enzyme are for glucuronic acid K , = 0.37 mM, galacturonic acid K , = 54 pM and NAD' (with glucuronic acid) K , = 80 pM. 8. A weak reverse reaction could be observed with glucaric acid lactones at acidic pH. 9. NADH is competitive with NAD+. The inhibitor constant is Ki = 60 pM. 10. The NADf binding site seems to be of lower specificity than the uronic acid binding site. The first step of uronic acid metabolism in bacteria is, in most instances, the conversion of uronic acids to the corresponding keto-uronic acids by a uronic acid isomerase [l - 31. A different reaction was discovered in Pseudomonas syringae [4] and Agrobacterium tumejaciens [ 5 ] . In these organisms an inducible NAD+-linked uronic acid dehydrogenase catalyzes the oxidation of the uronic to the dicarboxylic acids according to the following scheme. The uronic acid dehydrogenases were partly purified and characterized ~ ~ 7 1 . Glucuronic acid +


Galacturonic acid

uronic a c i d + dehydrogenase

formed unknown product X is converted to the endproduct in a non-enzymic step. NADH is formed in stoichiometric yield from the uronic acids. In this paper the enzyme from glucuronic-acidadapted Pseudomonas, which is specific for glucuronic and galacturonic acid, was investigated with the intention to develop an optical test [8] for enzymic determination of uronic acids [9]. MATERIALS AND METHODS Chemicals


X +NADH+H' +


Glucaric acid Mucic acid In alkaline solution the equilibrium of this reaction is far on the product side. Probably, the enzymically Enzymes. Alcohol dehydrogenase (EC 1.I. 1. I) ; aldolase (EC; catalase (EC; deoxyribonuclease I (EC; lactic dehydrogenase (EC; ribonuclease A (EC; uronic acid dehydrogenase (EC

Chemicals were purchased from the following sources : Aldolase, catalase, lactic dehydrogenase, DNase I, RNase A, ovalbumin, chymotrypsinogen A, cytochrome c, NAD+ grade 11, NADH grade 11, NADPH, ATP, ADP, AMP, 3': 5'-cyclic AMP (Boehringer, Mannheim, FRG) ; bovine serum albumin (Behring Werke, Marburg, FRG) ; D-galacturonic acid, D-glucuronic acid lactone, D-glucaric acid 1,4lactone (Sigma, St. Louis, Missouri, U.S.A.); D-glucuronic acid (Serva, Heidelberg, FRG) ; D-glucaric acid 3,6-lactone (K & K laboratories, Plainview, New York, U.S.A.); nutrient broth, yeast extract (Difco,

Uronic Acid Dehydrogenase


Detroit, Michigan, U.S.A.). All other substances were reagent grade from commercial sources. Hydroxyapatite was prepared according to Levin [101. D-Glucaric acid (dicyclohexylammonium salt) was prepared from the water-insoluble calcium salt as described by Fish [ll]. Reduced 3-acetylpyridineadenine dinucleotide was obtained from 3-acetylpyridine - adenine dinucleotide (Sigma) by reduction with alcohol dehydrogenase according to Fisher [12] with subsequent chromatography on DEAE-SH-cellulose. A molar extinction coefficient of 7.8 x lo6 at 365 nm was assumed [13].

in an Eppendorf photometer at 334 nm or 366 nm at 25 "C. The standard assay contained in a l-cm cuvette 5 pmol NAD' and 1 pmol D-glucuronic acid in 3 mlO.05 M sodiumdiphosphate/HCl buffer, pH 8.0. The reaction was started by addition of 0.1 ml enzyme solution containing up to 0.1 U of uronic acid dehydrogenase. One unit of enzyme is defined as that amount of enzyme that catalyzes the reduction of 1 pmol of NAD+/min at 25 "C. The specific activity is 1 unit/mg of protein.


Step I :Crude Extract. 500 g of wet cells were suspended in 11 0.01 M ice-cold buffer A and treated 4 times for 2 min each with a Branson sonifier B 12 (Danbury, Connecticut, U.S.A.) at full power (150 W) in a cooled 150-ml rosett vessel. During this step a temperature of 20 "C was not exceeded. The extract was then cooled to 4 "C and the following steps were performed at that temperature. After centrifugation (20 min at 27 000 x g ) the sediment was discarded and the supernatant adjusted to pH 8.0 by addition of 0.1 N NaOH. The supernatant was treated for 15 h with DNase I (1000 U/mg) and RNase A (40 U/mg), each 2 mg/100 ml solution. Step 2: Ammonium Sulfate Fractionation. To 1 1 of the nuclease-treated supernatant 243 g of finely ground (NH,),S04 were slowly added (40 saturation). The precipitate was removed by centrifugation (20 min at 12000 x g ) and discarded. The supernatant was brought up to 65 % saturation by adding 168 g/1 (NH4)2S04 and centrifuged again for 20 min at 12000 x g . The sediment containing the uronic acid dehydrogenase was dissolved in about 400 ml of 0.01 M buffer A and dialyzed for 20 h against 3 x 5 1 of the same buffer. Step 3 : DEAE-Cellulose Chromatography. The dialyzed solution (about 500 ml) was applied to a column 5 x 50 cm packed with DEAE-SH-cellulose and equilibrated with 0.01 M buffer A. The enzyme was eluted by a concentration gradient formed by 21 0.01 M and 2 1 0.1 M buffer A. Uronic acid dehydrogenase was eluted at 0.025 M buffer A. All fractions with more than 15 U/18 ml were combined (about 600 ml) and dialyzed for 12 h against 10 1 of 0.01 M buffer A. Step 4 : DEAE-Sephadex Chromatography I. A column 3 x 20 cm filled with DEAE-Sephadex A-50 and equilibrated with 0.01 M buffer A was loaded with the enzyme solution. The uronic acid dehydrogenase was eluted by a gradient formed by 800 ml 0.01 M and 800 mlO.15 M buffer A at a buffer concentration of 0.04 M. The active fractions (more than 15 U) were collected (about 200 ml) and concentrated in a ultrafiltration system TCF 10 (Amicon, Oosterhout, Holland) membrane PM 10 at 1.8 atm (182 kPa) pressure to a volume of about 30 ml.

Buffers used in enzyme preparation were : buffer A : sodium diphosphate/HCl buffer, pH 8.0; buffer B :potassium phosphate buffer, pH 7.2. All buffers contained 0.02 o/, sodium azide as preservative. Organism

The source for the isolation of uronic acid dehydrogenase was Pseudomonas syringae van Hall ATCC 13394 (Rockville, Maryland, U.S.A.). For storage a cell suspension in nutrient broth with 15% glycerol added was kept in the freezer at -20 "C. Cultivation of' Bucteriu

The Pseudomonas syringae strain was cultivated as described by Bateman [6] with the following modifications. Medium A was replaced by nutrient broth. The carbon source in medium B was D-glucuronic acid lactone ( 5 g/1 medium) adjusted to pH 8.0 by adding 10 N NaOH. The 500-ml culture was used to inoculate 20 1 of the same medium. The cells were grown at 25 "C with aeration (10 1 air min-' 20 1-') to an A5,* of 1.9. The bacteria were harvested at 38 000 rev./min in a continuous-flow high-speed centrifuge (Padberg/Lahr, FRG). The yield was about 90 g wet weight/20 1 medium. In the bacteria stored at -20 "C no loss of uronic acid dehydrogenase activity was observed within 3 months. Determination of Protein

Protein was determined in the crude extract by the biuret method [14] at 546 nm, in all other cases by the method of Lowry [15] at 578 nm. Bovine serum albumin was used as a standard in both determinations. Assay of Uronic Acid Dehydrogenase Activity

The activity of uronic acid dehydrogenase was determined by optical test [8]. The increase of NADH as product of uronic acid oxidation was measured

Enzyme Preparation


G. Wagner and S. Hollmann

Step 5 : Gel-Chromatography. The concentrated solution was poured on to a Sephadex G-100 column (5 x 90 cm) that had before been equilibrated with 0.01 M buffer B, which was also used for elution. Fractions of 6 ml in 10 min were collected and active fractions greater than 20 U were combined (about 150 ml). Step 6 :Hydroxyapatite Chromatography. The protein solution obtained from gel chromatography was applied to a hydroxyapatite column (3.5 x 18 cm) and eluted with 0.01 M buffer B. Uronic acid dehydrogenase does not adsorb to hydroxyapatite under these conditions. The active material was combined (about 170 ml). Step 7: DEAE-Sephadex Chromatography II. The eluate was applied to a DEAE-Sephadex A-50 column (1.6 x 12 cm), which was equilibrated with 0.01 M buffer A. The column was first washed with 2 vol. of 0.01 M buffer A, then the enzyme was eluted with 100 ml of 0.05 M buffer A. The pure fractions were collected (about 25 ml). Disc Electrophoresis. Analytical disc electrophoresis was performed on 3 mm thick polyacrylamide slab gels (9.5 x 8 cm) in an flat-gel electrophoresis apparatus (Desaga, Heidelberg, FRG). The gel systems according to Maurer 1161 were system 1 (pH 8.9, 7.5% acrylamide) and system 6 (pH 7.5, 7.5 acrylamide). Between 25 - 50 pg of protein/sample was applied. The run was made at 0 "C and 40 mA. The protein was stained with amido black 10 B. Electrophoresis on Sodium Dodecylsulfate Gels The apparatus was that used for disc electrophoresis. Gel system (1O'X acrylamide, 0.2 % dodecylsulfate) and pretreatment of uronic acid dehydrogenase and the reference proteins were as described by Weber [17]. Protein samples between 5-25 pg were applied. Coomassie brilliant blue R 250 was used for staining. Gel Chromatography The molecular weight of uronic acid dehydrogenase was determined by gel chromatography according to Whitaker [18] on a Sephadex G-100 column 2 x 90 cm, equilibrated with 0.05 M Sorensen buffer pH 7.5. 5 mg of the calibration proteins and 1.6 mg uronic acid dehydrogenase (80 U/mg) were applied in 3 ml buffer. Fractions of 2.4 ml were collected. The position of the proteins was ascertained by their absorption at 280 nm and by positive biuret reaction. Ultracentrifugation The sedimentation velocity of purified uronic acid dehydrogenase was studied in a model L2 50B preparative ultracentrifuge with installed schlieren optics


accessory (Beckman, Miinchen, FRG). The enzyme (2.7 mg/ml) in 0.02 M Sorensen buffer, pH 7.2, in a single-sector cell sedimented at 49 500 rev./min at 20 "C. Photographs of the schlieren pattern were taken at 10-min intervals.

RESULTS Purification of' Uronic Acid Dehydrogenase Uronic acid dehydrogenase was purified 338-fold (Table 1). The specific activity of the pure enzyme is 81 U/mg. On disc electrophoresis this enzyme behaved as a single protein in two different systems (Fig. 1). On ultracentrifugation the purified uronic acid dehydrogenase sediments as a single symmetrical schlieren peak. Stability The pure enzyme (1.5 mg protein/ml 0.05 M sodium diphosphate-HC1 buffer, pH 8.0) with 30% glycerol loses 5 % of its activity within 1 month at 4 "C. No loss of activity could be observed at the same enzyme stored frozen with 0.1 % bovine serum albumin at - 20 "C. Molecular Weight Estimation Estimation by Gel Chromutogruphy. The molecular weight determined by gel chromatography on Sephadex G-100 according to Whitaker [I81 from four different runs was 59000 f 1500 (Fig. 2). Estimation by Ultrucentrifugation. From the schlieren optical pattern a szo,wvalue of 4.4 S was estimated at a protein concentration of 2.7 mg/ml. From that an approximate molecular weight of 59100 for a globular protein can be calculated if a frictional coefficient of 1.3 and a partial specific volume of 0.73 cm'/g are assumed [19]. Estimation by Sodium Dodecylsu!iate Electrophoresis. In sodium dodecylsulfate electrophoresis uronic acid dehydrogenase shows one single protein peak. A molecular weight of 30500 f 3000 could be determined by comparison of the mobility or uronic acid dehydrogenase with the mobilities of proteins with known molecular weight [17] (Fig. 3). Denaturation of Uronic Acid Dehydrogenase Uronic acid dehydrogenase was preincubated at 30 "C in 8 M urea and in 30 mM sodium dodecylsulfate. After 30 min both enzyme samples were completely inactive. After a 24-h dialysis the sodium dodecylsulfate enzyme remained inactive, the urea enzyme regained 10% of its original activity.


Uronic Acid Dehydrogenase

Table 1. Purification of uronic acid dehydrogenase Purification step


Total protein

Total activity

Specific activity



mg 36 750 9 320 1300 630 189 44 17




8 790" 8495 6 595 5 580 4 490 1930 1385

0.24 0.91 5.1 8.9 23.8 44 81

100 97 75 63 51 22 16

1250 490 620 210

Crude extract 65 % Ammonium sulfate fraction DEAE-cellulose chromatography DEAE-Sephadex I Sephadex G-100 Hydroxyapatite DEAE-Sephadcx I1

155 170 23

Purification factor

1 4 21 37 99 183 338

The reaction mixture contained 1 mM KCN




Fig. 1. Analytical dkc electrophoresis. (A) Gel system 1,35 pg protein applied; (B) gel system 6, SO pg protein applied



3 4 5 6 7 8 9 Molecular W g h t

Fig. 3. Determination of the molecular weight of the uronic acid dehydrogenase subunits. From each of the pretreated proteins 15 pg was applied. The proteins are: 1 = cytochrome c (12500), 2 = chymotrypsinogen A (2SOOO), 3 = uronic acid dehydrogenase, 4 = lactic dchydrogenase (34000), 5 = ovalbumin (45000), 6 = catalase (60000), 7 = bovine scrum albumin (67000)


0.6 2.5


i 4





0.4 0.3

0.2 0.1










Log Molecular wight

Fig. 2. Estimation .f molecular weight ojuronic acid dehydrogenase by gel chromatography on Sephadex G-100. Calibration proteins: 1 = cytochrome c (12500), 2 = chymotrypsinogen A (25000), 3 = ovalbumin (45000). 4 = uronic acid dehydrogenase, 5 = bovine serum albumin (67000), and 6 = aldolase (158000) used for voidvolume estimation

Fig. 4. Absorption spectrum of uronic acid ~l~,k.l~sdrogc.nast.. Uronic acid dehydrogenase (1 mg/ml 0.02 M sodium diphosphate buffer, pH 8.0) was recorded in a double-beam Actd 111 spectrophotometer (Beckman, Munchen)

G. Wagner and S. Hollmann


Fig. 5. Lineweaver-Burk plot for K, determination of uronic acids. The assay mixture contained in 0.5 ml 75 mM sodium diphosphate buffer, pH 8.0,2.5 pmol of NAD+ and 15 pg of uronic acid dehydrogenase (81 Ujmg). The reaction was initiated by addition of 0.25 ml of 0.1 3 mM glucuronic (0)or galacturonic acid (0)and measured at 334 nm at 25 "C ~

Fig. 6. Lineweaver-Burkplot for K, determinaiion of NAD' withglucuronic acid. The assay mixture was a solution of 0.5 ml75 mM sodium diphosphate buffer, pH 8.0, containing 2.5 pmol of glucuronic acid and 15 pg enzyme (81 Ujmg). The reaction at 25 "C was started by addition of 0.25 ml of 0.1 - 1 mM NAD'

Absorption Spectrum

Reducing Agents

The absorption spectrum of uronic acid dehydrogenase is a typical protein spectrum with one single peak at 278 nm (Fig. 4).

Reducing agents, such as 2-mercaptoethanol, dithiothreitol and dithioerythritol in a concentration of 1 mM are without influence on the uronic acid dehydrogenase reaction. A protective function against thermal destruction of the enzyme could be observed.

Influence of Sulfhydryl-Group Inhibitors The inhibition of uronic acid dehydrogenase by p-chloromercuribenzoic acid is linear in a range from lo-'- 1 O - j M p-chloromercuribenzoic acid. 1 mM p-chloromercuribenzoic acid inhibits the uronic acid dehydrogenase activity to 60% of its original value. The inhibition can be reversed completely by an excess of L-cysteine. A similar inhibition is observed with N-ethylmaleimide.

Influence of Temperature The temperature optimum or uronic acid dehydrogenase is 37 "C. Increasing this temperature leads to a complete loss of activity within 3 min. From 0 "C to 15 "C the pure enzyme is stable for 24 h, at 25 "C the activity falls to 85 % within 2 h, at 37 "C 90 % of the original activity is lost in 60 min. After 20 h at 35 "C the enzyme is inactive in any case.

Uronic Acid Dehydrogenase


Fig.7. Inhibition by N A D H . The assay mixture contained in 0.5 ml of 75 mM sodium diphosphate buffer, pH 8.0, 2.5pmol glucuronic acid, 15 pg uronic acid dehydrogenase (81 U/mg) and NADH so that the final concentrations were: I = 0 mM, I1 = 0.1 mM, 111 = 0.15 mM, IV = 0.2 mM, V = 0.25 mM, VI = 0.3 mM, VII = 0.4 mM. After starting the reactions by addition of 0.25 ml of 0.1 - 1 mM NAD', the increase of absorbancc at 334 nm was measured at 25 "C

Eflect of'Metal Ions

Without effect on uronic acid dehydrogenase reaction are 1 mM cations (except Hg2+)and complex formers such as EDTA, arsenate, sodium azide, and KJFeCN,]. 1 mM KCN, which was added to the uronic acid dehydrogenase assay to block unspecific NADH oxidases in the crude extract, reduces the activity of the pure enzyme to about 85 %.

Table 2. Inhibition ojuronic acid dehydrogenase reaction by N A D t rehted inhibitors 7.5 pg of uronic acid dehydrogenase (81 U/mg) was preincubated for 15 min at 25 "C in 0.1 ml of 0.1 M sodium diphosphate buffer, pH 8.0, containing the inhibitor (0.5 mM). The uronic acid dehydrogenase reaction was started by the addition of a 2 0 4 aliquot of the preincubated enzyme to 1 ml of standard assay mixture containing 0.5 mM inhibitor. The increase of absorption at 334 nm was read at 25 "C Inhibitor (0.5 mM)

Percentage original activity

No inhibitbr Adenine Adenosine AMP 3': 5'-Cyclic AMP ADP ATP NADH NADPH

100 78 53 63 66 92 91 39 87

x Kinetic Studies

Kinetic studies were performed with uronic acid dehydrogenase isolated from glucuronic-acid-adapted bacteria. The K , values of the pure enzyme were determined according to Lineweaver [20] (Fig. 5, 6). Glucuronic acid: K , = 0.37 mM; galacturonic acid: K , = 54 pM ;NAD+ (glucuronic acid) :K , = 80 pM. The turnover number for glucuronic acid is 4800/min, for galacturonic acid 1600/min if a molecular weight of 60 000 is assumed.

3,6-lactone 3 % of that of the forward reaction at that pH. Reverse Reaction

In previous investigations the reverse reaction of uronic acid dehydrogenase with glucaric acid could not be established [6,7].In this study a slow reverse reaction was observed at pH 6.0 using glucaric acid lactones as substrates. The reaction velocity with glucaric acid 1,Clactone is 4%, with glucaric acid

Inhibition by NADH

Uronic acid dehydrogenase is competitively inhibited by NADH (Fig. 7). The inhibitor constant determined according to Dixon [21] is K i = 60pM. The NADH analogue, reduced 3-acetylpyridineadenine dinucleotide, is not inhibitory.

G. Wagner and S. Hollrnann

Inhibition by NAD' Constituents and NAD' -Related Compounds

The substances were applied in 0.1 mM concentration. Of no effect were D-ribose, D-ribose 5-phosphate, pyrophosphate, nicotinic acid, nicotinamide, NADP' and guanine. Substances with inhibitory effect are listed in Table 2. Influence of Substances Structurally Related to Uronic Acid

No influence of a wide variety of related substances (compounds between C2 and C,) as substrates or inhibitors of uronic acid dehydrogenase could be observed.

DISCUSSION Uronic acid dehydrogenase in Pseudomonas syringae can be induced by growth on glucuronic acid, galacturonic acid and glucaric acid. In cells grown on glucose no uronic acid dehydrogenase can be detected. The native enzyme has a molecular weight of about 60000 as determined by gel chromatography. The exact determination by ultracentrifugation was not possible in the preparative ultracentrifuge. This enzyme dissociates in the presence of urea or sodium dodecylsulfate into two inactive subunits of molecular weight of 30000. The subunits are able to reaggregate to a certain extent to the active form. The absorption spectrum with a maximum at 278 nm shows no evidence for a prosthetic group. The catalytic function of the enzyme seems to be independent of -SH groups because the specific inhibitors react with uronic acid dehydrogenase only in relatively high concentrations. Metal ions in the presence or absence of complexing agents are without influence on enzyme activity with the exception of H 2 + ,which denaturates the protein. It is assumed that the inhibitory effect of KCN on the pure enzyme is not due to a cation complex formation. The oxidation of uronic acids in uronic-acid-induced cells is catalyzed by one enzyme only. This enzyme is highly specific for glucuronic and galacturonic acid with a higher affinity for galacturonic acid. Related compounds and smaller parts of the uronic acid molecule are without inhibitory effect on enzyme activity and, thus, seem not to act with the substrate binding site. The coenzyme binding site of the enzyme is less specific for NAD' analogues, and adenosine-containing compounds are inhibitors of uronic acid dehydrogenase. The adenosine ribose seems to be important for coenzyme binding, since the inhibition decreases with increasing phosphate group number at the ribose moiety. as in NADP' and the adenosine phosphates.


The nicotinamide group of the coenzyme has no significant effect. In the preliminary investigations [6,7] the reverse reaction could not be demonstrated. This may depend on the fact discussed by Chang [22] that glucaric acid is not the primary product of the reaction but instable glucaric acid lactones are formed as proposed in the mammalian system by Marsh [23]. On the other hand, the oxidation of the glucuronic acid aldehyde group to the glucaric acid carboxyl group is thermodynamically highly favoured at neutral pH and cannot easily be reversed [24]. In fact, in this study the reverse reaction could be observed to a certain extent at acidic pH with glucaric acid 1,Clactone and 3,6-lactone. Both lactones showed the same reactivity. The possibility that the unavailable glucaric acid 1,4 :3,6-dilactone is the actual product must be considered. The assumption of Chang [7] that the uronic acid dehydrogenases induced by different uronic acids were not identical was not subject of this paper. The possibility of using the enzyme for quantitative determination of free and conjugated glucuronic acid is described elsewhere [9]. We thank Miss Anna Maria Unterreiner for skilful technical assistance.

REFERENCES 1. Ashwell, G., Wahba, A. J. & Hickman, J. (1958) Biochim. Biophys. Acta, 30, 186- 187. 2. Kilgore, W. W. & Starr, M. P. (1959) J. B i d . Chem. 234, 2227 - 2235. 3. McRorie, R. A,, Williams, A. K. & Paync, W. J. (1959) J. Bacteriol. 77,212 - 216. 4. Kilgore, W. W. & Starr, M. P. (1959) Nature (Lond.) 183, 1412- 1413. 5. Zajic, J. E. (1959) J. Bacteriol. 78, 734-735. 6. Bateman, D. F., Kosuge, T. & Kilgore, W. W. (1970) Arch. Biochem. Biophys. 136,97- 105. 7. Chang, Y. F. & Feingold, D. S. (1969) J. Bucteriol. 99, 667673. 8. Warburg, 0. &Christian, W. (1936) Biochem. Z. 287,291 - 328. 9. Wagner, G. & Hollrnann, S. (1975) J . Clin. Chem. Clin. Biochem. submitted for publication. 10. Levin, 6. (1962) Methods Enzymol. 5, 27-32. 11. Fish, D. C. & Blumenthal, H. J. (1966) Methods Enzymol. 9, 53 - 56. 12. Fisher, H. F., Conn, E. E., Vennesland, B. & Westheimer, F. H. (1953) J. Biol. Chem. 202,687-697. 13. Kaplan, N. 0. & Ciotti, M. M. (1956) J. B i d . Chem. 221, 823 - 832. 14. Beisenherz, G., Boltze, H. J., Biicher, T., Czok, R., Garbade, K. H., Meyer-Arendt, E. & Pfleiderer, G. (1953) Z. Naturforsch. 86,555- 577. 15. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J . Biol. Chem. 193, 265-215. 16. Maurer, H. R. (1971) in Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis, p. 44, Walter de Gruyter, Berlin and New York. 17. Weber, K. & Osborn, M. (1969) J . Biol. Chem. 244,4406-4412.

596 18. Whitaker, J. R. (1963) A n d . Chem. 35, 1950- 1953. 19. Elias, H.-G. (1969) Ulfruzenfrifugen-MeZhoden, Beckman Instruments, pp. 125- 126. 20. Lineweaver, H. & Burk, D. (1934) J . Am. Chem. SOC.56, 658 - 666.

G. Wagner and S. Hollmann: Uronic Acid Dehydrogenase 21. 22. 23. 24.

Dixon, M. (1953) Biochem. J . 55, 170-171. Chang, Y.F. & Feingold, D. S. (1970) J . Bucferiol.102,85-96. Marsh, C. A. (1963) Biochem. J . 87, 82-90. Rose, I. A. & Rose, 2. B. (1969) Compr. Biochem. 17,127- 131.

S. Hollmann and G. Wagner, Institut fur Physiologische Chemie I der Universitat Dusseldorf, D-4000 Dusseldorf 1, UniversitatsstraBe 1, Federal Republic of Germany

Uronic acid dehydrogenase from Pseudomonas syringae. Purification and properties.

Eur. J. Biochem. 61, 589-596 (1976) Uronic Acid Dehydrogenase from Pseudomonas syringae Purification and Properties Gunter WAGNER and Siegfried HOLLM...
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