Biochimie, 69 (1987) 157- 161 157 © Soci~t~ de Chimie biologique/Elsevier, Paris

Short communication

One step purification of Escherichia coli t3-glucuronidase

Carlos BLANCO and Georges NEMOZ

Laboratoire de Microbiologie et Laboratoire de Chimie Biologique, LN.S.A. Bat 406, 20 av. Albert-Einstein, 69621 Villeurbanne Cedex, France

(Received 13-11-1986, accepted after revision 19-11-1986)

Summary - fl-glucuronidase was purified by affinity chromatography on thiophenyl-glucuronide cou- pled to Sepharose. The enzyme was more than 95°70 pure. This enzyme is a tetramer composed of identical 74 kDa monomers. The amino-terminal sequence determined was: N H 2 - M e t - L e u - A r g - P r o - V a l .

~-glucuronidase I affinity chromatography / amino-terminal sequence

R~sum6 - Pur i f i ca t ion de la fl-glucuronidase d'Escherichia coi l La fl-glucuronidase d'Escherichia coli a 6td purifide par chromatographie d'affinit6 sur du thioph6nyl-glucuronide greffd sur Sdpharose. La puret6 de l'enzyme est supdrieure ~ 95 %. Cette enzyme est composde de quatre sous-unit6s identiques de 74 kDa. La sdquence amino-terminale d6terminde correspond ~: NH2-Met-Leu-Arg-Pro-Val .

fl-glucuronidase I chromatographie d'affinltd I sdquence amino.terminale

I n t r o d u c t i o n

fl-glucuronidase (EC 3.2.1.31) (UID), the enzyme responsible for the degradation of various polysac- charides or the cleavage of glucurono-conjugates, is widely distributed in animals, plants, insects and bacteria. It has been isolated from a number of mammalian species, including cows [1], rats [2, 3] and rabbits [4]. Commercial enzyme is generally extracted from beef liver, Helix pomatia or Escherichia coli.

The bacterial UID has been studied less than the eukaryotic one. Purification of bacterial UID was reported for Staphylococcus [5], Streptococcus [6] and E. coil strains ML30 [7] and K12 [8]. The reported purifications are incomplete and involve several steps, giving low overall yields. In the case of E. coil UID, the purification reported by F. Stoeber [7] involved 4 steps and the yield was 14o/0 for a 10-fold purification. Using the procedure reported by G. Novel [8], after 4 steps, we were able to obtain a yield of 30070, with UID representing 60070 of the final proteins (as determined by us).

Since affinity chromatography has generally been proven to be a very efficient technique for enzyme purification, we developed a new affinity procedure to improve efficiency and yields of UID purifica- tion. Enzyme purification by affinity chromato- graphy, as described by Cuatrecasas et al. [9], re- quires the use of a selective ligand linked to a gel matrix. This ligand may be a specific competitive inhibitor of the enzyme. Among the various g~ucuronide analogs synthesized by F. Stoeber [7], thiophenyl-~-D-glucuronide (TPU) proved to be a competitive inhibitor with a high affinity for UID. The linkage of TPU to amino groups of the gel by hs carboxyl group creates a TPU-amide, which is expected to show a still greater affinity for the en- zyme, since amide derivatives of several other glucuronides have a higher affinity than the cor- responding glucuronide [7]. TPU was thus retain- ed as an affinity ligand, and covalently bound to a Sepharose matrix.

The affinity procedure reported in this paper yielded an 85070 pure UID in one step, and a prepa- ration with a purity greater than 95070 in two steps.

158 C. Blanco and G. Nemoz

Materials and methods

Chemicals A-H Sepharose 4B was obtained from Pharmacia Fine Chemicals, EDC-HCI from Sigma Chemical Co. TPU was synthesized by G. Luthaud accoiding to the pro- cedure described by F. Stoeber [7]. All other chemicals were reagent grade.

Bacterial growth conditions and cell extracts Cultures of E. coil strain S~200 [10] containing plasmid pCB12, a derivative of pCB2 [111 bearing UID structural gene uidA, were grown at 37°C in L.B. rich medium [12] containing 25 pg/ml of ampicillin.

Bacteria were collected by centrifugation and disrupted in a French pressure cell at 25 psi in buffer A: 10 mM Tris-HCl buffer, pH 7, containing 10 mM 2-mercap- toethanol. The cytoplasmic juice was collected after cen- trifugation for 15 rain at 12000 rpm.

Preparation of affinity column A-H Sepharose 4B (2 g) was thoroughly washed accord- ing to the supplier's recommendations, and suspended in 10 ml of deionized water; 100 mg (300 mM) of TPU were dissolved in 4 ml of water and added to the A-H Sepharose suspension. EDC-HCI (18 mg in 4 ml of deionized water) was added dropwise. The pH was brought to 4.5 with HC1 and the reaction allowed to pro- ceed for 48 h at room temperature with continuous gen- tle mechanical stirring. The pH was frequently readjusted to 4.5 during the first 24 h. An additional overnight in- cubation in presence of 2 mM acetic acid was performed in order to block any remaining activated groups.

The resulting mAxture was aitemative!y washed with 0.1 M sodium acetate, pH 4, and 0.1 M Tris, 0.5 M NaCI, pH 8.3, five times, and finally washed and stored in col- umn buffer A.

Chromatography procedure Chromatography was performed as indicated in the legend of Fig. 1 and in Table I. A gradient elution was followed by extensive washes of the column with 3 M KCI in buffer A. This procedure allows a good regeneration of the affinity column which can be reused several times.

A nalyticai procedure UID was assayed according to Novel and Novel [13]. Pro- tein was determined according to Lowry et aL [14]. SDS-polyacrylamide gel electrophoresis was perform- ed as described by Laemmli [15]. Polyacrylamide gradient gels were purchased from Pharmacia and used according to the manufacturer's recommendations.

Determination of amino-terminal amino acid sequence Purified UID (95% purity) was dialyzed against water to eliminate all salts, and lyophilized. 7 mg of lyophi- lized enzyme were subjected to automatic Edman degradation by J. Bonicel (Laboratoire de Chimie Bact6rienne du CNRS, Marseilles).

Results

Purif ication o f the enzyme

After application of the cell extract sample to the column, more than 90°70 o f the total absorbance at 280 nm appeared in the effluent following the load- ing o f the column. This absorbance is due to nucleic acids and proteins that have no affinity for the gel. No UID activity was detected in this first peak o f effluent fraction (Fig. 1A). After washing the col- umn with buffer A, UID was eluted by a gradient of 0 - 0 . 4 M NaCI in buffer A (Fig. 1A).

Enzymatic activity was eluted at 0.2 M NaC1 in a single peak containing 96070 of the total UID ac- tivity. The different fractions e!uted were analyz- ed by SDS-polyacrylamide gel electrophoresis, and the tracks scanned in a Vernon photoanalyzer. The purity of the enzyme was thus estimated to be 80-85070 in the purest fractions (No. 5 -11 , Fig. 1A).

Fractions 5-11 were pooled, concentrated to 5 ml and dialyzed against buffer A: 0.1 M NaC1. The concentrated sample containing 90°70 of the total UID activity was again applied to TPU-Sepharose

Table I. Purification of UID.

Fraction Volume UID Total assayed (ml) total activity proteins

(mU) (mg)

UID Yield Purification UID specific activity (%) fold purity (mU/mg) (~/o)

Crude extract 5 12.0 x 10 ~ 200 6.0 × 103 100 1 First chromatography 21 10.8 x 105 20 5.4 × 104 90 9 85 Re-chromatography 33 9.5 x l0 s 15 6.4 x 104 80 10.6 95

One unit of UID is defined as the amount of enzyme hydrolyzing 1/zmol of paranitrophenyl-fl-v-glucuronide/min. The purity of UID was evaluted by SDS-PAGE experiments.

Purification of fl-glucuronidase 159

, 3 , s , , s , [NaCl]* I m

e

t Z ~ 4. S 5 ' 6 9 10 11 ,~[$' ls~-

lk !_ • -/ / ~ ~ - ~ ~ ~ - . - l U

) II

Fig. 1. A. Chromatography of E. coil crude extracts. TPU-Sepharose gel was packed into a 10 ml column, washed and equilibrated with buffer A at room temperature. The flow rate was fixed at 40 ml /h . Enzyme samples (5 ml) containing 200 mg of protein were applied to the column. The column was washed with 500 ml o f buffer A. 10 ml fractions were collected. The column was eluted with a linear 0 - 0 . 4 M NaC1 gradient in buffer A. Fractions of 3 ml were collected, fl-Glucuronidase activity ( o - - - o ) was assayed in each fraction according to Novel and Novel [13]. Absorbance of the effluent was monitored at 280 nm ( ~ ) . The SDS-polyacrylamide (807o) gel electrophoregram of peak fractions stained with Coomassie blue is shown in the insert. B. Re-chromatography of/3-glucuronidase fractions. Fractions obtained in the experiment described in Fig. IA and containing ~-glucuronidase activity were pooled, concen- trated to 5 ml, dialyzed against buffer A - 0.1 M NaC1, and loaded on the column equilibrated with the same buffer, The column was washed with 500 ml of the same buffer. The enzyme was eluted with a 0 .1-0.3 M NaCI gradient in buffer A. Elution conditions are the same as above, fl-glucuronidase was assayed in each fraction ( o - - - o ). The effluent was monitored for UV absorbance at 280 nm ( ........ ). The SDS-polyacrylamide (807o) gel electrophoregram o f peak fractions is shown in the insert.

column, the column was washed with the same buffer (50 column volumes) and UID was eluted with a gradient of 0.1-0.3 M NaCI in buffer A (Fig. I B). Enzymatic activity was only detect- ed in a single peak, eluted at 0.2 M NaCI, and containing 80°70 of total initial UID activity. The purity of the enzyme, as judged by SDS- polyacrylamide gel electrophoresis was more than 95070 (Fig. IB). The amount of enzyme

recovery was 15 mg from 200 mg of protein in the crude extract.

Determination of the molecular weight of [3-glucu- ronidase monomer UID was subjected to electrophoresis in an SDS-polyacrylamide gel along side a track con- taining molecular markers (Fig. 2). Its molecular weight was estimated to be 74000.

160 C. Blanco and G, Nemoz

kOa Mh/ UlD

• .J

96 m

5 8 ~

55,¢__

z .5~

669

1

3o - B

1,3 _ _ ~

Fig. 2. Determination of fl-glucuronidase monomer molecular weight. Purified enzyme was subjected to electrophoresis in 15o70 polyacrylamide gel in the presence of SDS, alongside molecular markers: (96) phosphorylase b, (68) bovine serum albumin, (58) catalase, (55.4) glutamate dehydrogenase, (45) ovalbumin, (30) carbonic anhydrase. The position of//-glncuronidase is indicated by the arrow heads, standard molecular markers are 103 units.

Fig. 3. Determination of ~-glucuronidase molecular weight. Purified enzyme was subjected to electrophoresis in a polyacrylamide gradient gel (4-30o70). Molecular weight markers (MW) are thyroglobin (669), ferritin (440), catalase (230), LDH (140), bovine serum albumin (67) and ovalbumin (43). The posi- tion of fl-glucuronidas¢ is shown by bars (UID track).

Determination o f the molecular weight o f native fl-glucuronidase The molecular weight of native UID was determin- ed by electrophoresis in a polyacrylamide gradient gel (4-30070). Two peptides of 130 kDa and 260 kDa were detected (Fig. 3). We assume that they respectively correspond to a dimer and a tetramer composed of 74 kDa subunits, and that the dimer results from the degradation of the tetramer.

Determination o f amino-terminal amino acid sequence The amino terminal sequence determined with 95°7o pure UID, by means of automatic Edman degrada- tion was: N H 2 - M e t - L e u - A r g - P r o - Val.

Discussion

The affinity chromatography on TPU-Sepharose reported here is a convenient procedure for a rapid purification of UID with good yields in enzyme recovery (9007o or 80070) with a high purity (8507o and

> 95°70 respectively). This method could be useful for the purification of UID from other sources and would facilitate the purification of this enzyme from tissues where it is present in low amounts.

The molecular weight of the E. coli UID monomer, 74 000 is identical to that of mouse liver or kidney purified to homogenity [15,16] (69000-73 000). Mouse enzyme is a tetramer com- posed of identical subunits [15, i7, 18]. E. col, ~ en- zyme seems to be a tetramer composed of identical subunits of 74 kDa. The theorical values expected for a dimer and a tetramer composed of 74 kDa subunits would be 148 kDa and 296 kDa respec- tively. The variation observed with regard to ex- perimental values may be du~ to the method used. This molecular weight of native UID must be con- firmed by other methods.

Acknowledgements

We are indebted to F, Stoeber for encouragement and support of this work, to J. Bonicel for expert determina-

Purification o f fl-glucuronidase 161

tion of the peptidic sequence and to G. Luthaud for technical assistance. We thank C. Van Herrewege for help in the preparation of illustrations.

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