Eur. J. Biochem. 93, 301-311 (1979)

Properties of Two Molecular Forms of P-Glucuronidase from the Mollusc Littorina littorea L. Trinidad DIEZ and Jose A. CABEZAS Departamento de Bioquimica, Facultades de Ciencias y Farmacia, Universidad de Salamanca, and Consejo Superior de Investigaciones Cientificas, Salamanca (Received September 11, 1978)

The occurrence of two molecular forms, I and 11, in the P-glucuronidase of the liver (hepatopancreas) from the marine mollusc Littorina littorea L. has been demonstrated for the first time. The two forms have been purified 355-fold and 1262-fold, respectively. Form I and I1 of P-glucuronidase behave differently on DEAE-cellulose chromatography, polyacrylamide gel disc electrophoresis, isoelectric focusing (pH 5.5 and 4.2, respectively), optimum p H (4.4 and 3.4-4.1, respectively), thermal stability, K , (1.2 mM and 0.5 mM with p-nitrophenyl p-D-glucuronide, 0.3 mM and 0.15 mM with phenolphthalein P-D-ghcuronide as substrates for form I and 11, respectively) and V. Their molecular weight, estimated by gel filtration through Sephadex G-200, was about 250000 for both forms. Several subunits were separated by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate. This p-glucuronidase is a glycoprotein, but sialic acid(s) were not detected. The enzyme was very active on synthetic substrates and also on hexasaccharides and tetrasaccharides containing glucuronic acid residues with p 1* 3 linkages ; it had practically no activity on certain glycosaminoglycans. Hg2' and glucaro-l,4-lactone were very effective inhibitors of this enzyme ; the latter by a competitive mechanism.

8-Glucuronidase occurs in mammalian organs, molluscs, insects, plants and bacteria; a review article on the distribution,. properties and role of this enzyme was published in 1955 [l]. Over the last two decades several papers have appeared, concerning the Pglucuronidase from human [2 - 91, bovine [lo] and rodent sources [I 1 - 231. The critical topic of multiple forms of this enzyme in mammalian tissues has been studied by several authors, using chromatography on DEAE-cellulose, polyacrylamide-gel electrophoresis and electrofocusing procedures. The presence of multiple forms of P-glucuronidase in organs such as human liver [6,9], bovine liver [lo], rat liver [7,15,16,19,20,23] and preputial gland [22], mouse kidney [ I l l and rabbit brain [21] has been demonstrated. Enzymes. 8-Glucuronidase (EC 3.2.1.31); 8-galactosidase (EC 3.2.1.23); B-glucosidase (EC 3.2.1.21); x-galactosidase (EC 3.2.1.22); cc-glucosidase (EC 3.2.1.20); a-L-fucosidase (EC 3.2.1.51); a-mannosidase (EC 3.2.1.24); /I-xylosidase 01- exo-1,4-fi-xylosidase (EC 3.2.1.37); /I-N-acetylglucosaminidase (EC 3.2.1.30); fl-N-acetylgdlactosaminidase (EC 3.2.1.53). Trivial Name. Naphthol AS-BI, 7-bromo-3-hydroxy-2-naphth0-anisidine.

Although the occurrence of this enzyme in certain marine molluscs was reported by Dodgson et al. as early as 1953 [24] and it is known that molluscs are rich sources of glycosidases, we have not yet found any reference to the occurrence of multiple forms of this enzyme in marine molluscs. It seems interesting to study P-glucuronidase because the deficiency of this enzyme, which is involved in the degradation of some glycosaminoglycans (mucopolysaccharides), is associated with mucopolysaccharidoses in man [2-51; because the role of 8-glucuronidase in the conversion of benzopyrene glucuronides to carcinogens has been described recently [25]; and because of the long-established use of this enzyme as an analytical tool for the hydrolysis of glucuronides. This paper deals with the purification and properties of the p-glucuronidase from the marine mollusc Littorina littorea L. The presence and characterization of two molecular forms of the enzyme in this mollusc are reported for the first time. Preliminary reports of some of this work have been presented [25a, b].

302

MATERIALS AND METHODS Materials The liver (hepatopancreas) of the marine mollusc Littorina littorea L. (perinwinkle), from live animals, was employed. Glucuronic acid, p-nitrophenol, p-nitrophenyl BD-glucuronide, phenolphthalein 6-D-glucuronide, pnitrophenyl glycosides, naphthol AS-BI glucuronide, salt fast Garnet GBC, hyaluronic acid from human umbilical cord, chondroitin 4-sulphate, heparin, monosaccharides, acrylamide, DEAE-cellulose, bovine serum albumin, ovalbumin, cytochrome c, immunoglobulin, tris(hydroxymethyl)aminomethane, sodium azyde, hyaluronidase from bovine testes, hyaluronidase of the P-N-acetylglucosaminidase type from Apis mellifera venom and sodium dodecylsulphate, were purchased from Sigma Chem. Co. (St Louis, Mo., U.S.A.). Blue Dextran and Sephadex G-200 were from Pharmacia (Uppsala, Sweden). Ampholines were from LKB Produkter A.B. (Bromma, Sweden). All other products, from Probus (Badalona, Spain) were of the highest purity commercially available. Collodion bags were from Sartorius-Membranfilter GmbH (Gottingen, F.R.G.). Assay of Enzymes The P-D-glucuronidase, P-D-glucosidase, B-N-acetylglucosaminidase, B-D-galactosidase, a-D-mannosidase, a-L-fucosidase, P-N-acetylgalactosaminidase, BD-xylosidase, a-D-galactosidase and a-D-glucosidase activities were determined as previously described [26,27]. The reaction mixture consisted of 1.4 ml of 0.05 M citric acid/sodium phosphate, pH 4, containing 1.4 ymol of p-nitrophenyl glycoside (except for P-D-xylosidase activity which was measured with the o-nitrophenyl derivative) and 0.1 ml of the enzyme solution. p-Nitrophenyl or phenolphthalein glucuronide were employed as substrates for glucuronidase. After incubation at 37 "C for a preset time (generally 10 min), 1.5 ml of 0.2 M Na2C03 was added to stop the reaction. The released p-nitrophenol (or phenolphthalein) was measured at 400nm (or 552nm, respectively). All buffer solutions contained 0.02 % NaN3 as preservative. 1 unit of enzyme (U) was defined as the amount of enzyme which liberated 1 ymol p-nitrophenol (or phenolphthalein)/min under the assay conditions. Specific activity was expressed as U/mg protein. Protein Determination Protein was determined by the method of Lowry et al. [28] with bovine albumin as standard. In some cases, protein was estimated by absorption at 280 nm.

Two Molecular Forms of P-Glucuronidase from a Mollusc

Purification of B-D-Glucuronidase All procedures were carried out at 4 "C unless otherwise indicated. Step 1 :Preparation of the Crude Extract. Approximately 13 g of livers (hepatopancreas) from about 300 g of whole animals was homogenized in distilled water (1/6, w/v) in a mortar with sand. The suspension was allowed to settle for 2 h and the crude extract was centrifuged for 20 min at 12000 x g ; the precipitate was discarded. Step 2: Ammonium Sulphate Fractionations. Solid (NH&S04 was added with stirring to the supernatant fraction, to 45 % saturation. After allowing it to settle for 17 h, the resulting precipitate was removed by centrifugation as above and was discarded. Solid (NH4)2S04 was added to the supernatant fraction to 60 % saturation, the precipitate was allowed to settle for 7 h and the mixture was centrifuged as above. Step 3 : First Gel Filtration on Sephadax G-200. The precipitate from step 2 was dissolved in the minimum volume (2-2.5 ml) of 0.005 M citric acid/ sodium phosphate buffer (pH 5.5) in 0.005 M NaCl and was applied to a column (2 x 100 cm) of Sephadex G-200, which had been equilibrated with the same buffer solution, which was also used for elution. 5-ml fractions were collected at a flow rate of 1015 ml/h. Step 4 : Chromatography on DEAE-Cellulose. The contents of tubes containing P-glucuronidase activity were pooled and applied to a column (2 x 30 cm) of DEAE-cellulose medium mesh equilibrated with the above-mentioned buffer solution. Elution was started with this solution, followed by a continuous gradient of 0.1 -0.2 M NaCl in 0.005 M citric acid/sodium phosphate buffer (pH 5 ) ; 3-ml fractions were collected at a flow rate of 20-2.5 ml/h. Two peaks, I and 11, with B-glucuronidase activity were separated. Step 5 : Repeat Gel Filtration on Sephadex G-200. The contents of tubes containing B-glucuronidase activity (peaks I and 11) were separately pooled and concentrated by ultrafiltration by Amicon PM30 membranes at a Nz pressure less than 5 atm (506 . 625 Pa) to a volume of 3 ml. These fractions were applied to columns of Sephadex G-200 which were eluted at the same conditions than in step 3; 3-ml fractions were collected. Step 6: Isoelectric Focusing. Fractions containing P-glucuronidase activity from step 5 were separately pooled, subjected to dialysis against water for 3 h and electrofocused by the method of Vesterberg and Svenson [29] in a 110-ml electrofocusing column. The ampholite concentration was 1 % (v/v) with a pH range from 4.0 to 6.0 for the molecular form I, and 3.5 to 5.0 for the form 11, in a sucrose gradient; voltage was 350 V. The run was carried out for 18 h after which 3-ml fractions were collected and the pH

303

T. Diez and J. A. Cabezas

of each fraction was measured at 2 "C with a digital pH meter (Radiometer pH M-62). Electrophoresis The course of purification was followed by polyacrylamide-gel disc electrophoresis employing essentially the method of Ornstein and Davis [30,31] with 7 % acrylamide gels, 3 mA per column, and Tris/ glycine buffers, pH 8.3. The proteins in the gels were stained with Coomassie brilliant blue, according to Weber and Osborn [32]; for glycoproteins, the periodic acid-Schiff procedure was employed as previously described [33]. P-Glucuronidase activity was located in the gels by incubation of the gels in 100 mg/l solution of the glucuronide of naphthol AS-BI for 90 min at 37 "C followed by salt fast Garnet (1 mg/l ml) for 11 min; a slight modification of the method of Dean [34]. Polyacrylamide-gel disc electrophoresis with sodium dodecyl sulphate was carried out according to the method of Weber et al. [35] with 7 % and 5 % acrylamide gels. The protein bands were visualized with Coomassie brilliant blue and the enzyme activity was demonstrated with the stain naphthol AS-BI P-D-glucuronide, as described by Dean [34], in a sample not subjected to the treatment of Weber and Osborn ~321.

Estimation of Molecular Weight

The molecular weight of both forms was estimated on columns (2 x 95 cm) of Sephadex G-200, equilibrated and eluted with 0.005 M citric acid/sodium phosphate buffer (pH 5.5) in 0.005 M NaC1, using the following standards : cytochrome c (molecular weight, 12400), myoglobin (17 000), ovalbumin (45 000), bovine serum albumin (67 000), immunoglobulin (160000) and catalase (248000). Molecular form I was also studied by ultracentrifugation analysis. Sedimentation equilibrium an velocity runs were performed in a Beckman L-3-75 ultracentrifuge equipped with an ultraviolet scanner, according t o the procedure of Sussman [36]. For sedimentation-coefficient determination runs were done at 24660 and 39200 rev./min at 20 "C; molecular weight was determined by sedimentation equilibrium at 10950 rev./min for 20 h.

areas. The method of Lustig and Langer, as described by Winzler [39], was employed for total-hexose determination. Hexosamines were determined by the ElsonMorgan procedure as modified by Rimington and described by Winzler [39]. The occurrence of sialic acids was assayed by the modified resorcinol procedure [40,41] and by the thiobarbituric acid method [42, 431. Amino Acid Analysis

The amino acid composition of the P-glucuronidase forms I and I1 from the purified enzyme (step 6) was determined using a JEOL (mod. JLC-5 AH) amino acid analyzer following a 20-h hydrolysis in 6 M HC1 as described by Moore and Stein [44]. Thermal Stability Determination

The thermal stability of both forms of the Pglucuronidase was determined with fractions from step 4 of the purification procedure by pre-incubation for times ranging from 5 - 90 min at temperatures of 40, 50 and 60 "C. Also a portion of the pool with form I1 P-glucuronidase activity (step 5 ) was used for thermal stability determination ; the incubation was performed at temperature ranging between 35 and 65 "C for 5 min. In both types of experiments, p-nitrophenyl P-Dglucuronide was used as substrate in 0.05 M citric acid/sodium phosphate buffer, pH 4.0. Determination of pH Optimum and Stability at Variousp H Values

Samples of purified enzyme from step 5 were incubated withp-nitrophenyl P-D-glucuronide for 30 min at pH values between 2.6 and 8.5 using the following buffers : 0.05 M citric acid/sodium citrate, 0.05 M acetic acid/sodium acetate, 0.05 M Tris-HC1 and 0.05 M citric acid/sodium phosphate. The stability of the enzyme at various pH values was determined by pre-incubation of a purified fraction from step 5 in 0.2 M Tris/sodium citrate buffer ranging from pH 2.2 to pH 8.7 at 37 "C for 2 h ; 0.02 % NaN3 was employed as preservative. The control was a similar but non-incubated preparation. Determination of K,

Analysis of the Carbohydrate Composition of P-Glucuronidase

Samples of the purified enzyme from step 6 were used. Hexoses were identified according to the method of Sweeley et al. [37] slightly modified [38], and were quantitatively determined by measurement of peak

The Michaelis constant and maximal velocities were determined by incubation of the purified enzyme from step 3 and also fractions from step 5 with p nitrophenyl glucuronide and phenolphthalein glucuronide at concentrations ranging from 66.6 pM 13 mM and 66.6 pM- 5.3 mM, respectively, for 10 min

304

at the pH optimum. The value for the apparent K, and V was calculated from Lineweaver-Burk [45] and Eadie-Hofstee (as described by Penasse) [46] plots. Hydrolysis of Natural Substrates by ,B-GZucuvonidase Hyaluronic acid (as sodium salt) and chondroitin 4-sulphate were employed. Also several hyaluronic acid and chondroitin 4-sulphate derivatives, obtained after hydrolysis either with hyaluronidase from bovine testes of with hyaluronidase of P-N-acetylglucosaminidase type (from bee venom), were used. The higher glucuronic acid-containing oligosaccharides resulting from the former treatment and the hexasaccharides and tetrasaccharides of the latter were employed as substrates for the fl-glucuronidase assay. 4.8 mg of hyaluronic acid or chondroitin 4-sulphate were incubated with 0.84 U of the molecular form I1 of /3-glucuronidase (previously determined with p nitrophenyl p-D-glucuronide) in a total volume of 6 ml for 60 h at 37 "C. When hyaluronidases were employed, 12 mg of hyaluronic acid and 2.4 mg of enzyme were incubated for 72 h at 37 "C and the resulting oligosaccharides were subjected to the activity of 0.82- 1.37 U of P-glucuronidase for 21 - 72 h at 37 "C. Other assays were carried out as follows: 60 mg of chondroitin 4-sulphate were incubated with 3.3 U of p-glucuronidase; and 1.66 mg of heparin with 4. I U of /3-glucuronidase. Incubation time was between 24 and 46 h . The products liberated were separated by ultrafiltration through collodion bags and concentrated. For identification of these products, descending chromatography on Whatman No. 1 paper was employed, with butan-1-ol/pyridine/O.1 M HC1 (5/3/2, v/v/v) as solvent. The liberated products were located by the method of Trevelyan et al. [47]. Free glucuronic acid was determined by the carbazol reaction [48] after ultrafiltration through collodion. Influence of the Effectors and Determination of Inhibitor Constants Cations, CN-, acetate and EDTA were tested at 2 mM and 10 mM concentrations. Also ascorbic acid and monosaccharides at 10 mM and 100 mM concentrations, and heparin at 0.25 mM and 0.50 mM concentrations were assayed for inhibitory activity. In all cases a control of the same composition (except for the effector) was run. Inhibitor constants, Ki, were determined by the method of Dixon [49], with p-nitrophenyl p-D-glucuronide as substrate at concentrations ranging from 0.2-9.3 m M ; ~-glucaric-1,4-lactonewas used as an

Two Molecular Forms of 8-Glucuronidase from a Mollusc

inhibitor at concentrations ranging from 0.002 0.040 mM. The enzyme preparation (purified fraction from molecular form 11) was incubated with the substrate and inhibitor in 0.05 M citric acid/sodium phosphate, pH 4.0, for 10 min at 37 "C. The inhibition type was determined according to Lineweaver-Burk [45] and Eadie-Hoffstee plots [46].

RESULTS Purification and Criteria ,for Homogeneity The following glycosidase activities, expressed as units of total activity, were found in 13 g of the crude extract : P-glucuronidase (142 U), /3-~-glucosidase (77 U), P-N-acetylhexosaminidase (59 U), P-D-galactosidase (48 U), a-D-mannosidase (32 U), a-L-fucosidase (24 U), j-N-acetylhexosaminidase (21 U), pD-mannosidase (32 U), a-L-fucosidase (24 U), p-Nacetylhexosaminidase (21 U), P-D-xylosidase (9 U), a-D-galactosidase (6 U) and a-D-ghcosidase (3 U). The described procedure, based on precipitation on ammonium sulphate (initial experiments determined the optimal waiting period), gel filtration through Sephadex G-200, chromatography on DEAEcellulose and repeat gel filtration on Sephadex G-200, led to an enriched P-glucuronidase preparation, free from other glycosidases except for a-L-fucosidase, a-D-mannosidase and b-D-glucosidase, which were present at less than 5 % of the total activity of pglucuronidase (form 11). The elution profile of a-glucuronidase, separated into two forms (I and 11) by chromatography on DEAE-cellulose (step 4), is shown in Fig. 1. When both pools of P-glucuronidase purified fractions from step 4 were independently subjected to a repeat gel filtration on Sephadex G-200, a peak corresponding to each form was obtained (Fig. 2). The final preparations obtained after electrofocusing were practically free of other glycosidases, as determined with p-nitrophenyl glycosides as substrates. A summary of the purification is given in Table 1. The purity at each step of the enzyme purification was monitored by polyacrylamide-gel disc electrophoresis (Fig. 3). A protein band for the molecular form I was obtained from the pooled fractions from the last step of purification. The position of enzyme activity, determined in a parallel gel, completely coincided with the protein band (see below). Isoelectric focusing of the pooled fractions either from molecular form I or I1 also showed only one peak for each form (see later). In sedimentation velocity runs in the analytical ultracentrifuge only one single symmetrical peak was observed for form 1(form I1 was not analyzed).

305

T. Diez and J. A. Cabezas

F r a c t i o n number

Fig. 1. Chromatography of DEAE-cellulose. An aliquot portion of enzyme solution (68 mg protein), obtained after gel filtration on Sephadex G-200, was applied to a column (2 x 30 cm) of DEAE-cellulose. The column was equilibrated with 0.05 M citric acid/sodium phosphate (pH 5.5) in 0.005 M NaCl solution. The elution was started with this solution, followed by a continuous gradient of 0.1 -0.2 M NaCl in 0,005 M citric acid/sodium phosphate buffer (pH 5). 3-ml fractions were collected. (0)Proteins; (0)B-glucuronidase activity

I

A

B

30 40 50 Fraction number

30 40 Fraction number

50

Fig.2. Gel filtration on Sephadex G-200. Aliquots of 3 ml solution of the enzyme from DEAE-cellulose chromatography, peak I (9 mg) and I1 (32 mg), were separately subjected to a second gel filtration on Sephadex G-200. (A) From peak I ; (B) from peak 11. (0) Proteins; (0)p-glucuronidase activity

Table 1. Purification of fi-glucuronidase from 13 g of liver of L. littorea L . Conditions of purification and definitions of units are described in the text. Phenolphthalein 8-o-ghcuronide was used as a substrate. Numbers in parentheses represent the values for molecular form I1 of the P-glucuronidase when they are not the same than those of the form I Step and procedure

1. Crude homogenate 2. Ammonium sulphate precipitation 3. First filtration of Sephadex G-200 4. DEAE-cellulose 5 . Second filtration on Sephadex G-200 6. Isoelectric focusing

Total volume

Total protein

ml

mg

80 4.8

2640 230

0.58 0.62 (0.8)

57 43 15 6

68 9 1.2 0.11

Specific activity

Total activity

Purification

U/mg

U

-fold

Yo

0.016 0.18

42 41

1 11

100 99

39.4 5.6 (7.2)

36 39 (SO)

0.70 (3) 5.68 (20.3)

0.84 (3.6) 0.62 (2.2)

44 (187) 355 (1268)

Yield

94 13 (17) 2 (9) 1.5 ( 5 )

306

Two Molecular Forms of P-Glucuronidase from a Mollusc

Separation of Different Forms of the Enzyme

To avoid the risk of artifacts which might arise during the enzyme purification (especially after chromatography on DEAE-cellulose, step 4), P-glucuronidase-containing pooled fractions from step 3 were subjected to isoelectrofocusing. The column profiles are shown in Fig.4. Two peaks at pH 5.5 (peak I) and 4.2 (peak 11) were detected. These pH values were the same when pooled p-glucuronidase-containing

fractions (forms I and I1 from step 5) were separately subjected to electrofocusing. The corresponding profiles exhibit only one peak for form I and another peak for form 11, the pIvaIues of which are coincident with those of the sample from step 3 (Fig.4). Disc electrophoresis followed by detection of Pglucuronidase by using naphthol AS-BI P-D-glucuronide shows that one major band appeared when a sample of peak I from step 4 (DEAE-cellulose chromatography) was employed (Fig. 5). Also one band was obtained when a sample of peak I1 of the step 4 was used, but its mobility is different from that of peak I (Fig.5). On the contrary, two well defined bands were separated when a sample of the enzyme preparation from step 3 (first filtration on Sephadex G-200) was employed. Furthermore, gel electrophoresis analysis with samples from peaks I and I1 were performed separately and the bands were stained by periodic acid/Schiff. The bands were coincident with those which exhibit /3-glucuronidase activity. Thus, both /3-glucuronidase forms are glycoproteins (see below).

Molecular Weight Fig. 3. Polyacrylamide-gel disc electrophoresis of the proteins from each step of b-glucuronidase (form I ) purifcution. (I) Step I ; (2) step 2; ( 3 ) step 3; (4) step 4; ( 5 ) step 5; (6) step 6

.A

+

5

30

The molecular weight of both forms of P-glucuronidase estimated by gel filtration on Sephadex (3-200 was about 250000 (Fig. 6).

t'

10

81, 6

4 2 0

0

10

20

50

30 40 F r a c t i o n number

60

.-.-z

7.

L

r

250

Y

0

10

20 30 40 F r a c t i o n number

50

0

10

20

30 40 Fraction number

50

Fig. 4. Isoelectric focusing o j P-glucuronidase. (A) /l-Glucuronidase from step 3 of purification procedure. (B) /l-Glucuronidaae (peak I ) from step 5 of purification. (C) b-Glucuronidase (peak 11) from step 5 of purification. (0)Proteins; (0)P-glucuronidase activity; (A) pH values

307

T. Diez and J. A. Cabezas

The molecular weight of form I was also determined by sedimentation equilibrium in an analytical ultracentrifuge. By plotting the logarithm of the concentration over the squared distances from the rotor center a linear function was observed (Fig.7). The value obtained for the molecular weight was 113500. This result corresponds to approximately one-half of that obtained by gel filtration on Sephadex G-200. The sedimentation coefficient for form I, at 24 660 and 39200 rev./min, was 10.5 S. Subunit Analysis

The treatment of both forms of P-glucuronidase with 1 % sodium dodecyl sulphate and 1 2-mercap-

toethanol for 3 min at 100 "C causes the dissociation of the protein in subunits, as determined by polyacrylamide-gel disc electrophoresis. Two different concentrations of acrylamide were used, 5 % and 7 %; the best resolution in the bands was achieved when the concentration was 5%. Three bands were well separated. Carbohydrate Composition

Determination by gas-liquid chromatography of the monosaccharides in a sample from the purified enzyme (form I, step 6) gave the following approximate values: glucose, 2 %; galactose, 0.6 %; mannose, trace amounts; hexosamines, 1.7 %. Total hexoses, as assayed by the Lustig and Langer procedure, also showed a positive result; their content in form I1 of the enzyme was higher than that in form I. The poor sensitivity of this technique could not give a more quantitative evaluation because of the low carbohydrate content in the molecule of the P-glucuronidase (as deduced by gas-liquid chromatography). Hexosamines, determined by the Elson-Morgan method, gave a value of 1.8% (of total weight) for form I, and 11.9 % for form I1 of the enzyme. We did not find sialic acid(s) in a sample from purified form I (step 6) of the enzyme or in form 11, using both the resorcinol and thiobarbituric-acid procedures. Taking into consideration the high sensitivity of the thiobarbituric acid reaction, this indicates that there can only be a very small amount of sialic acid in this glycoprotein. Amino Acid Composition

Fig. 5. Polyacrjbuide-gel disc electrophoresis ofthe p-glucuroniduse. Bands were detected by incubation with naphthol AS-BI b-Dglucuronide followed with salt fast Garnet: (1) sample from peak I separated in step 4; (3) sample from peak I1 separated in step 4; (2) sample from step 3

c

A

* m

The results of the amino acid analyses are shown in Table 2. Both forms of the enzyme have concentration; in conamino acids at a very trast, lysine, aspartic acid, proline, glycine, isoleucine

B

f

m '6

.c

.-

z

(II

3

5e6

'. p- G l u c u r o n i d a s e

ve

1 v,

t

5.6

..

p-Glucuronidase

v,

1%

Fig. 6. Molecular weight determination of P-glucuroniduse on a calibrated Sephudex C-200 column. (A) Form I of 8-glucuronidase; (B) form 11. Conditions and standard proteins are described in the text

Two Molecular Forms of P-Glucuronidase from a Mollusc

308

and leucine are found in significative different amounts in form I1 than in form I.

from 4.6 to 6.5 using buffer solutions obtained by appropriate mixtures of 0.2 M citric acid and 0.2 M Tris at the assay conditions.

Thermal Stability of Both Forms of the Enzyme Fig.8 shows that the activity of the form I of the enzyme is stable to pre-incubation for 90 min at 60 "C. In contrast, form I1 is not stable at 60 "C even for 5 min (Fig. 8). p H Optimum andpH Stability

The effect of pH on the activity of both forms of a-glucuronidase from step 5 is shown in Fig.9. The pH-activity profile indicates a pH optimum of 4.4 for form I, and two values, at 3.4 and 4.1, for form 11, in a buffer of 0.05 M citric acid/sodium phosphate. Stahl and Touster have also found a double pH optima in /?-glucuronidase from rat liver lysosomes 1141. Tris was an effective inhibitor. Yet, the remaining activity of the enzyme is maxima in the range of pH

Kinetic Characteristics of the Enzyme The rate of hydrolysis of two synthetic substrates @-nitrophenyl P-D-ghcuronide and phenolphthalein P-D-glucuronide) was measured. The enzyme was found to follow typical Michaelis-Menten kinetics. The plots of l / v versus l/[S] showed a straightline relationship. The Lineweaver-Burk plots gave the following values for the apparent K, and V :for form I (obtained after step 5 of purification) the K , was Table 2. Amino acid analysis ofthe form I and IIfrom ~-gluc.uronidase from the mollusc L. littorea L. These results are averages of duplicate determinations performed as described in the text. Insufficient material precluded the use of varying hydrolysis times. The aim of the analysis was to compare both forms Form I

Amino acid

I 3.5

I:

Form I1

mo1/100 mol

\ :

:\

,

!

4a

50

1D

42

4 4 46 rz

Fig. 7. Sedimentation equilibrium duta f o r b-glucuroniduse (form I ) . Experimental conditions are described in the text

IB

4OoC 50°C

5.7 2.3 1.4 11.9 7.2 5.9 9.6 5.6 8.7 8.2 0.7 7.4 2.2 4.7 8.6 4.4 5.5

6.0 2.4 1.6 12.7 7.0 5.8 9.6 5.9 8.2 8.2 0.9 7.5 2.1 4.2 7.9 4.5 5.4

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine

- 100 ._ .? %

c

75

c

m u

._

c m ._

.z

.m

50

50

.-m 5

:*

E

n a,

n

0

0

0

30

60 Time (min)

90

I 0

\ y ? 6 0 ° C 30

60 Time (min)

50

25

0 90

30

40

50

Temperature

60

70

("C)

Fig. 8. Thermal stability oj~-glucuroniduse.(A) Activity of the form I from step 4 of purification. (B) Activity of the form 11 from step 4. (C) Activity of the form I1 from step 5, after 5 min at temperatures ranging between 35 "C and 70 "C

309

T. Diez and J. A. Cabezas

1

3

5

7

9

- 2 - i O l

11

PH

Fig.9. Effect oj p H on both forms of the purified ,!-gluewoniduse (step S), in a buffer of 0.05 M citric acidlsodium phosphate. (0) Form I ; (0)form I1

-

1.2 mM and the V (as pmol x min-' x mg-l) 2.9 with p-nitrophenyl P-D-glucuronide as substrate, and 0.3 and 1.O, respectively, with phenolphthalein fl-D-glucuronide; for form I1 (separated also at the step 5) the K, and Vwere 0.5 mM and 7.1 pmol x min-' x mg-' , respectively, using the former substrate; and 0.15 and 4.4, respectively, with the latter. For the partially purified enzyme from step 3, the &,value was 0.76 mM and the V was 0.37 pmol x min-' xmg-' using pnitrophenyl P-D-glucuronide as substrate (Fig. 10). These results were confirmed when Eadie-Hoffstee plots were employed.

.

-

- 4 - 2 0

4

5

6

7

2

4

6

8

10

8

10

l/[S] (rnM-')

E

Hydrolysis of Natural Substrates

z

1.0

The purified enzyme from step 6 shows little activity on hyaluronic acid (about 1 % of glucuronic acid was liberated). However, after hydrolysis of this substance with hyaluronidase from bovine testes (to obtain glucuronic-acid-containing oligosaccharides), approximately 8 % of glucuronic acid was liberated, as measured by the carbazol reaction and paper chromatography. A higher activity of fl-glucuronidase was observed on hexasaccharides and tetrasaccharides obtained after treatment o f hyaluronic acid with hyaluronidase from bee venom (30 % liberation of glucuronic acid). Similar results were obtained on chondroitin 4sulphate (2 % liberation of glucuronic acid) and oligosaccharides obtained at the same conditions (23 % of glucuronic acid liberated). Practically no liberation of glucuronic acid was observed when heparin was used as a substrate. On the other hand, heparin was an inhibitor of this enzyme (see later).

s

0.4

The effect of several cations ( H g + , Ca2+,Mg2'and Mn2+, as chlorides; Cu2+, Ni2+, Co2+, Zn2'

3

5

m

Effect of Ions, Chelator, Acetate and Some Carbohydrates and Derivatives

2

1 / [ S ] (rnM-')

-6

-4

-2

0

2

4

6

12

14

1 / [ S ] (rnM-')

Fig. 10 Lineweaver-Burk plots of p-glucuronidase. (A) Partially purified enzyme from step 3. (B) Purified form I of the enzyme from step 5. (C) Purified form I1 of the enzyme from step 5. The effect of substrate on reaction rate was studied under conditions described in the text. (0)p-Nitrophenyl ,!-D-glucuronide substrate. (0)Phenolphthalein p-D-glucuronide substrate

and NHZ, as sulphates), CN-, sodium acetate and ascorbate, and EDTA, at 2 mM and 10 mM concentrations, was investigated. Hg", Cu2+ and Ni2+ were effective inhibitors of P-glucuronidase (85 - 95 %, 31 - 88 % and 11 - 36 % inhibition at both concentrations, respectively). D-Mannose, D-glucuronic acid, 8-D-gluconelactone, ~-glucaro-l,4-lactone,L-ascorbic acid, at 10 mM and 100 mM concentration, and heparin at 0.25 mM and 0.5 mM, were also assayed. Inhibition was found with D-glucaro-1,4-lactone (100 %), glucuronic acid (40 - 90 %), ascorbic acid (31 - 60 %) and heparin

310

Two Molecular Forms of 8-Glucuronidase from a Mollusc

I S ] =0.20mM IS] =0.26mM

-

1.6

I'

S] = 0.40mM

S]=O.6OmM

S l =9.33mM

- 10

-4 - 3 - 2 - 1

0

1

2

3

4

5

6

7

8

t/[Sl(mM-')

Fig. 11, Effect of ~-glucaro-1,4-lactoneas inhibitor of 8-glucuroniduse. (A) Dixon plot. (B) Lineweaver-Burk plot. Experimental conditions are described in the text

(35 %). ~-Glucaro-1,4-hctoneseems to be a partial competitive inhibitor (Fig. 11) (see Discussion). Its Ki was 2.5 pM.

DISCUSSION The mollusc hepatopancreas is a system rich in glycosidases, but the purification of a single glycosidase from these sources is difficult in some cases [27,50, 511, due in part to accompanying enzymes of similar characteristics. Besides, stirring or freezing and thawing treatments are dangerous for activities of enzymes such as purified glucuronidase from L. littoreu. Presumably, this enzyme is unstable to freezing because of subunit dissociation. Nevertheless, this enzyme maintains at 4 "C its activity without loss for at least a month. We have separated two glucuronidases from the visceral gland of the perinwinkle after chromatography on DEAE-cellulose. They were purified and characterized. This result has been confirmed by isoelectric focusing. The possibility of autolysis or artifacts seems to be excluded in our experimental conditions. Thus, the livers of L. littorea were extracted from live animals and immediately used for isolation of the enzyme; moreover, all steps of the purification were carried out at 4 "C, a temperature in which this enzyme is stable. The evidence for the presence of two genuine forms (at least) of fi-glucuronidase in the perinwinkle liver is based on the following facts: firstly, separation by three different procedures (chromatography on DEAE-cellulose, isoelectric focusing, and polyacrylamide-gel dis electrophoresis) ; secondly, their different values for optimum pH, K,, V, and thermal stability. The molecular weights of both forms seem to be very similar or identical. It may be deduced that the

different behaviour of these forms of glucuronidase in the separation procedures and their different kinetic and stability characteristics might be due to their electric charge. We have not found sialic acid(s) in their composition; but the different concentration of hexosamines in both forms could explain, at least in part, this behaviour. Besides, a higher concentration of some amino acids in form I1 has also been observed. One may then wonder if the above-mentioned different behaviour of both forms is due to a different content of hexosamines, of some amino acids, or to both. In this case, P-glucuronidase forms from L. littorea should show similarities with multiple forms (microsonial and lysosomal P-glucuronidases) from rat liver [23] and differences against P-glucuronidase forms from preputial gland of the female rat [22] which differ only by their carbohydrate content. It seems that the addition of the carbohydrate moieties is accomplished during the transport from endoplasmic reticulum to lysosomes [52]. The occurrence of subunits in P-glucuronidase from the hepatopancreas of L. littoreu has been detected by us employing electrophoresis in presence of sodium dodecyl sulphate. Using the same procedure, other authors have also found subunits in fi-glucuronidases from mammalian sources such as: rat liver [14], rabbit liver [34], mouse kidney [13], and, recently, human placenta [8]. The enzyme hydrolyses not only synthetic substrates but also some natural ones. It has practically no activity on macromolecules, but it is active on tetrasaccharides, hexasaccharides and higher saccharides containing glucuronyl residues with ol-+ 3 linkages. So it may be useful as an analytical tool for study of structures. Finally, from our kinetic assays it may be deduced that Hg2+ is an effective inhibitor, probably by interaction with the - SH groups of the enzyme. Glucaro1,4-lactone was also a very active inhibitor; it seems

31 1

T. Diez and J. A. Cabezas

that the Dixon and Lineweaver-Burk plots (Fig. 11) apply well to a partial competitive inhibition [46]. However, glucaro-l,4-lactone may be converted into the corresponding 1,5-1actone, according to the observation of several authors [53] in similar studies. This work forms part of a thesis carried out by T. Diez in this Department, supported by the Ministerio de Educacibn y Ciencia and 'C.S.I.C.' and by a grant of the Presidencia del Gohierno. We wish to thank Dr A. Reglero and Dr N. PCrez, members of this Department, respectively by the determination of neutral sugars by gas-liquid chromatography and the estimation of molecular weight of one form of the enzyme by ultracentrifugation, the latter at the Department of Biochemistry of the Faculty of Medecine of Lyon (France) (Prof. P. Louisot); and Dr A. Reglero for helpful discussions. Also the determination of amino acids by J. E. Guacianca in the Servicio de andisis del Centro de Investigaciones Biolitgicas del C.S.I.C., Madrid (Dr E. Mufioz), is acknowledged. Finally, we thank Mrs Colette Delamare for the correction of the manuscript and Mrs Rosario Reglero for the secretarial work.

REFERENCES 1. Fishman, W. H. (1955) Adv. Enzymol. 16, 361 -407. 2. Touster, 0. (1973) Mol. Cell. Biochem. 2, 169- 177. 3. Nicol, D. M., Lagunoff, D. & Pritzl, P. (1974) Biochem. Biophys. Res. Commun. 59, 941 -946. 4. Brot, F. E., Glaser, J. H., Roozen, K. J., Sly, N., Willian, S. & Stahl, P. D. (1974) Biochem. Biophys. Res. Commun. 57,l-8. 5. Sly, W. S., Greene, A. E. & Coriell, L. L. (1975) Cytogenet. Cell Genet. 15,410-412. 6. Glaser, J. H., Roozen, K. J., Brot, F. E. & Sly, W. S. (1975) Arch. Biochem. Biophys. 166, 536- 542. 7. Owens, J. W., Gammon, K. L. & Sahl, P. D. (1975) Arch. Biochem. Biophys. 166,258 - 272. 8. Brot, F. E., Bell, E. & Sly, W. S. (1978) Biochemistry, 17, 385- 391. 9. Hultberg, B. & Ockerman, P. A. (1972) Clin. Chim. Acta, 39, 49 - 58. 10. Plapp, B. U. & Cole, D. (1967) Biochemistry, 12, 3676-3681. 11. Swank, R. T. & Paigen, K. (1973) J . Mol. Biol. 77, 371 -389. 12. Keller, K. R. & Touster, 0. (1975) J . Biol. Chem. 250, 47654769. 13. Lin, Ch.-W., Orcutt, L. M. & Fishman, W. H. (1975) J . Biol. Chem. 250,4737-4743. 14. Stahl, P. D. & Touster, 0. (1971) J . Biol. Chem. 246, 53985406. 15. Potier, M. & Gianetto, R. (1973) Can. J . Biochem. 51, 973979. 16. Mameli, L., Potier, M. & Gianetto, R. (1972) Biochem. Biophys. Res. Commun. 46, 560-563. 17. Lucier, G. W. & McDaniel, 0. S. (1972) Biochim. Biophys. Acta, 261, 168 - 176. 18. Fishman, W. H., Goldman, S. S. & DeLellis, R. (1967) Nature (Lond.) 213,457 - 460. 19. Delvin, E. & Gianetto, R. (1970) Biochim. Biophys. Acta, 220, 93 - 100.

20. Himeno, M., Nishimura, Y..Tsuji, H. & Kato, K. (1976) Eur. J . Biochem. 70, 349-359. 21. Jungalwala, F. B. & Robins, E. (1968) J . Biol. Chem. 243, 4258 -4266. 22. Tulsiani, D. R. P., Keller, R. K. & Touster, 0. (1975) J. Biol. Chem. 250,4770-4776. 23. Tulsiani, D. R. P., Six, H. Sr Touster, 0. (1978) Proc. Nut1 Acad. Sci. U.S.A. 75, 3080-3084. 24. Dodgson, K. S., Lewis, J. I. M. & Spencer, B. (1953) Biochem. J . 55,253 - 259. 25. Kinoshita, N. & Gelboin, H. V. (1978) Science (Wash. D.C.) 199, 307-309. 25a. Diez, T. & Cabezas, J. A. (1976) 12" Journies Biochimiques Latines, abstract A23, Bordeaux, France. 25b. Diez, T. & Cabezas, J. A. (1977) 11th FEBS Meet. C-5-351819. 26. Cabezas, J. A. & Vazquez-Pernas, R. (1969) Rev. ESP. Fisiol. 25,147-152. 27. Reglero, A. & Cabezas, J. A. (1976) Eur. J. Biochem. 66, 379387. 28. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J . (1951) J . Biol. Chem. 193,265-275. 29. Vesterberg, 0. & Svenson, H. (1966) Acta Chem. Scand. 20, 820-934. 30. Ornstein, L. (1964) Ann. N. Y. Acad. Sci.121, 321 - 349. 31. Davis, B. J. (1964) Ann. N. Y . Acad. Sci. 121, 404-427. 32. Weber, K. & Osborn, H. (1969) J . Biol. Chem. 244, 44064412. 33. Porto, E. & Cabezas, J. A. (1970) Rev. ESP. Fisiol. 26, 335338. 34. Dean, R. T. (1974) Biochem. J . 138, 395-405. 35. Weber, K., Pringle, I. R. tk Osborn, M. (1972) Methods Enzymol. 26, 3 - 27. 36. Sussmann, A. R. (1975) Guide to Basic Applications of the Preparative UV Scanner, Beckman reprint BIR 75-4. 37. Sweeley, C. C., Bentley, R., Makita, M. & Weells, W. W. (1963) J . Am. Chem. Soc. 85, 2497 - 2705. 38. Reglero, A. (1971) Thesis for Graduation, Fac. of Sciences, Univ. of Salamanca. 39. Winzler, R. J. (1955) Biochem. Anal. 2, 279-301. 40. Svennerholm, L. (1957) Biochim. Biophys. Acta, 24, 604-611. 41. Miettinen, T. & Takki-Luukainen, I.-T. (1959) Acta Chem. Scand. 13, 856- 858. 42. Warren, L. (19551) J . Biol. Chem. 234, 1971- 1975. 43. Aminoff, D. (1961) Biochem. J . 81, 384-392. 44. Moore, S. & Stein, W. H. (1963) Methods Enzymol. 6 , 819831. 45. Lineweaver, H. & Burk, D. (1934) J . Am. Chem. SOC.56, 658 - 666. 46. Penase, L. (1974) Les Enzymes: Cinitique er Micanisme d'Action pp. 25, 75, 77, Masson et Cie., Paris. 47. Trevelyan, W. E., Procter, D. P. & Harrison, J. S. (1950) Nature (Lond.) 166, 444-445. 48. Dische, 2. (1947) J . Biol. Chem. 267, 189-196. 49. Dixon, M. (1953) Biochem. J . 55, 170-171. 50. Cabezas, J. A. & Reglero, A. (1977) in Avances de la Bioyuimica (Cornudella, L., Oro, J., Heredia, C. F. & Sols, A., eds) pp. 145- 155, Salvat, Barcelona. 51. PCrez, N. & Cabezas, J. A. (1977) Biochimie, 59,729-733. 52. Goldstone, A. & Koenig, N. (1973) Biochem. J. 132, 267-282. 53. Levvy, G. A. & Snaith, S. M. (1972) Adv. Enzymol. 36, 151181.

T. Diez and J. A. Cabezas, Departamento de Bioquimica, Facultades de Ciencias y Farmacia, Universidad de Salamanca, Salamanca, Spain