Vol.

THE JOURNAL 246, No. 17, Issue

OF Bm~ocncnr, CHEMISTRY of September 10, pp. 5398-5406, Printed in U.S.A.

1071

P-Glucuronidase
PURIFICATION,

of Rat Liver
SUBUNITS*

Lysosomes
(Received for publication, February 12, 1971)

PROPERTIES,

PHILIP D. STAHL~ AND OSCARTOUSTER

From the Department of Molecular Biology, Vanderbilt Uwiversity, Nashville, Tennessee 3?2?03

SUMMARY Previous work has established that rat liver contains several closely related forms of &glucuronidase differing in their subcellular distribution. The present report focuses on the most abundant form, which is the major one occurring in lysosomes. This /3-glucuronidasewas purified 8,400-fold, to the highest specific activity thus far reported. The preparation is homogeneousby all criteria applied. It contains a relatively high content of glutamic and aspartic acids and a very low content of sulfur-containing amino acids. The enzyme has a molecular weight of approximately 280,000, the value obtained depending upon the method used for the determination. Since gel filtration in the presence of urea, or polyacrylamide gel electrophoresis under denaturing conditions, yielded results suggesting that the enzyme consists of subunits of molecular weight of approximately 75,000, the native enzyme apparently is a tetramer.

@-Glucuronidase (EC 3.2.1.31) is a unique liver enzyme in that it occurs usually in appreciable activity in both the lysosomes and in the endoplasmic reticulum (1). It may therefore be of particular value in an investigation of the biosynthesis of lysosomal enzymes. The process by which the lysosomal enzymes are finally packaged into the organelles in which they are found is a subject of considerable contemporary interest (2). A common assumption, based largely on recent cytochemical observations, is that precursor proteins of many lysosomal enzymes are glycosylated, perhaps through the mediation of the Golgi system, before they take their final form(s) as found in the lysosomes (3). The strongest evidence that P-glucuronidase is a glycoprotein was contributed by Plapp and Cole (4, 5), who isolated multiple forms from bovine liver and found them to differ in glucosamine, but not amino acid, content. Ganschow and Paigen (6), in their pioneering genetic studies of fi-glucuronidase distribution in mouse tissues, concluded from K, values, thermal inactivation curves, and polyacrylamide gel electrophoresis that microsomal and lysosomal ,&glucuronidases of liver are identical. Subsequently, however, small differences * This investigation was supported in part by Grant GB-7425X from the National Science Foundation. $ Fellow of the Arthritis Foundation. Present address, Department of Physiology and Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.

in electrophoretic mobility were observed for the two enzymes (7, 8). In the interesting study of Kato et al. (9) on the gonadotropin-enhanced incorporation of [l%]glucosamine and [YZ]leutine into mouse kidney P-glucuronidase, the authors described results suggesting that the renal enzymes are glycoproteins and that the newly synthesized enzyme is transported from the endoplasmic reticulum to the lysosomes. However, the enzyme fractions studied were not highly purified. Similarly, Van Lancker and Lentz (10) concluded, from the incorporation of labeled amino acid into the P-glucuronidase of regenerating liver of hypoxic rats, that the microsomal enzyme is formed first and then is transferred into lysosomex. It is difficult to assess the degree of purification achieved for the two enzymes, which were stated to have identical catalytic and electrophoretic properties. On the other hand, Mills, Paul, and Smith (11) had earlier observed three pH optima for partially purified ox liver P-glucuronidase, and Moore and Lee (12) had found evidence, in a study of rat liver soluble proteins, for multiple @-glucuronidases which could be resolved chromatographically. In 1965, Sadahiro, Takanishi, and Kawada (13) reported that rat liver contains three P-glucuronidases separable by chromatography, one associated with the microsomal fraction, one with the mitochondrial fraction (which contained lysosomes), and the third with the cell supernatant fraction. The three enzyme preparations behaved identically toward various inhibitors, but they exhibited different thermal stabilities. Shortly thereafter, similar findings were reported from two other laboratories (14-16). After completion of the present study, Delvin and Gianetto (17) reported t,hat two forms of the enzyme occur in rat liver lysosomes. It is evident that biosynthetic and related studies of @-glucuronidase would rest on firmer foundations and provide deeper insights if pure preparations were employed, each form of the enzyme being obtained from purified subcellular fractions. Moreover, the specific chemical interrelationships of the forms can be disclosed only by structural studies on the pure forms of the enzymes. In the present report we present a procedure for purifying from rat liver the principal lysosomal form of the enzyme to a state of apparent homogeneity, and evidence that this enzyme is an oligomer.
EXPERIMENTAL PROCEDURE

Materials Male Wistar rats were purchased from Camm Research Institute, Wayne, New Jersey or Harlan Industries, Inc., Cumberland, Indiana. Chemicals were obtained through the following

Issue of September

10, 1971

P. D. Stahl

and 0. Touster

5399

sources: phenolphthalein mono-fl-glucuronide, o-nitrophenyl-pD-galactopyranoside, Tris, poly-D-lysine (mol wt 175,000), methylated serum albumin, naphthol-AS-BI-/-glucuronic acid, fast garnet GBC, and DEAE-cellulose (coarse grade) were from Sigma; ferritin was from General Biochemicals; ammonium sulfate, sodium dodecyl sulfate, bovine serum albumin, ovalbumin, apoferritin, and chymotrypsinogen were from Mani?; Whatman DEAE-cellulose (DE 32), and Whatman Chl-cellulose1 (CM 32) were from H. Reeve Angel and Company, Inc., Clifton, New Jersey; Sepharose 4B, Sephadex G-150, and Sephadex G-200 were from Pharmacia; Ampholine u-as from LKB Instruments, Inc., Rockville, Maryland; electrophoresis reagents were from Canal Industrial Company, Rockville, Maryland; and Escherichia coli P-galactosidase (320 units per mg), catalase (3330 units per mg), yeast alcohol dehydrogenase (320 units per mg), and E. coli alkaline phosphatase (350 units per mg) were from Worthington. All other chemicals were reagent grade from various commercial sources. Methods Enzyme Assays-The standard assay used for P-glucuronidase was that of Talalay, Fishman, and Huggins (18) modified as follows: 0.001 M phenolphthalein /!-glucuronide was incubated in 0.05 1~ sodium acetate buffer, pH 5.0, in a total volume of 1.0 ml, After incubation usually containing about 0.1 unit of enzyme. at 37 for 15 min, the reaction was stopped with 3.0 ml of a solution containing 0.133 M glycine, 0.067 M NaCl, and 0.083 M Na2C03 (19). Yeast alcohol dehydrogenase was assayed by a minor modification of the method of Vallee and Hoch (20). The following enzymes were assayed as described in the references given: ,&galactosidase (al), alkaline phosphatase (22), and catalase (23). Chloride was determined by the method of Schales and Schales (24), and protein by the method of Miller (25) with bovine serum albumin as a standard. Ferritin was estimated by measuring its absorption either at 527 nm or at 360 nm. One unit of /%glucuronidase is the activity that catalyzes the release of 1 pmole of phenolphthalein per hour from phenolphthalein glucuronide under standard assay conditions. Chromatography and Gel Filtration-All adsorbents were treated before use by methods described in the brochure (Form lL2) provided by the Reeve Angel Company. Gel filtration was performed in the cold room according to the directions (technical data sheet No. 6) provided by Pharmacia. Chromatographic fractions were concentrated with an Amicon ultrafiltration cell equipped with an XM-50 membrane. Electrofocusing was performed on the LKB 7100 column. Amino Acid Analysis-The enzyme samples were dialyzed against distilled water, lyophilized, and hydrolyzed under vacuum in 6 N HCl for the times indicated. Amino acid analyses were made on a Beckman model 120C amino acid analyzer equipped with a Beckman automatic sample injector and sample programmer and an Infotronics (model CRS-12,413) digital readout system. Preparative Gel Electrophoresis-Electrophoresis was performed on a Canalco prep-disc preparative electrophoresis unit with a PD 2/230 column. The gel was made about 1 to 2 cm longer than needed and was cut off to the desired length to reduce holdup of the eluting proteins at the gel surface (26). This was 1 The abbreviations used are: CM-cellulose, carboxymethylcellulose; SDS, sodium dodecyl sulfate; BSA, bovine serum albumin.

accomplished by placing a size 00 rubber stopper over the elution tube and a plastic cap (outer diameter 2.5 cm) over the stopper. Saran Wrap was then placed around both of these objects and fixed around the column with several rubber bands. The acrylamide monomer was pumped (with a Buchler polystaltic pump) into the column filling the space around the rubber stopper. The column was then filled to the desired height with monomer, which was allowed to gel. The Saran Wrap was removed and the gel was cut at the base of the column with a fine wire (diameter 0.005 inches), after which the stopper and cap filled with gel The enzyme was eluted from the cut gel in half were removed. the time when compared to an uncut gel. The gel system used was that described by Davis (27). Analytical Gel Electrophoresis-Polyacrylamide gels (5%) were prepared and run by the following methods at room temperature. The pH and current per gel are indicated: (a) pH 9.5 at 2 ma per gel, method of Davis (27) ; (b) pH 7.0 at 3 ma per gel, method of Igarashi and Hollander (28) ; (c) pH 4.3 at 2 ma per gel, method of Reisfeld, Lewis, and Williams (29); and (d) pH 2.0 at 4 ma per gel, method of Takayama et al. (30). The acid gels c and d were subjected to preliminary electrophoresis before use. This was accomplished at low current, 0.5 In the case of c, preliminary electo 1.0 ma per gel, overnight. trophoresis was performed before placing the stacking gel into position to avoid mixing of the separate buffer systems therein; d is a continuous system and does not utilize the stacking gel. Furthermore, the system of Takayama et al., d, unlike a, b, and c, is highly dissociative, having 5 M urea in the gels and 6 M urea in the sample. All analytical electrophoresis was performed in a Canalco g-place apparatus or a Buchler la-place apparatus. Unless otherwise stated, gels were stained for protein by the method of Chrambach et al. (31) with Coomassie brilliant blue R250. @Glucuronidase activity in gels was detected by subjecting the gel to preliminary incubation in a solution (5 ml per gel) of cold 0.0077, naphthol-AS-BI-glucuronic acid in 0.05 M acetate, pH 5.0. The substrate (naphthol-AS-BI-glucuronic acid) was first dissolved in a small volume of acetone-methanol (1: 1) before diluting with acetate buffer. After 3 hours in the cold room, the gels were transferred to fresh cold buffered substrate solution, which contained in addition 0.5 mg per ml of fast garnet GBC. On storage in the cold, the bands corresponding to enzymatic activity usually appeared in 3 to 6 hours when 100 to 200 milliunits of enzyme were used. (Much more rapid development of the bands occurs at room temperature.) The migration of standard proteins on gels was estimated with a vernier caliper. Molecular IV eight DeterminationsSucrose gradients (5 to 20 %) were prepared and used according to the method of Martin and Ames (32). Sepharose 4B columns (2.5 X 80 cm) for molecular weight determinations were used as described by Marrink and Gruber (33), except that 0.005 M Tris-chloride, pH 7.5, containing 0.1 M NaCl was used as buffer. Molecular weight determinations by electrophoresis in polyacrylamide gels were performed according to the method of Hedrick and Smith (34), except t,hat the buffer system of Davis (27) was used. Hedrick and Smith indicated that this system is as dependable as the one they described. Subunit molecular weight was determined by SDS gel electrophoresis as described by Shapiro, Vifiuela and Maize1 (35) and by chromatography on a Sephadex G-150 column (2.5 x 80 cm) equilibrated with 0.05 M sodium acetat,e, pH 5.0, containing 8 1\1nrea.

5400
RESULTS

Lysosomal

P-Glucuronidase

Vol.

246, No.

17

Purifxation

of p-Glucuronidase

Step I. Preparation of Extract for Chromatography-Sixty fasted male Wistar rats were killed by decapitation. The livers All were removed, cooled, and passed through a meat grinder. subsequent operations were carried out at 5. From the resulting slurry, a homogenate was prepared in 0.25 M sucrose (3 ml per g of tissue) with one stroke of a loose fitting Potter-Elvehjem homogenizer. The homogenate was centrifuged at 2,500 rpm in a Sorvall GSA rotor for 10 min (1 x lo4 x g min). The pellet was twice suspended in 3 volumes of 0.25 M sucrose, rehomogenized, and recentrifuged. The pooled supernatant fractions from all three centrifugations constituted the cytoplasmic extract. From this extract a mitochondrial-lysosomal (ML) pellet was prepared by centrifugation at 8,500 rpm for 40 min (4.7 x lo5 x g.min). The ML fraction (containing about 40% of the total ,&glucuronidase) was suspended with a loose fitting Dounce homogenizer in 0.005 M Tris-phosphate, pH 7.8 (5 ml per g of liver), and dialyzed overnight against the same buffer. The dialyzed suspension was centrifuged in a Spinco L-19 rotor at 19,000 rpm for 3 hours (6 X lo6 X g.min). To the supernatant solution (ML extract), which contained about 90% of the enzymatic activity of the ML fraction, was added solid ammonium sulfate to a final concentration of 30% at 5. (The pH of this solution was 7.0.) After 20 min, the solution was clarified by centrifugation at 10,000 rpm for 20 min (2 x lo5 x g .min). The solution was then adjusted to pH 6.0 with 0.1 N HzS04 and brought to 70% saturation by adding solid ammonium sulfate over a period of 20 min. After a further 20 min, the solution was centrifuged to yield a precipitate which contained all of the enzymatic activity. The pellet was dissolved in a minimum amount of 0.005 M Tris-phosphate, pH 7.8, and dialyzed exhaustively against the same buffer. The yield from the ammonium sulfate fractionation was usually about 90%. However, in some preparations, such as the one presented here, the yield was as low as 70%. Step II. DEAE-cellulose Chromatography-The dialyzed ammonium sulfate product, was applied to a DEAE-cellulose column (Sigma coarse grade) (Fig. 1). The column was washed with

80

120 160 FRACTION

200

240

FIG. 1. DEAE-cellulose chromatography of P-glucuronidase. Elution pattern of fi-glucuronidase (10,650 units, 2,695 mg) from a DEAE-cellulose column (3 X 100 cm). The column was washed sequentially with 0.10 M and 0.25 M N$CI in 0.005 M Tris-phosphate buffer, pH 7.8. The flow rate was 120 to 300 ml per hour. Fractions were 20 ml. The enzyme eluted with 0.25 M NaCl was pooled, dialyzed, and used in the next step of purification.

1.5 liters of buffer containing 0.1 M NaCl, which eluted a large protein peak and a small but variable amount of enzyme (3 to 20% of the activity applied). The bulk of the enzyme was then eluted from the column with 0.25 M NaCl in buffer. The pooled product, usually 75 to 99% of the activity applied and representing a a-fold increase in specific activity, was dialyzed against the starting buffer (0.005 M Tris-phosphate, pH 7.8) and applied to a second DEAE-cellulose column (Whatman DE 32, 2.5 X 40 cm), which was eluted with a linear salt gradient (500 ml of starting buffer and 500 ml of 0.4 M NaCl in this buffer) at 50 ml per hour. The product was obtained in a single peak in high yield (80 to 92y0), with a 1.5. to 2-fold increase in specific activity. The enzyme was pooled and dialyzed against 0.05 M acetate buffer, pH 5.6. A precipitate, which contained a little enzymatic activity, formed during dialysis; this precipitate was removed by centrifugation. Dialysis against acetate should be executed as quickly as possible (preferably less than 6 hours), since the dialyzed enzyme is less stable at pH 5.6 than at 7.8. Step III. CM-cellulose Chromatography-The dialyzed product from the DEAE-cellulose step was applied to a column (2.5 X 40 cm) of CM-cellulose (Whatman CM 32), which was equilibrated with 0.05 M sodium acetate, pH 5.6. The column was developed with a linear salt gradient (500 ml of the above buffer and 500 ml of 0.3 M NaCl in this buffer) at a rate of 50 ml per hour. The enzyme was eluted, as a single symmetrical peak, at about 0.04 M NaCl, between two large protein peaks. The yield of enzyme ranged from 50 to 80%; the specific activity was increased 6- to i-fold. Step IV. Gel Filtration-The pooled fractions from CM-cellulose chromatography were immediately dialyzed against a buffer that was 0.005 M in Tris-Cl, pH 8.0, and 0.005 M in NaCl. The sample was concentrated to about 5 ml with an Amicon ultrafiltration cell and subjected to Sephadex G-200 chromatography. Two columns (2.5 x 90 cm) obtained from Pharmacia, and fitted with flow adapters, were eluted in series with reverse flow at 10 ml per hour. The enzyme was eluted as a symmetrical peak just after the void volume but before the bulk of the protein. This step routinely provides a 2- to a-fold increase in specific activity with an 80 to 90% yield. Step V. Preparative Electrophoresis-The pooled product from Sephadex G-200 chromatography was concentrated to about 5 ml and further purified witha Canalco prep-disc electrophoresis unit. A slight modification was made in the preparation of the gels as suggested by Schenkein, Levy, and Weis (26) and described under Methods. The sample was made 10%7, with respect to sucrose, and 20 ~1 of 0.04% bromphenol blue was then added. A 5-cm 5a/, separating gel and l-cm stacking gel were used in a PD 2/230 column. After the sample was layered onto the stacking gel, a 7- to g-ma current was applied and elution was begun. The enzyme was routinely eluted in 20 to 25 hours with IO-min samples and 30 to 50 ml per hour elution rate (Fig. 2). The pooled product was concentrated and subjected to analytical electrophoresis by the method of Takayama et al. (30) as a check for purity. Routinely, the gel, when stained for protein, revealed one major band and several minor bands. The enzyme was then subjected to electrophoresis again through a 1.5-cm 7% gel (1.0 cm stacking gel) in the manner just described. The product from this step, usually eluting at 20 hours, had a specific activity of approximately 3000. If assayed in the presence of poly-n-lysine (1 mg per ml), a specific activity of about 4400 was usually obtained. (With 100 ,ug of

Issue of September

10, 1971

P. D. Stahl and 0. Touster

5401

60

80

100 120 140 160 180 200 220 240

FRACTION
FIG. 2. Preparative polyacrylamide gel electrophoresis of pglucuronidase. The enzyme (4800 units, 14.3 mg) dissolved in 10% sucrose (5.0 ml) containing 20 ~1 of 0.04% bromphenol blue, was subjected to electrophoresis on a column (5 cm) of 5yo acrylamide gel as described under Methods and Results. TABLE

Purification
Fraction

of (3-glucuronidase
Protein

I from rat liver lysosomes

EllZyIW activity Yield

-Pll%cation ratio

Homogenate Mitochondrial-lysosomal (ML) fraction.. ML extract.. Ammonium sulfate. Sigma DEAE-cellulose Whatman DE-32.
What,man CM-cellulose.. w 130,000 16,500 9,660 2,695 708 458 40.5 14.3 1.63 0.976 0.976

units ti mits/ml
46,500 18,200 16,000 10,650 10,550 9,670 6,000 4,800 3,940 2,950 4,250 0.36 1.10 1.65 3.95 14.9 21.1 148 336 2,400 3,024 4,360

__ r %
100 39 34 23 23 21 13 10 8 6

1 3 5 11 41 59 410 933 ) 660 ,400

Sephades G-200. 5% gel. 7% gel Minus polylysine.. Plus polylysinea..

FIG. 3. Analytical polyacrylamide gel electrophoresis of purified b-glucuronidase. The enzyme (4 pg) was subjected to electrophoresis by the following methods: A, pH 9.5, Davis (27) ; B, pH 7.1, Igarashi and Hollander (28); C, pH 4.3, Reisfeld et al. (29); and D, pH 2.0, Takayama et al. (30). For A and B, electrophoresis was t.oward the anode; for C and D, electrophoresis was toward the cathode. Gels were stained for prot.ein by the method of Chrambath et al. (31).

a Assayed with 1 mg per ml of poly-o-lysine. polylysine per ml, the specific activity was about yield from 5% and 7% gels varied from 75 to 95%. was much reduced when less pure preparations were electrophoresis. The entire purification scheme is in Table I, which contains the results of a typical experiment. Properties of Purified 3900.) The The yield subjected to summarized preparative

Lysosomal P-Glucuronidase

Behavior in Polyacrylamide Gel Electrophoresis-Purified /3glucuronidase was examined by analytical electrophoresis in gels of 5% acrylamide run under various conditions of buffer and pH. Fig. 3 shows the pattern of 4 pg of a /3-glucuronidase preparation analyzed by the following electrophoretic systems: Fig. 3A, Davis (27), pH 9.5; Fig. 3B, Igarashi and Hollander (28), pH 7.1; Fig. 3C, Reisfeld et al. (29), pH 4.3; and Fig. 30, Takayama et al. (30), pH 2.0. Another sample of enzyme, not shown here, which was subjected to electrophoresis by the method of Davis (pH 9.5) and stained for enzyme activity as described under Methods, revealed only a single band at the same position as the protein band. In split gel polyacrylamide electrophoresis (36), the purified enzyme and the activity in the 0.25 M salt eluate of the DEAE-cellulose column (Fig. 1) showed identical mobilities with 7% gels at pH 9.5 (27) and the stain for /!-glu-

curonidase. The first three systems (3A, 3B, 3C) revealed single bands when stained for protein by the method of (hrambach et al. (31). The fourth gel, prepared and run according t.o Takayama et al. (30), showed, in addition, a faint slower band. This system is highly dissociative, but the faint. band could represent a trace of undissociated oligomer. Effects of Enzyme Concentration end Time on Bctivity-With enzyme concentrations up to at least 0.06 kg per ml and under the standard assay conditions, phenolphthalein release was directly proportional to the enzyme concentration. With 0.024 pg of enzyme per ml, phenolphthalein release was linear for at least 30 min. E$ect of Substrate Concentration-The effect of substrate concentration on enzyme activity at pH 5.0 is seen in Fig. 4. At the lowest substrate concentration tested less than lOTo was hydrolyzed at 5 min. The reaction appears to obey normal Michaelis-Menten kinetics; the K, was 7.05 X 10d5 M (Fig. 4). Effect of pH-@-Glucuronidase from various sources has been shown to display double pH optima (37, 38). However, it was shown that, with partially purified lysosomal ,&glucuronidase, only a single optimum is obtained in the presence of high salt concentration (37). Our purified lysosomal @glucuronidase also displays dual pH optima (Fig. 5) when assayed withphenolphthalein glucuronide. The peaks are around pH 4.4 and 4.9 with a trough at 4.6. The dual pH optima are not observed in the presence of high salt concentration, or if the assays are run with lower substrate concentration (lOA M) as shown in Fig. 5. With

5402
this substrate concentration the pH optimum

Lysosomal
is approximately

/%Glucuronidase

Vol. 246, No. 17

4.3. Activation by Polycations-The purified fi-glucuronidase described in the present paper is activated about 25% by polylysine

. -I>

[iI I 0.2 I 0.4

(mM-'1 I 0.8 I 1.0

I 0.6 [S](mM)

FIQ. 4. Effect of substrate concentration on p-glucuronidase activity. Purified enzyme (0.075 rg) was assayed in 0.05 M acetate buffer, pH 5.0, with substrate concentrations ranging from 5 X KY6 M to 1 X 1C3 M. For each substrate concentration assays were made at 1-min intervals for 5 min to ensure that initial velocities were being determined.

and methylated serum albumin when these substances are added directly to the standard assay mixture (see Table I). They appear to be equally effective on a weight basis. Bernfeld (39) has described the activation of P-glucuronidase and several other enzymes by polycations and has proposed that activators promote subunit association. Stability-Purified enzyme (9 units per ml) was dialyzed overnight against 0.05 M sodium acetate buffer, pH 5.0, or 0.05 M Tris-Cl, pH 8.0. The samples were then maintained at cold room temperature and were assayed for /?-glucuronidase activity at various intervals. Fig. 6 shows that enzyme maintained in Tris buffer at pH 8.0 lost about 407, of its activity over 10 days of storage, but that it appears to be completely reactivated if 30 pg per ml of poly-n-lysine is included in the incubation mixture. On the contrary, enzyme stored at pH 5.0 in acetate buffer lost activity rapidly and could not be reactivated by polylysine. The enzyme was routinely stored at 5 in 0.005 M Tris-Cl, pH 8.0, at a concentration of 90 units per ml. At this concentration, there was little loss of activity upon prolonged storage. Isoelectric Point-Purified fl-glucuronidase was subjected to isoelectric focusing on an LKB model 7100 column. A pH gradient from 5 to 8 was induced across the column. The enzyme migrated to a position corresponding to pH 5.8. Amino Acid Composition-Amiilo acid analyses were performed on two preparations of purified enzyme (Table II). The enzyme has a high concentration of aspartic and glutamic acids and only a trace of cysteine. The enzyme is a glycoprotein, yielding 0.72% hexosamine after 24 hours of acid hydrolysis. Since hexosamines in glycoproteins are degraded during long acid hydrolysis (40), it is very likely that the hexosamine content is conFurther work is in progress on siderably higher than this value. the carbohydrate components of the enzyme. Molecular Weight of Oligomer

Sucrose Gradient Centhfugation-fl-Glucuronidase, @-galactosidase (mol wt 520,000), catalase (mol wt 250,000), and yeast alcohol dehydrogenase (mol wt 150,000), in 0.1 ml of 0.05 M

/.

\
t POLYLYSINE

I I 12345678

t POLYLYSINE ------0 2 1 1 1 9 lb

4
FIQ. 5. Effect

DAYS AT 4'C FIG. 6. Effect of pH on the stability of purified&glucuronidase. Enzyme (3.0 pg per ml) was dialyzed overnight against 0.05 M sodium acetate, pH 5.0, or 0.05 M Tris-Cl, pH 8.0, and the activity was then followed for 9 additional days by standard assay. On the last day, the enzymatic activity was determined by standard assay in the presence, and absence, of 30 pg of poly-n-lysine per ml.

PH
of pH on p-glucuronidase activity. Purified enzyme (0.037 pg) was assayed under standard conditions except that the pH was varied from 4.0 to 6.0. The substrate concentrations were 10-3 M (A) and 10- M (B). The enzyme activity is expressed as nanomoles of phenolphthalein released in 15 min.

Issue of September

10, 1971
TABLE

P. D. Stahl II
from
rat liver lysosomes

and 0. Touster 8-

5403

Amino

acid

analysis

of

p-glucuronidase

-iAmino acid

Preparation IIb ~tnole/mg /.cmole/mg

7P-GALACTOSIDASE

" w 3 %
A 2 Y 0

65-

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

0.199 0.157 0.237 0.541 0.317 0.303 0.507 0.341 0.366 0.231 Trace 0.391 0.055 0.176 0.493 0.169 0.172 0.040

0.200 0.111 0.232 0.595 0.322 0.288 0.543 0.287 0.402 0.264 Trace 0.417 0.054 0.223 0.487 0.212 0.256 0.010

TOTAL \ 4

VOL

31 100

I I 300 400 200 ELUTION VOLUME (ML.)

Hexosaminec ..............

Q This sample was hydrolyzed for 24 hours in 6 N HCl. b This sa.mple was hydrolyzed for 72 hours in 6 N HCI. c The limited quantity of enzyme available precluded carrying out hydrolyses for various times in order to obtain more accurate data on hexosamine content (see Results).

FIG. 8. Molecular weight of p-glucuronidase by Sepharose 4B chromatography. The following were chromatographed on a column (2.5 X 80 cm) of Sepharose 4B (0.005 M Tris-Cl, pH 7.5, in 0.1 M NaCl) : 500 pg of Escherichia coli alkaline phosphatase (mol wt 80,000), 2.5 mg of yeast alcohol dehydrogenase (mol wt 150,000), 300 rg of catalase (mol wt 250,000), 14 units of p-glucuronidase, 10 mg of ferritin (mol wt 480,000), and 500 pg of p-galactosidase (mol wt 520,000) in 0.5 ml. Void volume and total volume were determined with dextran blue and potassium ferricyanide, respectively. Fractions of 1 ml were collected. The elution volumes of the standard proteins were plotted against the log molecular weight, the line being calculated by the method of least squares (41).

/3-GALACTOSIDASE P-GLUCURONIDASE

BOTTOM

FRACTION
FIG. 7. Molecular weight of P-glucuronidase by sucrose density gradient centrifugation according to Martin and Ames (32). The following were layered on to a 5 to 20% gradient: 1.3 units of figlucuronidase, 508 units of P-galactosidase (mol wt 520,000), 608 units of catalase (mol wt 250,000), and 2,508 units of yeast alcohol dehydrogenase (mol wt 150,000) in 0.1 ml of 0.05 M Tris-Cl, pH 7.5. After centrifugation for 6 hours at 39,000 rpm in an SW 39 rotor, a-drop fractions were collected, enzyme concentrations were estimated, and sucrose densities were determined by refractometry.

Tris-Cl, pH 7.5, were layered on a sucrose gradient prepared by the method of Martin and Ames (32). Sedimentation of p-glucuronidase through the gradient gave a single peak (Fig. 7). From the sedimentation of each of the three marker enzymes through the gradient a value for the molecular weight of ,&glueuronidase was calculated with the equation of Martin and Ames. The average value for fl-glucuronidase, with values from all three

markers, was 313,000. However, the use of catalase only as a marker yielded a molecular weight of 265,000 for P-glucuronidase. Martin and Ames had in fact found that the use of catalase as the reference protein gave molecular weights 84 to 87% of those calculated with yeast alcohol dehydrogenase as reference. Gel Filtration on Xepharose 4B-/%Glucuronidase, E. coli phosphatase, yeast alcohol dehydrogenase, catalase, ferritia, and /3-galactosidase, in 0.5 ml of the buffered salt solution used for elution, were applied to a Sepharose 413 column (2.5 x 80 cm) which was eluted with reverse flow with 0.005 M Tris-Cl, pH 7.5, containing 0.1 M NaCl (collected at a rate of 8 ml per hour in l-ml fractions). fi-Glucuronidase was eluted as a single peak between catalase and ferritin. When elution volume is plotted against log molecular weight with the method of least squares (41), a line is obtained as shown in Fig. 8. The elution volume (262 ml) for P-glucuronidase corresponds to a molecular weight of 270,000. Polyacrylamide Gel Electrophoresis-A modification of the method of Hedrick and Smith (34) was employed in estimating the migration of P-glucuronidase and standard proteins on gels of varying acrylamide concentrations. The Tris-glycine buffer system and gel composition of Davis (27) were used in place of that described by Hedrick and Smith (34). Bovine serum albumin, ovalbumin, apoferritin, and P-glucuronidase were subjected to electrophoresis on gels the acrylamide concentration of which ranged from 4 to 12%. A slope was estimated for each standard from a graph relating its migration to acrylamide concentration (legend of Fig. 9). A plot of the slopes, by the method of least squares (41), for the standard proteins against their molecular weights gave the line shown in Fig. 9. The slope obtained for P-glucuronidase (15.8) corresponds to a molecular weight of 280,000.

Lysosomal

$-Glucuronidase

Vol. 246, No. 17

2o

BSA; \
l -

5 w
3

BSA2.

30

p-GLUCURONIDASE /

P :
2E

IO8-

OVALBUMIN2

.BSA3

e 2
z5

7643-

\+-GLUCURONIDASE BSA; OVALBUMINl

\

= Y d z SLOPE
FIQ. 9. Molecular weight of p-glucuronidase by polyacrylamide electroDhoresis. Twentv micrograms each of BSA (mol wt 68,000); ovalbumin (mdl wt 45;000), and apoferritin (mol wt 480,000), and 4 pg of &glucuronidase were subjected to electrophoresis on polyacrylamide gels of 4 to 12yo acrylamide according to Hedrick and Smith (34) (see Results). The gels were stained for protein (30), and the migration of each standard was expressed as a ratio (ZL,) to the migration of the tracking dye, bromphenol blue. (The gels were cut at the leading edge of the tracking dye before staining.) A slope was determined for each standard by plotting migration, 100 log (R, X lOO), against acrylamide concentration (34). The slopes for the standards, including the dimer (BSAZ) and trimer (BSA,) of bovine serum albumin, were plotted against the molecular weights of the standards, the line being calculated by the method of least squares (41).

CHYMOTRYPSINOGEN

0,2

0,4 Rc

0.6

0.8

I,0

Molecular

Weight of Subunits

Gel Filtration on Sephadex G-i50-P-Glucuronidase is inactivated about 80% when assayed in 5 M urea at pH 5.0. When enzyme in 5 M urea (0.05 M acetate buffer, pH 5.0) is dialyzed overnight against 20 volumes of 0.05 M Tris-Cl, pH 8.0, to remove the urea, 95 to 100% of the activity is recovered. A solution of 30 units of fl-glucuronidase containing bovine serum albumin, E. coli alkaline phosphatase, and cytochrome c were subjected to chromatography on Sephadex G-150 in 0.05 M sodium acetate, pH 5.0, containing 8 M urea. The enzyme was assayed by diluting out the urea. Although a strictly linear relationship between elution volume of marker proteins and log molecular weight was not obtained, the fl-glucuronidase was routinely eluted by a volume equal to that required to elute bovine serum albumin, a result indicating a molecular weight of about 68,000 for the subunit of the enzyme. Electrophoresis in Presence of Sodium Dodecyl Sulfate-P-Glucuronidase, bovine serum albumin, ovalbumin, and chymotrypsinogen were subjected to electrophoresis by the method of Shapiro et al. (35) in gels of 5% acrylamide containing 0.1% SDS, after incubating the samples at 37 for 90 min in 1% SDS, 1% mercaptoethanol, and 0.1 M sodium phosphate buffer, pH 7.1. In some cases, 6 M urea was added to the incubation buffer, or the gels, or both. The migration of each protein was expressed as a ratio (R,) relative to the migration of chymotrypsinogen (36). Fig. 10 shows a plot of the R, of various standards against log molecular weight. /3-Glucuronidase, in the absence of urea, migrates as a single band corresponding to a molecular weight of 75,000. The presence of urea in the incubation mixture results in the appearance of two bands for p-glucuronidase, the

FIG. 10. Determination of subunit molecular weight of 8-glucuronidase by electrophoresis on polyacrylamide gels containing sodium dodecvl sulfate (35). Twentv microerams each of BSA (mol wt SS,OOOj,ovalbumin (mol wt 45,&M), and chymotrgpsinogen (mol wt 25$00); and 4 pg of &glucuro&da& were e&h in&bated in 1% SDS. 1% mercautoethanol. and 0.1 M sodium ahosDhate. DH 7:i, for 60 &in at $7. The samples (0.1 ml), td which 2 ;f of 0.04% bromphenol blue were added, were then carefully layered onto, and subjected to electrophoresis in, acrylamide gels (5%) containing 0.1% SDS and 0.1 M phosphate buffer, pH 7.1. The migration of standard proteins (including the dimers of ovalbumin and BSA and the trimer of BSA), expressed as a ratio (R,) relative to the migration of chymotrypsinogen (36), was plotted against log molecular weight. Gels stained for protein by a modification (36) of the method of Chrambach et al. (31) gave a single band for p-glucuronidase, which corresponds to a molecular weight of 78,000. When urea (6 M) was added to the incubation mixture, a second component (arrow) was observed, corresponding to a molecular weight of 155,000.

molecular weights of which correspond to 78,000 and 155,000, respectively. When urea was added to both incubation mixture and gel, only the heavier species was observed.
DISCUSSION

It is now evident that rat liver contains several forms of /3glucuronidase. Whereas Sadahiro et al. (13) present evidence suggesting that each of several subcellular fractions contains a different chromatographic form of the enzyme, the results of Delvin and Gianetto (17) indicate that lysosomes contain a minor form as well as a major form. Our own unpublished studies suggest that there is more than one form in both microsomes and lysosomes, as shown by chromatography and gel electrophoresis. For example, in the fractionation of whole liver with the Sigma DEAE-cellulose column employed in our standard preparative procedure (see Results), the enzyme eluted with 0.1 M salt was found to contain several isozymes separable by gel electrophoresis. This fraction appears to contain all the microsomal forms and at least one minor lysosomal form. The 0.25 M salt eluate however, the source of the enzyme purified in the present study,

Issue of September

10, 1971

P. D. Stahl and 0. Touster

5405

appears to consist only of the major lysosomal form. Details on the purification and characterization of the 0.11~ salt fraction will be the subject of a subsequent communication. There is an extensive literature on the purification of fl-glucuronidases from a wide variety of sources (1). The highest activity thus far reported is for the enzyme purified from the preputial (clitoral) gland of female rats; the specific activity given in the 1970 report of Ohtsuku and Wakabayashi (42) was almost as high as that given in the present paper. It is interesting that only a 19-fold purification was required to obtain electrophoretically and ultracentrifugally homogeneous ,&glucuronidase from this very rich source of the enzyme. Two crystalline preparations have been reported, one from beef liver (43) and the other from digestive juice of Helix pomatia (44) ; both were of relatively low specific activity. Highly purified bovine liver P-glucuronidase was reported by Plapp and Cole (4), whose enzyme had less than 20% of the specific activity of the rat liver lysosomal enzyme described in the present paper. Finally, the very recent report of Delvin and Gianetto (17) on rat liver lysosomal @-glucuronidase deserves comment. Although the specific activity of their product is much less (10%) than that produced by the procedure described herein, good evidence was presented for the presence of one major and one minor lysosomal fl-glucuronidase in rat liver. Delvin and Gianetto employ a trypsin treatment in their purification procedure. On the basis of their results, as well as on our own unpublished data, it appears that lysosomal P-glucuronidases are not affected by trypsin. However, we have preliminary data indicating that the microsomal P-glucuronidases present in the 0.1 M salt eluate (Fig. 1) are altered by trypsin in their behavior on DEAE-cellulose columns and in polyacrylamide gel electrophoresis. This difference between the microsomal and lysosomal enzymes may have relevance to the chemical nature of the forms found in different subcellular sites and is therefore under further investigation. The double pH optima displayed by pure lysosomal fi-gluWhen dual pH curonidase are abolished by high ionic strength. optima have been demonstrated, phenolphthalein glucuronide was used as substrate (37, 38). However, Levvy, McAllan, and Marsh (38), with a highly purified rat preputial gland P-glucuronidase, found double pH optima with phenolphthalein glucuronide and a single pH optimum with phenyl glucuronide. Similarly, H&u and Gianetto (45) found that partially purified rat liver lysosomal /3-glucuronidase exhibited a single pH optimum when tested with several thioglucuronides. The influence of substrate concentration was not studied in these reports. As shown in Fig. 5, with low concentrations of phenolphthalein glucuronide as substrate, only a single pH optimum is observed. Preliminary experiments in our laboratory2 employing a variety of substrates suggest that both the structure and concentration of substrate Further study of this probinfluence multiplicity of pH optima. lem is in progress. Amino acid analyses, performed on two different preparations of enzyme after 24 and 72 hours of hydrolysis, respectively, gave similar results. The amino acid content, in general, is similar to that obtained with P-glucuronidase from rat preputial gland (42), bovine liver (4), and human liver (46), even though the latter two had much lower specific activity. Because of the limited amounts of enzyme available, extensive studies on the carbohydrate composition could not be carried out. However, as ex2 C.-C. Wang and 0. Touster, unpublished observations.

petted, hexosamine was detected during the a.na,lysis for amino acids. Molecular weight determinations by the sucrose gradient centrifugation method of Martin and Ames (32) yielded values dependent on which marker enzyme was used as standard. The use of yeast alcohol dehydrogenase and E. coli @-galactosidase as molecular weight markers consistently gave values much higher (-350,000) for fl-glucuronidase than when catalase was used as a marker (~265,000). Martin and Ames found that catalase as a standard gives lower molecular weights for unknowns than does alcohol dehydrogenase, but higher values than lysozyme. Because the molecular weight estimations by Sepharose 4B and gel electrophoresis were 270,000 and 280,000, respectively, it appears reasonable to assume that the molecular weight of rat liver lysosomal /?-glucuronidase is about 280,000. This value is consistent with those reported for bovine liver (4), human liver (46), and rat preputial gland (42), and, as pointed out below, can be correlated with the subunit data. Because of its large size, it seemed probable that the enzyme was composed of subunits. The inactivation of P-glucuronidase by urea at pH 5.0, and its ability to be fully reactivated by dialysis, suggested dissociation of the enzyme into subunits. This notion was substantiated when the urea-inactivated enzyme was chromatographed on a column of Sephadex G-150 (eluted with 8 M urea, pH 5.0). Since the enzyme (assayed after dilution of the urea) was eluted in the same volume required for serum albumin, it seemed likely that the P-glucuronidase subunits have a molecular weight very close to that of BSA, namely, about 68,000. A more exact analysis of subunit molecular weight was achieved by electrophoresis on SDS gels by the method of Shapiro et al. (35). After incubation of enzyme and standards for 90 min at 37 in 1 y0 SDS and 1% mercaptoethanol, electrophoresis produced a single protein band which corresponded (Fig. 10) to a molecular weight of 75,000. When 6 M urea also was added to the incubation mixture to promote dissociation, electrophoresis produced two protein bands, one heavily and one lightly stained. The former corresponds to a molecular weight of 155,000 and the latter to 78,000. The presence of urea in both the incubation mixture and the gel resulted in the detection only of the larger species. This very surprising finding, that urea stabilizes a possible dimeric form of the enzyme at pH 7.1, is interesting in view of the dissociation produced by urea at pH 5.0, where a monomer was apparently produced. A similar effect of urea was observed by Jacobson and Pfuderer (47) for alcohol dehydrogenase. These data suggest a monomeric unit with a molecular weight of 68,000 to 78,000 and a dimer of about 155,000. On the basis of the molecular weight determinations on the undissociated enzyme, its oligomeric structure appears to be a tetramer. Whether or not each subunit contains carbohydrate, or identical carbohydrate chains, remains to be determined, and it will be of considerable interest to learn the chemical relationships of the various forms of P-glucuronidases to each other. In this regard, the recent work of Salnikow, Moore, and Stein (48) is quite relevant. The deoxyribonucleases of bovine pancreas, although of similar enzymatic activity, were found to differ in amino acid as well as carbohydrate content. Acknowledgments-We gratefully acknowledge the technical assistance of Mrs. Jane P. Griffin, as well as the help of Mr. Scott Orman in the preparation of enzyme. Special indebtedness is due Miss Lilah Clack for carrying out the amino acid analyses.

5406
We are also Dulaney for grateful to Dr. useful discussions Chi-Cheng during

Lysosomal
Wang and Dr. investigation. John T.

p-Glucuronidase
24. 25. 26. 27. 23. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

Vol. 246, No.

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this

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SCHALES, 0.. AND SCHALES. S. S.. J. Biol. Chem.. , 140. , 879 (1941): MILLER, G. L., Anal. Chem., 31,964 (1959). SCHENKEIN, I., LEVY; M., AND WEIS, P., Anal. Biochem., 26. 387 (1968). DAVIS, B. J., Ann. N. Y. Acad. Sci., 121,404 (1964). IGARASHI, M., AND HOLLANDER, V. P., J. Biol. Chem., 243, 6084 (1968). REISFELD, R. A., LEWIS, U. J., AND WILLIAMS, D. E., Nature, 196, 281 (1962). T.~K.~YAM.~ K:, MACLENNAN, D. H., TZAGOLOFF, A.. AND STONER, C. fi., Arch.. Biociem. Bidphys., 114, 223 (1966). CHRBMBACH, A., REISFELD. R. A.. WYCKOFF. M.. AND Zacc.4~1. J., Anal. kochem., 20, i50 (1967). MARTIN, R. G., AND AMES, B. N., J. Biol. Chem., 236, 1372 (1961). MARRINK, J., AND GRUBER, M., FEBS (Fed. EUT. Biochem. Sot.) Lett., 2, 242 (1969). HEDRICIC, J. L., AND SMITH, A. J., Arch. Biochem. Biophys., 126, 155 (1968). SHAPIRO, A. L., VIWUELA, E., AND MAIZEL, J. V., Biochem. Biowhus. Res. Commun.. 28. 815 (1967). DUNKED, A. K., AND RTJ&K&T, k. RI, J. Biol. Chem., 244, 5074 (1969). DELVIN. E.. AND GIANETTO, R., Can. J. Biochem.. I 46. _ 389 (1968j. LEVVY, G. A., MCALLAN, A., AND MARSH, C. A., Biochem. J., 69.22 (1958). BER~FEL~, P., in E. A. BALAZS AND R. W. JEANL~Z (Editors), The amino sugars, Vol. ZZB, Academic Press, New York, 1966, p. 213. FLETCHER, A. P., NEUBERGER, A., AND RATCLIFFE, W. A., Biochem. J., 126, 417 (1970). BATSON. H. C.. Introduction to statistics in the medical sciences. Burgess Publishing Company, Minneapolis, 1956, p. 57. OHTSUKA, K., AND WAKABAYASHI, M., Enzymologia, 39, 109 (1970). BONNICHSEN, R., Acta Chem. Stand., 18,1302 (1964). ALFSEN, A., AND JAYLE, M. F., Bull. Sot. Chim. Biol., 40,2143 (1958). H~Tu, C., AND GIANETTO, R., Can. J. Biochem., 48, 799 (1970). Mus.4, B. U., DOE, R. P., AND SEAL, U. S., J. Biol. Chem., 240, 2811 (1965). JACOBSON, K. B., AND PFUDERER. P.. . J. Biol. Chem., 246.3938 (1970). . SALNIKOTV, J., MOORE, S., AND STEIN, W. H., J. Biol. Chem., 246, 5685 (1970).