multiple forms of β-glucuronidase in rat liver lysosomes and microsomes

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 166, 258-272 (1975)

Multiple Forms of ,8-Giucuronidase in Rat Liver Lysosomes

and M icrosomes 1.2

J. W. OWENS3 K. L. GAMMON, AND P. D. STAHL

Department of Physiology and Biophysics, Washington University Medical School, St. Louis, Missouri 63110

Received July 29, 1974

Multiple forms of &glucuronidase have been demonstrated using sucrose gradient and polyacrylamide gel isoelectric focusing techniques in 6 M urea. Microsomal @-glucuronidase, a membrane-bound enzyme, was solubilized from lysosome-free, Ca*+-precipitated microsomes by detergents and isolated by chromatography on columns of rabbit anti-rat preputial gland j3-glucuronidase antibody bound to Sepharose. The enzyme has a p1 of 6.7. Polyacrylamide gel isoelectric focusing resolves the microsomal enzyme into three components, each of which is protease sensitive. The protease-modified microsomal enzyme is very similar to several forms of P-glucuronidase in lysosomes. The lysosomal @-glucuronidase, isolated from osmotically shocked lysosomes, is very heterogeneous after isoelectric focusing over the range p1 5.4-6.0. The lysosomal enzyme can be resolved into 10-12 bands by polyacrylamide gel isoelectric focusing. The more acid forms of the lysosomal enzyme are neuraminidase sensitive, suggesting they may be sialoglycoproteins.

/3-Glucuronidase is an acid hydrolase typically found in lysosomes. Unlike most other lysosomal enzymes, however, P- glucuronidase displays a unique subcellular distribution and is associated with both the liver lysosomal and microsomal fractions (1). The precise function of the microsomal enzyme, whether it serves a role as precursor to lysosomal /3-glucuronidase (2, 3) or some physiological function (4) in microsomal membranes, remains unclear.

In addition to the unique subcellular distribution, P-glucuronidase displays extensive multiplicity. Multiple forms of fi- glucuronidase have been demonstrated in the human (5), bovine (6), mouse (7), and rat (8) tissues by a variety of techniques. Using rat liver, Sadahiro et al. (9) have suggested three forms of P-glucuronidase, each of which has a different subcellular

1 Supported by HEW grants CA 12858 and GM 21096.

2 Preliminary accounts of this work were presented at the 9th International Congress of Biochemistry, Stockholm, Sweden (1973) and the 4th Subcellular Methodology Symposium, Guilford, England (1973).

3 Predoctoral fellow of the National Science Foun- dation.

localization. Moreover, Potier and Gia- netto (8) have demonstrated by ion- exchange chromatography and gel electrophoresis that rat liver lysosomes contain a number of forms of P-glucuronidase. Clearly, biosynthetic and turnover studies on rat liver P-glucuronidase would be more meaningful if the number of forms of the enzyme and their subcellular localization were known.

In the present study, the multiplicity of /?-glucuronidase from rat liver lysosomes and microsomes has been resolved by isoelectric focusing techniques. Microsomal fi- glucuronidase has been isolated from washed, lysosome-free microsomes and partially purified using a novel antibody-Sepharose chromatography technique. Protease and neuraminidase modification of microsomal and lysosomal p- glucuronidase, respectively, are also reported.

METHODS

Subcellular fractionation. Female Wistar rats (200 g), fasted overnight, were used throughout the study. Microsomes were prepared by two methods. Salt- washed microsomes were prepared by a modification

258

Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

MULTIPLE FORMS OF &GLUCURONIDASE 259

of the method of Weihing et al. (10). The procedure included the following wash steps: 0.15 M NaCl, 1.0 M NaCl, and 0.005 M Tris-Cl (pH 7.5). All washes were executed by resuspending the high-speed pellet 4:l (ml/g liver) in the appropriate wash solution.

Calcium-precipitated microsomes were prepared as described in Fig. 1. The procedure was adapted from that of Kamath and Rubin (11). Rat liver was minced, homogenized in 3 vol of cold 0.25 M sucrose using a loose-fitting Potter-Elvehjem homogenizer, and the resulting slurry was centrifuged at 1.2 x 10’ g .min in a IEC 870 rotor. The pellet was twice resuspended and recentrifuged, and the supematants were pooled yielding a crude extract (CE). The CE was centrifuged at 1.5 x 1O”g. min. The resulting mitochondrial-l:ysosomal (ML) pellet was resuspended in 2 vol of 0.25 M sucrose and recentrifuged at the same speed. The washed ML pellet was resuspended (4 ml/g liver) in 0.25 M sucrose. The supernatants from the two preceding spins were pooled and made to 8 mM Ca2+ by adding 1 M CaCl, dropwise. After 5 min, the solution was distinctly turbid and was centrifuged. at 1 x lO’g.min. The resulting supernatant (S,) corresponds to the cytosol fraction. The pellet (P,), or crude Cal+-precipitated microsomes, was twice resuspended (4 ml/g liver) in 5

mM Tris-Cl, pH 7.5, containing 8 mM CaCl, and centrifuged at 1 x lO’g.min. The corresponding supernatants, S, and S,, respectively, were saved for analysis. The pellet, after the second Tris-Caz+ wash, was resuspended in the same volume of 5 mM Tris-Cl without calcium and centrifuged at high speed (6 x 10Bg.min) in a Spinco L3-50 centrifuge. The final pellet (washed micmsomes or P,) and supernatant (S,) were analyzed with all other incidental fractions for marker enzyme and protein. The data generated by assay of marker enzymes and protein in S,, S,, and S,, respectively, have been combined for convenience under the heading (Wash) (Table I), The final pellet (P,) was then resuspended in 5 mMTris-Cl, pH 7.5, or 0.25 M sucrose for further treatment. Values for original homogenate were obtained by combining data from the cytoplasmic extract (CE) and nuclear fraction (N) as described by de Duve (1).

Lysosomes were prepared by the method of de Duve et al. (1) and Triton WR-1339-filled lysosomes by the method of Leighton et al. (12). Lysosomal /3-glucuronidase was solubilized by suspending the isolated lysosomal pellet (L) in 5 mM Tris-Cl, pH 7.5 (4 ml/g liver) followed by incubation at 5°C for 15- 30 min and centrifugation at 6 x 10Bg.min.

Antibody-Sepharose chromatography. Rat prepu-

CRUX LIVER EXTRACT (CE)

1.5x105 g.min

9mM CaC12. 5 mM bir Cl pH 7.5

PELLET 52

Reumpmded 1:4 9 mM CoC12, 5 mM Trir Cl pH 7.5

A

1x104 g.*n

PELLET 53

Rmnpended 1:4 5 mM kis Cl pH 7.4

3.7 x104 g.mim

A P2 54

FIG. 1. Flow chart for the preparation of washed, Ca*+-precipitated microsomes (P,) from crude liver extract (CE).

260 OWENS, GAMMON AND STAHL

TABLE I

MARKER ENZYMES AND PROTEIN ANALYSIS ON FRACTIONS AFTER SUBCELLULAR FRACTIONATION~

Marker

fl-Glucuronidase &Galactosidase Glucose-B-phos-

phatase Protein

Absolute Percentage values values

Fractionation of liver extract Fractionation of P,

CE + N N ML P, S, Re- P, P, Wash Re- covery covery

68.7 100 33.3 32.9 28.7 5.1 101 .o 100 75.9 18.0 93.9 293.1 100 32.3 40.6 16.1 9.7 98.5 100 57.3 62.7 120.0 695.4 100 31.3 5.9 42.7 7.3 87.2 100 97.2 3.8 101.0

233.3 100 28.0 14.8 15.2 29.6 87.6 100 80.7 24.7 105.4

D Marker enzyme and protein analysis on fractions after subcellular fractionation as described in Fig. 1. The data are presented in two parts: (a) Fractionation of liver extract, and (b) fractionation of P,. Absolute values for enzyme and protein are units/gram liver and mg/gram liver, respectively. Percentage distribution for enzyme and protein were calculated with CE + N = 100% and P, = 100% for liver fractionation and P, fractionation, respectively. Recovery for liver fractionation was determined by totaling activities in N, ML, P,, and S, compared with activity in CE + N. Recovery for fractionation of P, was determined by totaling activities in Wash and P, compared with activity in P,. CE = crude liver extract; N = nuclear fraction; ML = mitochondrial-lysosomal fraction; P, = crude calcium-precipitated microsomes; S, = cytosol fraction; P, = washed calcium- precipitated microsomes; Wash = pooled supernatants (S,, S,, S,) generated by washing calcium-precipitated microsomes.

tial gland /3-glucuronidase was used as antigen. The enzyme was purified by a modification of the method of Ohtsuka and Wakabayashi (13). The product, homogeneous by disc gel electrophoresis, had a specific activity of 2200-2400 U/mg. White New Zealand female rabbits were injected subcutaneously with antigen in complete Freund’s adjuvant (diluted 50:50). The first dose of 8-glucuronidase (0.9 mg) was given as several injections over the back. A second dose (0.7 mg) was administered similarly 20 days later. The animals were bled by ear vein 11 days after the second injection.

Gamma globulin was purified from the serum using standard techniques (ammonium sulfate precipitation and DEAE-cellulose chromatography) as out- lined by Weir (14).

Antibody-Sepharose was prepared by activating Sepharose 2B with cyanogen bromide (15) using 4.5 g CN Bd15 ml packed Sepharose. The antibody, dialyzed against 0.2 M sodium citrate, pH 6.5, was added to the activated Sepharose, and the mixture was gently rocked at 4°C for 16 hr. The gel was washed extensively with saline or Tris-Cl (0.005 M),

pH 7.5, before use.

Isoelectric Focusing Experiments

Sucrose gradient isoelectric focusing was per- formed using a Model 110 LKB isoelectric focusing apparatus as described by Russell and Geller (16). All reagents, except the cathode solution, contained 6 M urea. In the absence of urea, resolution of p-glucuronidase was poor and unpredictable. After 40-hr equili-

bration, the columns were eluted into l-ml fractions. Each fraction was assayed for enzymatic activity and selected fractions for pH. pH measurements were made at room temperature and were corrected for the presence of urea by subtracting 0.4 units from each reading as suggested by Ui (17).

Polyacrylamide gel isolectric focusing (PAGIF) was carried out in a jacketed Savant analytical electrophoresis unit at 4°C. Gels were prepared, using riboflavin as a catalyst, in 8.5 x O.&cm glass tubes. The gels (3.4% acrylamide) contained 6 M urea and 1.25% ampholine (pH 5-7 or pH 6-8). The lower electrode solution was 6 M urea containing 0.1 M H,SO,. The upper electrode solution was 1.0% NaOH. Samples containing j3-glucuronidase (usually 0.05-0.2 units) in 5 mM Tris-Cl, pH 7.5, were mixed with an equal volume of 9.5 M urea before being placed over the gel. The presence of urea was essential for PAGIF. In the absence of urea, the enzyme, presumably in an undissociated form, remained near the top of the gel. Power was never allowed to exceed 2 W and voltage was gradually increased over several hours to a maximum of 400 V. Gels were removed after a constant amperage was reached (2-4 hr). Gels were stained for /3-glucuronidase activity according to Stahl and Touster (18) except that the incubations were carried out in 30 ml of substrate reagent per gel.

Enzyme Assays and Analytical Procedures

&Glucuronidase and /Y-galactosidase were assayed as described by Stahl and Touster (18) and Craven et al. (19), respectively. Glucose 6-phospha-

MULTIPLE FORMS OF ,%GLUCURONIDASE 261

tase was assayed according to de Duve et al. (1) with phosphate determined by the semidine method (20). Protein was determined by the method of Miller (21). Bacterial neuraminidase was purified using the affinity chromatography method Illiano and Cuatrecasas (22). Neuraminidase was assayed using bovine sub- maxillary mucin as substrate in 0.1 M Na acetate, pH 5.5, containing 10 mM CaCl,. One unit equals 1 pmole sialic acid released per minute. Sialic acid re- lease was quantified using the thiobarbituric acid method of Warren (23). Trypsin and chymotrypsin modification of microsomal 8-glucuronidase was per- formed by diluting stock solutions of protease (1 mg/ ml in 1 mM HCl) with buffer and &glucuronidase such that the final concentrations were 57 mM Tris- Cl, pH 8.1, 10 mM CaCl,, 1 unit 8-glucuronidasel 0.1 ml incubation mixture. Protease was varied from 2 to 200 pglml. All reagents were analytical grade purchased from local vendors and Sigma Chemical Company, St. Louis, MO. Ultrapure urea was obtained from Schwarz-Mann, Orangeburg, NY.

Electron Microscopy

P, (microsomal) fractions suspended in 0.25 M

sucrose were spun at 6 x 10’g .min. The pellets were cut into l-mm pieces and fixed with glutaraldehyde (1%) in 0.05 M sodium phosphate buffer for several hours followed by postfixation in osmium tetroxide.

RESULTS

Lysosomal P-Glucuronidase

Multiplicity of lysosomal p-glucuronidase: effect of neuraminidase. p- Glucuronidase was prepared from a purified lysosomal fraction solubilized by os- motic shock. Solubilization was effected by suspending the lysosomal pellet in 0.005 M Tris-Cl pH 7.5 (4 ml/g liver) and centrifug- ing after about 30 min at high speed. This procedure solubilizes in excess of 90% of lysosomal P-glucuronidase. Fifty units of a solubilized I, fraction were placed on an LKB 110 isoelectric focusing column in 6 M urea (Fig. 2A). The results indicate extensive heterogeneity in lysosomal @- glucuronidase with various peaks whose p1 ranged from pH 5.5 to 6.0. Treatment of a similar sample, prepared in the same way, with bacterial neuraminidase for 4 hr at 37°C and pH 5.0 followed by isoelectric focusing in 6 M urea produces a shift in the focusing pattern. The results (Fig. 2B) indicate that the more acid forms of enzyme (p1 = 5.6) have been modified, pre-

sumably shifted to more basic isoelectric points.

Polyacrylamide gel isoelectric focusing. Much more resolution can be obtained by PAGIF in 6 M urea. Isoelectric focusing of solubilized lysosomal P-glucuronidase on gels followed by specific enzymatic stain, resolves the enzyme into a large number of forms, perhaps as many as 12 (Fig. 3A). This pattern has been observed using enzyme isolated (1) by Triton X-100 extraction of purified lysosomes, (2) osmotically solubilized ,&glucuronidase from lysosomes taken from livers of 5-day-old rats or adult 5-day starved rats and (3) Triton WR-1339-filled lysosomes isolated by the flotation technique (12) from livers of rats treated with detergent for 1,3, and 5 days.

Moreover, an experiment was run to determine if specific forms of P-glucuronidase could be solubilized under conditions which permit slow breakage of lysosomes in vitro with solubilization of their contents. A lysosomal fraction was incubated in 0.25 M sucrose containing 0.05 M Na acetate, pH 5.0, at 37°C and samples were taken at various times (0, 5, 10, 30, 60 min). After high-speed centrifugation 4, 6.4, 8.0, 19.3, and 93.3% of the total P-glucuronidase activity, respectively, was released into the supernatant. Total activity contained in the original particles was determined by treating a sample with Triton X-100 (0.2%) before assay. Gel isoelectric focusing of the enzyme released at various times produced essentially identical patterns after the enzyme stain. These results suggest that both labile and stable lysosomes contain similar multiple forms of /3-glucuronidase.

The multiple forms of lysosomal /3- glucuronidase were obtained for further study by preliminary separation on liquid isoelectric focusing columns in 6 M urea as described in Fig. 2A, followed by fractionation. Six pooled fractions were obtained from an LKB 110 column. After extensive dialysis against 0.005 M Tris-Cl, pH 7.5, gel isoelectric focusing of the six fractions is shown in Fig. 3 (B-G). Treatment of the individual fraction B (Fig. 3B) with purified neuraminidase followed by PAGIF shows a shift of the activity toward the


FIG. 2. A. Isoelectric focusing (pH 5-7) of 50 units of solubilized lysosomal &glucuronidase. The column was eluted at 1 ml/min and the fractions were assayed for P-glucuronidase (0) and pH (0). All procedures were according to Methods. B. isoelectric focusing (pH 5-7) of 90 units of solubilized lysosomal 8-glucuronidase treated with neuraminidase. A solubilized lysosomal fraction was incubated with bacterial neuraminidase (0.1 units/unit 8-glucuronidase) for 2 hr at 37°C. A second aliquot of neuraminidase (0.1 units/unit &glucuronidase) was added after 2 hr and the reaction was allowed to proceed for 2 hr. The reaction mixture was then made 6 M in urea and placed on the column. Recovery of P-glucuronidase after incubation was 65%. After equilibration, the column was eluted, assayed for p-glucuronidase (0) and pH (0). All procedures were according to Methods.

basic pole (Fig. 3 H-I). Neuraminidase alone had no appreciable effect on p- glucuronidase activity. Neuraminidase treatment generates forms similar to those found in the more basic fractions. While the bulk of the &glucuronidase activity found in fraction B was modified by neuraminidase digestion, complete conversion was not possible even after extensive neuraminidase treatment.

Cytosol /3-Glucuronidase

After homogenization and subcellular fractionation, about 5% of liver p-

glucuronidase is found in the supernatant or cytosol fraction. Since Sadahiro et al. (9) have suggested the presence of a chromatographically distinct form of P-glucuronidase associated with the supernatant fraction, 15 units of /3-glucuronidase from post- microsomal supernatant prepared from rat liver by the method of de Duve et al. (1) were focused on an LKB 110 column in 6 M urea. The enzyme was first partially purified by passage over a Sephadex G-200 column (5 mMTris-Cl, pH 7.5). The results indicate that enzyme found in rat liver supernatant fractions is very similar, if not

MULTIPLE FORMS OF /3-GLUCURONIDASE 263

FIG. 3. Polyacrylamide gel isoelectric focusing of lysosomal @-glucuronidase before (A) and after fractionation as described in Fig. 2A. Six fractions were collected, dialyzed and refocused on gels (B-G). Fraction B was incubated in the absence (H) and presence of neuraminidase (0.05 unit/unit, /3-glucuronidase) for 2 hr followed by overnight dialysis against 5 mM Tris-Cl, pH 7.5.

identical, to liver lysosomal P-glucuronidase suggesting that cytosol P-glucuronidase is due to breakage of organelles during the fractionation procedure.

Microsomal P-Glucuronidase

Subcellular fractionation. Liver from female Wistar rats, fasted overnight, was homogenized and fractionated as described in Methods. Washed microsomes were prepared in two ways. The method used at the outset of this study was a modification of the method of Weihing et al. (10). For convenience, the bulk of the experiments reported in this study, and all of the fractionation results reported here, were done with microsomes prepared by a modification of the method of Kamath and Rubin (11). Qualitatively, the results were the same. After the preparation of washed Caz+-precipitated microsomes, marker enzymes were assayed in the incidental fractions. /3-Galactosidase was used as a lysosome marker and glucose-6-phosphatase for microsomes. The entire fractionation scheme is shown in Fig. 1. Enzyme analy- ses (after treatment of the fractions with 0.2% Triton X-100) made on all the fractions, appear in Table I. The enzyme in each fraction (N, ML, P, and S) is expressed as a percentage of that found in the starting homogenate (CE + N). The enzyme in the wash fractions and PZ, respectively, is expressed as a percentage of that found in P,.

/3-Glucuronidase distributes about

equally between the mitochondrial-lysoso- ma1 (ML) fraction and the crude microsomal fraction P, with a small amount of activity found in the soluble fraction. After extensive washing of the P, fraction with low ionic strength buffer (0.005 M Tris-Cl, pH 7.5), the bulk of the @-glucuronidase (76%) remains associated with the membrane fraction. fl-Galactosidase, on the other hand, shows a distribution typical of a lysosomal hydrolase with only about 16% of its activity present in crude microsomes, better than half of this activity being removed by washing with low ionic- strength buffers, presumably reflecting lysosome breakage. Glucose-6-phosphatase, as expected, is largely present in the P, fraction and the bulk of this activity (97%) remains associated with the membranes during the washing procedure. A significant portion of all the enzymes remains associated with the nuclear fraction, presumably associated with unbroken cells.

Latency. The question of lysosome con- tamination of washed microsomes was further examined by testing the effect of Triton X-100 on the activity of ,8- glucuronidase in the ML, P,, and P, fractions. The fractions were suspended in 0.25 M sucrose and assayed in the same using the standard assay. The effect of Triton X-100 was tested by adding the detergent, to a final concentration of 0.2%, to the fractions 15 min prior to assay. Figure 4 shows the effect of the detergent, an agent which is known to lyse lysosomes, on the

OWENS, GAMMON AND STAHL

0 40

SC PROTEIN

FIG. 4. Comparison of latent and nonlatent 8- glucuronidase activity in cell fractions. Cell fractions, prepared as described in Fig. 1, were suspended in 0.25 M sucrose and assayed in the presence of the same. @-Glucuronidase activity is expressed as relative specific activity to CE + N. The width of each bar corresponds to the percentage of protein of the liver homogenate from which the fractions were isolated. The crosshatched areas indicate activity in the absence of Triton X-100 (nonlatent). The open area is additional (latent) activity expressed in the presence of a total concentration of 0.2% Triton X-100 added to sample 15 min prior to assay.

activity of /3-glucuronidase. The ML fraction is activated 4.4-fold, the P, fraction 1.3-fold, and the P, fraction was un- changed. These results indicate that the P, fraction is essentially nonlatent and supports the proposal that the fraction is nearly lysosome-free. Moreover, an electron micrograph of this fraction shows very few lysosomelike structures (Fig. 5).

Extraction of P,. The @glucuronidase associated with P, can be quantitatively solubilized with 0.25% Triton X-100 or 0.1% Na deoxycholate, the former was used principally in these experiments. Fig- ure 6 shows the effects of various Triton concentrations on the solubilization of P- glucuronidase. The activity remaining in the high-speed supernatant fraction after treatment with various Triton concentrations indicates about 0.2% Triton X-100 is effective in solubilizing > 90% of the activity. Moreover, incubation of crude membranes (P,) with 6 M urea solubilizes about half the P-glucuronidase while trypsin treatment is ineffective. The preparation of acetone powder from washed microsomes also solubilizes the enzyme upon suspension of the powder in aqueous media.

Isolation of Microsomal fl-Glucuronidase

Purification of detergent-solubilized P- glucuronidase was achieved with the use of a /?-glucuronidase-specific antibody-sepharose column. Antibody to rat preputial gland P-glucuronidase was raised in rabbits as described in Methods. The rat preputial gland is an extremely rich source of /3- glucuronidase and milligram quantities of highly purified enzyme can be readily obtained. IgG, isolated from rabbit antiserum to pure rat preputial gland /3-glucuronidase and coupled to Sepharose 2B, will adsorb P-glucuronidase from preputial gland, liver lysosomes, or microsomes. Figure 7 shows a typical preparation of detergent-solubilized enzyme passed over a rat antibody-Sepharose column. The bulk of the protein passes through the column unre- tarded, while the 8-glucuronidase is ad- sorbed. After extensively washing the column, either with 5 mM Tris-Cl, pH 7.5, or 0.15 M NaCl, the &glucuronidase can be eluted from the resin with 6 M urea. The enzyme, having been dialyzed against 5 IIIM Tris-Cl, pH 7.5, is fully reactivated, and the column, having been washed with 0.15 M saline, is ready for reuse. The antibody-Sepharose column routinely pro- vides a purification in the neighborhood of 80- to loo-fold with a recovery of 60-80%. Urea also elutes some macromolecular ma- terial from the column which can be removed by chromatography on Sephadex G-200. (This procedure has permitted the isolation of microsomal P-glucuronidase in pure form. The characterization of the purified enzyme will be the subject of a subsequent communication).

Isoelectric Focusing Experiments: Micro- somal P-Glucuronidase

Triton X-100 extracts of washed microsomes and antibody-Sepharose column- purified @glucuronidase were analyzed by isoelectric focusing in 6 M urea using the LKB 110 column and polyacrylamide gels. The results were qualitatively the same with both enzyme preparations; however, electrofocusing of crude microsomal extracts often results in precipitation of protein near the anode, making elution diffi-

MULTIPLE FORMS OF 84LUCURONIDASE 265

FIG. 5. Electron micrograph of washed, Ca2+-precipitated rat liver microsomes as prepaed in Methods (x 73,000).

cult. For this reason, all of the electrofocus- column. After equilibration and elution, ing experiments described here are with the fractions were assayed for enzyme ac- antibody column-purified enzyme. tivity and pH. A single form of the enzyme

Isoelectric focusing of semipurified mi- was observed (p1 = 6.7). crosomal @-glucuronidase is described in Polyacrylamide gel isoelectric focusing of Fig. 8. Sixty-five units were placed on the the same sample was achieved as described


in Methods. After equilibration, ‘the gel, when stained for enzymatic activity, re- veals three to six closely related forms (Fig. 8, insert). The most basic three forms (Ml, M2, M3) are routinely more intensely stained, the second three can be eliminated if the membranes are extracted rapidly or in the presence of urea.

Individual bands have been isolated

% TRITON X-100

FIG. 6. Solubilization of fi-glucuronidase activity from washed microsomes (P,) by Triton X-100. Sam- ples of P, were resuspended in 5 mMTris-Cl, pH 7.5 (4 ml/g liver). Triton X-100 was added from a 10% stock solution to give various concentrations. The samples were then spun at 6 x 10eg. min in a Spinco L3-50. The supernatant (0) was poured off and the pellet (0) resuspended in 5 mM Tris-Cl, pH 7.5 + 0.2% Triton X-100. Assays were according to Methods.

from focused gels by slicing into sections after staining followed by elution with 6 M urea. Refocusing experiments with single forms (Fig. 9) supports the conclusion that the multiple forms observed are not ar- tifacts of the gel system.

P-Glucuronidase from Smooth and Rough Endoplasmic Reticulum

Rough and smooth endoplasmic reticulum (24), Golgi (25), and plasma membranes (26) were isolated by standard techniques. Smooth and rough endoplasmic reticulum contain considerable P- glucuronidase activity. Golgi and plasma membranes, while having low relative specific activities, contain enough activity, after detergent extraction, for polyacrylamide gel isoelectric focusing. The banding patterns from these organelles, after staining for enzymatic activity, were no different from enzyme isolated from whole microsomes.

Proteolytic Modification of Microsomal p- Glucuronidase

An observation made several years ago by Stahl and Touster (18) indicated that

2 4 6 8 10 12 14 16 ,I) 20 22 24 26 !

7

6

FIG. 7. Purification of j3-glucuronidase using antibody-Sepharose. fl-Glucuronidase was solubilized from P, suspension in 5 mM Tris-Cl, pH 7.5, and 0.2% Triton X-100 (4 ml/g liver), followed by centrifugation at 6 x 10qg.min in a Spinco L3-50. The supernatant (sample) was poured off and applied to a rabbit anti-rat preputial P-glucuronidase antibody-Sepharose column as prepared in Methods. After the sample was applied, the column was washed with 50 ml of 0.15 M

NaCl and then 75 ml of 6 M urea. Fractions were collected and assayed for @-glucuronidase (0) and protein (0) according to Methods.

MULTIPLE FORMS OF j3-GLUCURONIDASE

FIG. 8. (A) Isoelectric focusing of antibody-purified microsomal 8-glucuronidase. Antibody column-purified @-glucuronidase was prepared as described in Fig. 7. After dialysis against 5 mM

Tris-Cl pH 7.5, and concentration, 65 units of B-glucuronidase were focused in a sucrose density gradient containing 6 M urea with pH 5-7 Ampholine. The column was eluted and fractions were assayed for B-glucuronidase (0) and pH (0). (B) 0.2 units antibody column-purified P-glucuronidase focused in a polyacrylamide gel according to Methods using pH 68 Ampholine.

microsomal (3-glucuronidase, while being chromatographically distinct from the lysosomal enzyme, was sensitive to trypsin; the former becoming much more acidic after trypsin modification without loss of enzymatic activity.

Trypsin and chymotrypsin modification of purified microsomal fl- glucuronidase. Microsomal /3-glucuronidase, purified by antibody-Sepharose chromatography, was incubated with vary- ing amounts of trypsin and time at pH 8.1 in 57 mM Tris-Cl and IO mM CaCl,. The reaction was stopped by addition of an equal volume of 9.5 M urea followed by polyacrylamide gel isoelectric focusing. The results shown in Fig. 10 indicate that trypsin markedly lowered the isoelectric points of microsomal fi-glucuronidase. Moreover, trypsin-modified microsomal fl- glucuronidase and the most basic forms of lysosomal P-glucuronidase (Fig. 3G) appear very similar. The effect of chymotrypsin was essentially the same. By mixing trypsin- or chymotrypsin-modified p- glucuronidase and the five most basic forms (Fig. 3G) from lysosomal P-

the modified microsomal bands except one co-focus with as many lysosomal forms, suggesting that they are very similar, perhaps identical (Fig. 11). This point is further demonstrated by an experiment in which the three major bands of microsomal /3-glucuronidase were first isolated as described earlier with subsequent modification with trypsin. The trypsin-modified individual bands of microsomal p- glucuronidase were electrofocused with fraction G (Fig. 3G) from lysosomes. Figure 12 shows that the most basic band from microsomes (Ml) co-focuses with the most basic band of lysosomes (Ll), band 2 (M2) from microsomes focuses between band 2 (L2) and 3 (L3) of lysosomes, and band 3 of microsomes (M3) co-focuses with band 4 (L4) of lysosomes.

Lysosomal modification of microsomal P-glucuronidase. Incubation of purified microsomal P-glucuronidase with a liver lysosomal fraction at pH 4.5 and 37°C in the presence of 0.2% Triton X-100 (the latter insures complete unmasking of lysosomal hydrolases) followed by high-speed centrifugation, dialysis, and polyacrylam-

glucuronidase, it can be shown that all of ide gel isoelectric focusing is shown in Fig.


FIG. 9. Isolation of individual forms of microsomal @-glucuronidase. Antibody column-purified @- glucuronidase was focused in polyacrylamide gels on a preparative scale (20 units/gel) according to Methods in pH 6-8 Ampholine. The gels were briefly stained (4 min). The gel section containing each band was excised with a scalpel. Sections from a set of gels were pooled in 6 M urea, homogenized with a Polytron homogenizer, and centrifuged after 3 hr. Samples of the supernatant were focused analytically and stained according to Methods. A, Control; B, band Ml; C, band M2; D, band M3.

13. At 6 hr of incubation, there is almost complete conversion of microsomal fi- glucuronidase to the lysosomal forms.

DISCUSSION

The dual subcellular localization of /3- glucuronidase in liver tissue was first suggested by the early experiments of Walker (27) and later verified by the classic studies of de Duve et al. (1). The precise chemical and biosynthetic relationship of microsomal P-glucuronidase to lysosomal P- glucuronidase is still unclear; however, a number of studies have been made which suggest that in some species a close struc- tural and/or biosynthetic relationship ex- ists between the enzyme found in these two subcellular sites. In the rat, for example, Sadahiro and co-workers (9) have presented evidence for three forms of p- glucuronidase, one associated with the microsomal fraction, a second with the mitochondrial fraction (which contained lyso-

somes), and a third which was associated with the cytosol fraction. Unfortunately, the purity of their organelles was not evalu- ated and the basis for their claim of multiple forms was stepwise elution from DEAE-cellulose. Fractions were not moni- tored by gel electrophoresis. The experiments reported here indicate the cytosol 8- glucuronidase to be very similar to lysoso- ma1 P-glucuronidase, suggesting that the former arises from lysosomal breakage during tissue fractionation. More recently, Potier and Gianetto (8) have shown multiple forms of /3-glucuronidase in rat liver lysosomes by a combination of ion- exchange chromatography and gel electrophoresis. In the rabbit, Dean (281 has recently reported that microsomal and lysosomal P-glucuronidase are very similar, if not identical. Paigen and colleagues ini- tially found no differences in the electrophoretic mobility of P-glucuronidase from mouse liver lysosomes and microsomes (29). More recently, they have reported electrophoretic differences between microsomal and lysosomal /3-glucuronidase and have presented evidence in favor of a peptide which binds the microsomal enzyme (7). Unlike the present study with rat enzyme, the multiplicity of mouse microsomal /3-glucuronidase was abolished by urea. The microsomal membranes used in the latter study contained a large amount of lysosomelike /3-glucuronidase enzyme. Plapp and Cole (6) studied bovine ,B- glucuronidase isolated from whole liver without subcellular fractionation. They reported extensive heterogeneity due to unequal distribution of sugar residues; however, their procedure included an autolytic step.

The present study was designed to determine the number of forms of rat liver P-glucuronidase and their subcellular localization. A necessary prerequisite for de- termining the multiplicity of P-glucuronidase was the preparation of purified organelles. Since liver microsomal fractions are generally considered a crude fraction and are contaminated with other organelles, it was first necessary to prepare lysosome free microsomes. Using low ionic-strength buffers and a Ca2+ precipitation technique

MULTIPLE FORMS OF B-GLUCURONIDASE 269

FIG. 10. Alterations in the isoelectric focusing pattern of microsomal j3-glucuronidase due to trypsin. Antibody-Sepharose-purified @-glucuronidase (1 unit/O.7 ml) was trypsinized, focused on polyacrylamide gels in pH 6-8 Ampholine, and stained according to Methods. A, control; B, 10 pg trypsin 4 min; C, 10 kg 10 min; D, 50 c(g 15 min; E, 100 c(g 15 min; F, 100 wg 30 min; G, 200 pg 30 min; and H, 200 fig 120 min.

adapted from the method of Kamath and Rubin, a microsomal fraction was prepared which (1) was enriched in glucose-6-phosphatase, but not P-galactosidase (2) re- tained a significant amount of P-glucuronidase activity which was solubilized, but not activated, with detergents and (3) showed few lysosome-like structures when examined by electron microscopy.

The lack of latency of fi-glucuronidase in washed microsomal membranes raises an interesting point about the localization of the enzyme. The structure of microsomal membranes, with ribosomes attached to the outer surface, suggests that the contents of isolated microsomes correspond roughly to the cisternae of smooth and rough endoplasmic reticulum in. uiuo. p- Glucuronidase, if enroute to the Golgi apparatus for packaging or secretion, would be expected to occur on the inside of microsomal vesicles. Since the latter are thought to be impermeable to charged molecules in excess of 100 A4, (30), one might expect P-glucuronidase using phe- nolphthalein glucuronide as substrate (Mr 512) to display latency. Additional observations from our laboratory suggest that P-glucuronidase is truely nonlatent. From the present experiments, it is unclear where microsomal P-glucuronidase is local- ized. Aside from the question of cisternal or cytosol-sidedness, these studies, as well as earlier reports (31, 32), support the notion

that microsomal P-glucuronidase is membrane bound. A recent report by Mameli and Gianetto (31) suggests that microsomal P-glucuronidase can be solubilized by extensive sonication; however, we have not been able to reproduce their results using Ca” -precipitated microsomes.

Antibody to rat preputial gland p- glucuronidase bound to Sepharose has pro- vided a convenient method for the purification of rat and human @-glucuronidase (33). Antibody column-purified rat liver microsomal P-glucuronidase focuses as a single peak after isoelectric focusing in sucrose and urea, but can be resolved into three bands by PAGIF. Since the individual bands from lysosomes and microsomes can be isolated and refocused, it is likely that the extensive multiplicity observed is not artifactual.

The protease sensitivity of microsomal P-glucuronidase is of some interest in view of the enzyme’s membrane localization. Whether the trypsin-sensitive portion of the enzyme, like cytochrome b, (34), is im- portant in membrane binding, must await large-scale purification and binding studies. Moreover, the similarity of protease- modified microsomal /3-glucuronidase and certain forms of lysosomal p-glucuronidase suggest that one mechanism for the packaging of liver P-glucuronidase is autophagy, particularly since liver autophagic vacuoles have been shown to contain assorted frag-


Frc. 11. Comparison of lysosomal fl-glucuronidase with micmsomal P-glucuronidase by isoelectric focusing in polyacrylamide gels with pH 6-8 Ampholine. Trypsinization, focusing, and staining were according to Methods. A, microsomal P-glucuronidase control; B, microsomal @-glucuronidase incubated with 200 peg trypsin for 120 min; C, microsomal &glucuronidase incubated with 200 ~g trypsin for 120 min t fraction G (Fig. 3G) of lysosomal fi-glucuronidase; D, fraction G (Fig. 3G) of lysosomal /3-glucuronidase. The five most basic hands of lysosomal @-glucuronidase have been designated Ll to L5 in order of increasing acidity.

ments of endoplasmic reticulum (31). The multiplicity of lysosomal /3-

glucuronidase is very extensive. Since the enzyme is a glycoprotein (18), it is not unlikely that the heterogeneity arises from unequal distribution of sugar residues on the protein backbone. In addition to heterogeneity which may arise biosyntheti- tally (35), lysosomes are enriched in all the enzymatic machinery which, by their ac- tion on P-glucuronidase, for example, may generate heterogeneity and ultimately, complete digestion. This notion is further suggested by the partial sensitivity of the more acid forms of /3-glucuronidase to neuraminidase digestion. The inability to completely convert the more acid forms of the enzyme to more basic isoelectric points may be due to the inaccessibility of certain

sialyl residues. There is a report (36) that certain mucopolysaccharides may avidly bind lysosomal enzymes. Since lysosomes are rich in mucopolysaccharides, this effect could mask certain sialyl residues and could possibly account for some of the heterogeneity observed.

Caution must be used in the interpreta- tion of experiments employing neuraminidase purified by the affinity chromatography method of Cuatrecasas and Illiano (22). A recent study of Rood and Wilkinson (37) indicates other proteins (e.g.. phos- pholipase C) are co-purified using the same resin.

Curiously, the extensive heterogeneity of lysosomal /3-glucuronidase was observed in lysosomes prepared in several ways after various treatments (e.g., starvation). Moreover, the same multiplicity was observed in /3-glucuronidase released from particles which burst readily in response to stressful incubation conditions (pH 5.0,

FIG. 12. Comparison of lysosomal fl-glucuronidase with isolated bands of microsomal 8-glucuronidase after trypsinization. Individual hands were incubated with 200 ~(g trypsin for 120 min and focused in polyacrylamide gels in pH 6-8 Ampholine. Trypsini- zation, focusing, and staining are according to Methods. A, fraction G (Fig. 3) from lysosomal @-glucuronidase; B, trypsinized microsomal band Ml; C, trypsinized microsomal band M2; D, trypsinzed microsomal band M3.

MULTIPLE FORMS OF @-GLUCURONIDASE 271

FIG. 13. Modification of microsomal @-glucuronidase by treatment with lysosomes. A lysosomal fraction was prepared according to Methods. The L fraction was resuspended (2 ml/g liver wt) in 50 mM sodium acetate, pH 4.5, and 0.2% Triton X-100. A 1.5-ml sample of the L fraction and 1.5 ml of antibody- purified microsomal @-glucuronidase (7 units/ml in 5 mM Tris-Cl, pH 7.5, were mixed at time zero and placed in a 37°C shaking water bath. At appropriate times 0.5-m) aliquots were withdrawn, stopped with 0.5 ml 0.5 M Tris-Cl, pH 7.5, and dialyzed against 5 mM Tris-Cl, pH 7.5. The aliquots were focused on polyacrylamide gels according to Methods. A, control; B, 2 hr; C, 4 hr; and D, 6 hr of incubation.

37°C) when compared with that released from particles which burst much more slowly. These findings suggest that extensive multiplicity of P-glucuronidase is found throughout the liver-vacuolar apparatus. This observation is not unexpected since it is known that indigestible sub- strates taken up by liver cells (i.e., detergents) ultimately permeate the entire lysosomal system (12).

Our final point deserves comment. All of the isoelectric focusing experiments reported here were done in 6 M urea. Rat liver /3-glucuronidase is a tetramer which dis- sociates into monomers in the presence of urea (18). Therefore, it is possible that the forms of the enzyme observed on gels or columns are monomeric forms. If this be the case, the multiplicity of the lysosomal @-glucuronidase tetramer is more extensive than suggested.

ACKNOWLEDGMENTS

The expert technical assistance of Jane Somsel Rodman is gratefully acknowledged. Thanks are also due to Guy Lemcoe for the electron microscopy and photography.

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multiple forms of β-glucuronidase in rat liver lysosomes and microsomes

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