Enzyme therapy

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

179,

397-408

(19?i+)

Enzyme Therapy Differential in Viva Retention @Glucuronidases and Evidence

of Bovine Hepatic, Renal, and Splenic for Enzyme Stabilization by Intermolecular Exchange’

M. B. FIDDLER* * Dight

Institute

for Human

Genetics Minnesota,

R. J. DESNICK**

AND

and t Pediatrics Minneapolis, Received

August

and Genetics and Cell Minnesota 55455

t Biology,

University

of

27, 1976

The in vivo fates of P-glucuronidase partially purified from bovine hepatic, renal, and splenic tissues were compared. Following administration to p-glucuronidase-deficient mice, hepatic and renal enzymes were primarily recovered in murine liver lysosomes (70% of dose by 30 min) with a subsequent decline to undetectable levels by 24 h. However, recovery of the splenic activity was markedly prolonged, 25% of dose was recovered in murine liver at 24 h with a decline to undetectable levels at 68 h, which was three times longer than that observed for the hepatic or renal enzymes. When administered entrapped in autologous erythrocytes, prolonged retention was again observed only with splenic enzyme, suggesting that recognition processes were not responsible for the splenic retention. No physical or kinetic differences were observed when these enzymes were further purified and characterized by gel filtration, ion-exchange, and polyacrylamide or cellulose acetate electrophoreses. Mixing of the splenic with hepatic or renal enzymes prior to administration resulted in an apparent enhanced retention of the hepatic and renal enzymes, suggesting a unique subunit structure of the splenic isozyme which might enhance the in vivo stability of the hepatic or renal enzymes by subunit exchange. These results indicate that subtle differences in the enzymes derived from multiple tissue sources may be recognized in uivo and that tissue source should be a consideration for enzyme therapy.

tion of uptake of periodate-treated p-Nacetyl-n-glucosaminidase by cultured skin fibroblasts implicated the role of carbohydrates in recognition and uptake processes. Other recent observations have also suggested that as yet uncharacterized features of a protein may directly affect the efficiency of cellular uptake. For example, p-glucuronidase activity isolated from human platelets exhibited a 50-fold greater efficiency of uptake by normal human cultured fibroblasts compared to the same activity isolated from other human tissue sources (6). In addition, human splenic pglucuronidase activity was resolved into two components, one which exhibited a higher efficiency of uptake by cultured fibroblasts than the other (7). The basis of this differential recognition process has

Among the requisites for enzyme therapy is the need for purified enzyme which is maximally stable for efficient substrate metabolism. However, the factors affecting the uptake, distribution, and intracellular retention of an administered enzymatic activity have not been identified. Investigations of the fate of enzymes and other proteins in both tissue culture and animal systems have suggested that posttranslational modifications as well as the primary amino acid sequence may participate in cell-protein recognition processes (l-5). The altered in uivo clearance of circulating glycoproteins following neuraminidase treatment (2, 4) and the preven1 This therapy.

is the seventh

paper

in a series

Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

on enzyme 397

ISSN

0003-9661

398

FIDDLER

AND

not been determined; however, the higher uptake forms of the enzyme exhibit a lower pI upon isoelectric focusing (8). We have encountered another parameter of enzyme processing, intracellular stability, which has not been extensively studied and which may reflect tissue specific modifications of an enzyme. Development of an in uiuo animal system has facilitated the systematic investigation of the fate of intravenously administered enzymatic activity (9). We have previously demonstrated that commercially prepared bovine hepatic p-glucuronidase was rapidly cleared from circulation (tl,z - 3 min) with a concomitant uptake almost solely by the lysosomes of the murine liver by 30 min; a decline of recovered hepatic activity followed until undetectable levels were observed by 24 h postinjection. Since we were intrigued with the previous findings of differential uptake of various human p-glucuronidase activities, we undertook to determine the uptake, distribution, and clearance of partially purified bovine hepatic, renal, and splenic ,&?-glucuronidase in our in uivo system, the last of which exhibited a prolonged intracellular retention. MATERIALS

AND

METHODS

Enzyme assays. Assay and discrimination of bovine and murine /3-glucuronidase activities and processing of tissues from C3H/HeJ Gush mice have been described in detail previously (9, 101. A unit of enzymatic activity was defined as that amount of activity which hydrolyzed 1 nmol of substrate/h. Purification of bovine tissue pglucuronidase activity. p-Glucuronidase activity was purified from fresh or frozen (-70°C) bovine liver, spleen, or kidneys by a sequence of conventional chromatographic methods; affinity methods were not used in order to avoid the introduction of any step which might select a specific molecular species of the enzymatic activity. Organs were washed with 0.15 M NaCl, cleaned of membranes, and homogenized 1:l (w/v) in the stock buffer, 0.05 M sodium phosphate, pH 6.5 containing 0.02% sodium azide. The homogenate was centrifuged at 10,400g for 30 min in a Sorvall RC-5 centrifuge at 4°C. The supernatant was removed and saved while the pellet was resuspended in a volume of 0.5% sodium cholate equal to that removed. Following centrifugation as above, the second supernatant was combined with the first and filtered through gauze. Methanol:butanol (1:l) was then added to the supernatant in a ratio of 1:3 (v/v),

DESNICK the mixture was shaken vigorously and centrifuged as above, and the precipitate was discarded. Ammonium sulfate was added to the supernatant to 50% saturation; this solution was allowed to stand for 1 h at 4°C and then centrifuged at 10,400g for 30 min. The solid phases were collected over gauze, resuspended in stock buffer, vortexed to homogeneity, and recentrifuged as above. The supernatant, concentrated if necessary by Amicon ultrafiltration, was passed through Sephadex G-200 and eluted with stock buffer, and peak fractions were pooled. The enzyme was then preparatively chromatographed over DEAEZ-cellulose (DE-52, Whatman), equilibrated with stock buffer, and developed in a single peak at approximately 0.1 M KC1 with a 0 to 0.5 M KC1 linear gradient in stock buffer. Alternatively, the post-Sephadex activity was chromatographed on DEAE-cellulose equilibrated at pH 5.77 and developed with a linear pH gradient of 0.05 M pyridineacetate from pH 5.6 to 4.85, essentially as described by Plapp and Cole (11). Most studies were carried out with the postDEAE enzyme, which represented a 340-fold purification to a specific activity of approximately 60,000 nmolihlmg of protein; protein determinations were done by the method of Lowry et al. (12) using bovine serum albumin as standard. The yield of enzymatic activity was approximately 8% of initial total activity, which probably accounted for the relatively low“fold purification” figure; both the yield and degree of purification may be improved by the use of affinity chromatographic methods (see Discussion). Where indicated, more highly purified enzyme was obtained by further chromatography over CMcellulose (CMC; CM-52, Whatman) as described by Plapp and Cole (11); this resulted in a further 1.7fold increase in specific activity. Finally, highly purified enzyme was also obtained by preparative polyacrylamide electrophoresis of the post-DEAE activity; the system of Clark (13) as modified by Swank and Bailey (14) was scaled up to accommodate large amounts of protein. Gels were sliced and p-glucuronidase was eluted by sonication in the presence of 0.05 M sodium phosphate, pH 6.5. Dialysis experiments. Post-DEAE enzyme was subjected to dialysis against 0.05 M sodium acetate, pH 4.6, 0.05 M sodium phosphate, pH 6.5, or 0.05 M Tris-HCl, pH 8.5 for 24 h at 4°C with three changes of buffer. Electrophoresis. Electrophoreses of the three enzymes were conducted in several systems. Polyacrylamide electrophoresis at pH 7.1 was carried out by the method of Swank and Bailey (14), using 7% acrylamide gels. Gels were stained with napthol* Abbreviations used: .DEAE-, diethylaminoethyl-; CM-, carboxymethyl-; 4-Mu-p-n-glucuronide, 4-methylumbelliferyl-p-n-glucuronide; Con A, concanavalin A; SDS, sodium dodecyl sulfate.

FATES

OF

BOVINE

TISSUE

ASBI-glucuronide substrate (Sigma, St. Louis, MO.) as described by Ganschow and Bunker (15). Polyacrylamide disc electrophoreses were run at pH 9.5 by the method of Ganschow and Bunker (151 and at pH 4.3 as described by Reisfeld et al. (16). Cellulose acetate gel electrophoreses (Cellogel, Kalex Scientific) was carried out under the following conditions: (i) pH 7.0 in 0.03 M sodium phosphate, (ii) pH 8.0 in 0.03 M Tris-HCl, (iii) pH 8.5 in 0.03 M Tris-HCl, (iv) pH 8.5 in 0.02 M Tris-HCI, 0.5 M NaCl, and (v) pH 9.0 in 0.02 M borate. Bands of enzymatic activity were assayed with 4-MU-p-n-glucuronide as substrate at pH 4.6, followed by rinsing in 0.1 M glycine carbonate, pH 10.7 and visualization under an ultraviolet lamp. Analytical ion-exchange chromatography. Behavior of the three enzymes on DEAE ion-exchange resin was explored following the procedure of Ikonne and Ellis (17). Enzyme samples, 2 ml, were applied to 0.9 x 15-cm columns equilibrated with 0.01 M sodium phosphate, pH 6.0; after 20 ml of the 0.01 M buffer had been pumped through the column (flow rate, 60 ml/h), a linear KC1 gradient (O-O.5 M) was applied. Fractions 2.0 ml, were collected and assayed for enzymatic activity. DEAE chromatography of the three enzyme preparations was also run at pH 8.1 using 0.005 M TrisHCl and developed, in initial runs, by a two-step gradient of 0.02 M NaCl and 0.1 M NaCl and, on subsequent runs, by a 0.1 to 0.5 M NaCl linear gradient. Binding studies of bovine pglucuronidase to Con A-sepharose. In order to assess possible differences in carbohydrate content or conformational availability of carbohydrates among the three enzymes, all three post-DEAE preparations were tested for their affinity for immobilized concanavalin A, which binds available mannose and glucose residues. Con A-Sepharose (Pharmacia), 1.0 ml, was placed in a lml disposable syringe with a polyethylene diffusion disc as a bed support. The column was preequilibrated with 0.1 M sodium phosphate, pH 6.0 containing 0.5 M NaCl and 0.01 M MgCI,, and 10 ml of each enzyme preparation was applied. Effluents were recycled through the column three times. Release of bound enzyme from the column was assessed following elution with 100 ml of 1.0 M a-methyl-n-glucoside in 0.1 M sodium phosphate buffer, pH 7.0, containing 0.5 M NaCl and 0.01 M MgCl,. The conditions for binding and elution have been investigated previously for other lysosomal enzymes (18; R. J. Desnick, unpublished results) and are used here as a standardized procedure for comparison of the three enzyme preparations. Gel filtration properties and SDS polyacrylamide electrophoresis. Gel filtration was used as a means of crudely assessing the molecular weight differences of the three enzymes. Samples were chromatographed on Sepharose 4B (Pharmacia, Piscataway,

p-GLUCURONIDASES

399

N.J.) with 0.02 M sodium phosphate, pH 6.5, as the eluting buffer. Because P-glucuronidase is a multimeric protein, molecular weight differences in the subunits of the purified hepatic, renal, and splenic enzymes were assessed by SDS-polyacrylamide electrophoresis according to Weber and Osborn (19); gels were stained with Coomassie blue. Kinetic properties. Effect of pH on the catalytic activity of each of the enzyme preparations was determined using 0.05 M sodium acetate buffers over a pH range of 3.6 to 5.6. Effect of substrate concentration (4-MU-/3-n-glucuronide) was assessed for each enzyme at pH 4.6. In vivo experiments. Bovine hepatic, renal, or splenic p-glucuronidase (1000-2500 units representing approximately 0.2 mg of protein) in 0.2 ml of 0.15 M NaCl was injected into the tail vein of individual mice. Bovine and murine activities were determined in murine tissue homogenates obtained from sacrificed mice at various intervals as described above. The partially purified hepatic and splenic enzymes were entrapped in autologous murine erythrocytes by hypotonic exchange as previously described (10). Erythrocyte-entrapped activity was administered intravenously to several mice which were sacrificed at 30 min, 2 h, 16 h, and 1, 3,4, 5, 7, and 8 days and livers were removed and assayed for the thermolabile bovine activity as described above. In vivo mixing experiments. Several in vivo mixing experiments were carried out with preparations of the three enzymes: (i) Equal activities of bovine hepatic and splenic, renal and splenic, or hepatic and renal activities were mixed, allowed to stand at room temperature for 5 min, and then administered intravenously to several mice. After each administration, mice were sacrificed at 24 h postinjection and their livers were analyzed for bovine and murine activities. (ii) Hepatic or renal enzyme was administered intravenously to several mice followed 1.0 or 3.5 min later by the administration of an equal amount of splenic activity. (iii) Hepatic or renal activity was administered to several animals followed 30 min later by the administration of splenic activity. In a second series of in vivo mixing experiments, one of the three enzyme preparations was pre-incubated at 60°C for 60 min to destroy enzymatic activity, rapidly cooled to room temperature, and then mixed with one of the other two preparations prior to administration. Mice were sacrificed at 24 h postinjection for determination of thermolabile bovine activity. In vitro mixing experiments. To assess the additivity of enzymatic activity of the three enzyme preparations, various activity levels of each enzyme were mixed at 37°C in vitro both in the presence and absence of the murine liver homogenate and immediately assayed. Assays of the mixtures were com-

400

FIDDLER

AND

pared to the expected total activities calculated as the sums of the individual activities. Other in vitro experiments. In order to assess the degradation rates of the three enzymatic activities in uitro, several protocols were explored. Mixtures of enzyme plus liver homogenate or enzyme plus a lysosomally enriched subcellular fraction from murine liver were incubated in 0.3 M sucrose buffered to pH 5.5 with 0.01 M acetate at 37°C in screw-capped vials for up to 24 h. Samples were withdrawn at 15 and 30 min, and 1, 2, 4, and 24 h and assayed for bovine and murine activities. Similarly, each enzyme was administered to a mouse which was then sacrificed at 30 min. Hepatic tissue was removed and subcellularly fractionated. The lysosomally enriched fraction containing the majority of the administered enzyme was then incubated at 37°C and sampled as above. The purified enzymes were incubated with 20 pg/ ml of bovine pancreatic trypsin or protease (Sigma, St. Louis, MO.) at pH 8.1 in 0.05 M Tris-HCl with 0.012 M CaCl, for up to 3 h and monitored every 10 min for loss of activity. A similar protocol was also carried out using porcine pepsin at pH 3.0. Control studies of enzyme in buffer were run simultaneously. In order to promote possible mixing of enzyme subunits prior to administration to animals, each enzyme preparation and each possible combination of enzymes were subjected to denaturation in 6 M urea for 24 h; this was followed by dialysis against 0.05 M acetate at pH 4.6, 0.05 M phosphate at pH 6.5, or 0.05 M Tris-HCl at pH 8.0, and then monitored for enzymatic activity. RESULTS

In Vivo Fate of pGlucuronidases from Bovine Liver, Kidney, and Spleen The blood clearance, tissue uptake and subcellular localization of bovine hepatic /3-glucuronidase prepared in this laboratory from fresh tissue were identical to that previously reported for the commercially prepared enzyme (9). Approximately 70% of dose was recovered in murine liver by 30 min with a decline to undetectable levels by 24 h postinjection. A low level of activity, less than 6% of dose, was recovered in kidney at early time points. Similarly, the tliz for the clearance of enzymatic activity from the circulation was approximately 3.5 min. Figure la illustrates the uptake and distribution of bovine p-glucuronidase activity purified from renal tissue. The renal activity was rapidly cleared from plasma (tliz =3 min) with a decline to undetectable

DESNICK

B

P

100

RENAL

,,,,i

SPLENK

ENZYME

ENZYME

0

b.

J

FIG. 1. (a) Fate of renal P-glucuronidase intravenously administered to p-glucuronidase-deficient mice. Bovine renal fi-glucuronidase activities recovered from murine organs and plasma are expressed as percentages of injected dose. (b) Fate of bovine splenic p-glucuronidase intravenously administered to /3-glucuronidase-deficient mice. Recovered bovine activity expressed as above.

levels by 30 min. A concomitant rapid uptake of the enzyme was observed in hepatic tissue with a maximum of approximately 74% of dose by 30 min. This recovery was followed by a decline to undetectable levels by 24 h. Subcellular fractionation of hepatic tissue at 30 min revealed 85-87% of recovered activity in the mitochondrial/lysosomal-enriched fraction with the remainder recovered in the nuclear fractious. Recovery of the administered activity in murine kidneys was an average of 11% at 1 h with a subsequent decline to less than 2% by 2 h postinjection. Figure lb illustrates the fate of /3-glucuronidase purified from bovine splenic tissue. The splenic activity was rapidly cleared from the circulation (tl,z =2 min) with approximately 70% of the administered activity recovered in the liver at 30 min. It should be noted that the blood clearance and hepatic uptake were somewhat more rapid than that observed for the hepatic or renal enzymes. More significantly, however, was the clearance of

FATES

OF

BOVINE

TISSUE

splenic activity from hepatic tissue. In contrast to that of administered hepatic or renal activity, the splenic activity was more slowly cleared; 25% of dose was recovered at 24 h and activity could be reliably detected up to 62 h postinjection, almost three times longer than that observed for the hepatic and renal enzymes. Subcellular fractionation of hepatic tissue at 2 and 24 h postinjection demonstrated a primarily lysosomal localization of the splenic activity as observed for the hepatic and renal enzymes. A low level of splenic p-glucuronidase activity (~10%) was recovered in murine spleen for up to 2 h (not shown); however, this finding was inconsistent and values ranged from 0 to 11% (n = 6, ii = 6%). Less than 10% of the splenic activity was recovered in renal tissue at early time points. Figure 2 compares uptake and retention of bovine hepatic (91, renal, and splenic pglucuronidase activities by murine liver. Note the prolonged retention of the splenic activity25% of dose at 24 h-which was markedly different from the undetectable levels observed for the hepatic and renal enzymes at 24 h. The rate of activity loss, determined by a least-squares analysis of the data, best fits first-order decay kinetics. This supports the proposition that the increased stability of splenic enzyme is not the result of a process extrinsic to the enzyme or enzyme preparation (e.g., “overloading” of the lysosome), as would have been suggested by a zero-order decay curve. 100 $83 B Pt 60 2 2

40

8 2

20

60 2 i-ml” : 0 30

4

6 8 IO I6 2228 34 40 4652 5864 hours Ttme Post lnjectlon

FIG. 2. Comparison of the murine liver uptake and retention of intravenously administered bovine hepatic, renal, and splenic p-glucuronidase-deficient mice.

P-GLUCURONIDASES

Characterization and Splenic

401 of Bovine Hepatic, Renal /3-Glucuronidase (Table I)

The surprising finding of prolonged liver retention of the bovine splenic activity stimulated a series of investigations to characterize the differences in the p-glucuronidase activities from the three enzyme sources, which may provide insights into the basis of the differential stability of the splenic versus the hepatic or renal enzymes . Since it was possible that a molecule which conferred resistance to intracellular degradation was copurified with the splenic /3-glucuronidase activity, two additional steps of purification beyond the DEAE ion-exchange chromatography were carried out. Neither chromatography over CM-cellulose nor exhaustive dialysis against pH 4.6, 6.5, and 8.5 buffers resulted in the removal of the hypothetical “stability” factor as determined by in vivo time course studies. Characterization of the molecular weights of the three enzymes were carried out by gel filtration and SDS-polyacrylamide electrophoresis. All three enzymes eluted at the same inclusion volume when chromatographed over Sepharose 4B. SDS-polyacrylamide gel electrophoreses were accomplished with the three enzymes which had, as a final step, been purified by polyacrylamide preparative electrophoresis. As plotted in Fig. 3, a major protein band was observed for all three enzyme preparations at a molecular weight of 71,000; this corresponds to a possible tetramerit structure for the reported characterization of bovine hepatic P-glucuronidase as a protein with a molecular weight of approximately 280,000 (11, 20). No apparent differences in the size of this hypothesized subunit could be detected. Despite the final purification procedure by preparative electrophoresis which evidenced only one protein staining band on analytical polyacrylamide gels at pH 7.1, several other protein bands were also observed on SDS-polyacrylamide gels. All three p-glucuronidase preparations had a band at molecular weights of 18,000-19,000; the renal and splenic sources both showed bands at molecular weights of 16,500,56,000-60,000,

402

FIDDLER

6

?

AND

i

5 ab x4

\

.OVALEUMIN

\

Liver Kidney Spleen

l

B A

IO

lOIl_iL Mobility FIG. 3. SDS-polyacrylamide of bovine hepatic, renal, dase preparations.

and

gel electrophoresis splenic /3-glucuroni-

and 86,000; in addition, the splenic and hepatic sources both showed bands at molecular weights of 31,000-34,000. Because of the 10% error inherent in this system, it was tentatively concluded that no protein staining band was unique to the splenic preparation, as might be expected if a proteinaceous “stability” factor was present. However, it should be noted that it is not possible to label the proteins as identical, since the protein bands detected were derived from different sources. Electrophoreses of the three enzymes utilizing the systems described under Materials and Methods failed to resolve any differences in electrophoretic mobility. At pH 8.0 and 8.5 in the cellulose-acetate system, a suggestion of greater electronegativity of the splenic enzyme was observed but could not be further resolved by manipulation of ionic strength, current, or time of run and it was, therefore, concluded that no differences existed among the enzymes in any of the systems used. Electrophoresis of the three bovine tissue homogenates prior to purification in the pH 7.1 polyacrylamide system showed one major band of activity and one minor,

DESNICK

more electronegative, band. Upon purification, activity was only observed at the site of the major band. Profiles of the three enzymes on DEAEcellulose were identical; all three activities were eluted at 0.09 M KC1 when run at pH 6.0. The columns run at pH 8.1, as described under Materials and Methods, did not reveal multiple peaks as reported for human splenic p-glucuronidase (7); all three enzymes were eluted by a linear gradient at 0.17 M NcAl. The hepatic, renal, and splenic activities were all 98-100% bound by Sepharose-Con A and all three were released approximately 70% by 1.0 M a-methyl-n-glucoside. Finally, the kinetic characteristics of the three partially purified (post-DEAE) enzymes with the artificial substrate were almost identical. Apparent Michaelis constants were 8.1 x lo-“, 8.2 x 10m5, and 4.7 x lo+ M for the hepatic, splenic, and renal activities, respectively. All three showed broad pH profiles with slight optima at pH 4.6. The results of these physical and kinetic characterizations are summarized in Table I. Administration of Erythrocyte-Entrapped Hepatic and Splenic Activities In order to test the possibility that the structure of the splenic enzyme initiated a cellular response upon recognition at the membrane for uptake which is markedly different than that of the hepatic enzyme - thereby resulting in a slower rate of intralysosomal degradation-these two activities were individually entrapped in autologous murine erythrocytes prior to administration to the /3-glucuronidase deficient mice. By administering enzymatic activity in erythrocytes, the uptake of activity was dependent upon the erythrocyte and not the enzyme protein itself. Table II shows the data for the murine liver recovery of the two activities 3 to 8 days following injection; the uptake and distribution at earlier time points was identical for the two enzyme-entrapped preparations. However, the hepatic activity was undetectable by 5 days (lo), whereas the splenic activity was maintained up to 8 days, again ex-

FATES

OF

BOVINE

TISSUE TABLE

COMPARISON

OF KINETIC

AND PHYSICAL AND SPLENIC

I

CHARACTERISTICS fl-GLUCURONIDASES

Hepatic pH optimum Apparent K, Affinity for Sepharose-Con A Elution from DEAE-cellulose At pH 6.0 At pH 8.1 Subunit molecular weight band on SDS-polyacrylamide) Electrophoretic mobility In polyacrylamide In cellulose acetate

Percentage

dose recovered hepatic tissue

in

Hepatic enzyme

Splenic enzyme

17 10 0 0 0

22 18 15 10 0

a prolonged intracellular

of carand

reten-

In Vivo Mixing Experiments As illustrated in Fig. 2, the differential clearance rates of the hepatic or renal activities versus the splenic p-glucuronidase activities permitted the use of an in vivo bioassay to determine the fate of mixtures of these enzyme preparations. At 24 h postinjection, the bovine hepatic and renal activities were undetectable in murine liver while 25% of the splenic activity was reproducibly observed. Thus, in mixtures of splenic and hepatic or splenic and renal activities only 25% of the splenic activity,

RENAL,

source

4.6 4.7 X lo-5 100% bound 66% eluted

Splenic M

0.09 M KC1 0.17 M NaCl 71,000

All three All three

(1 Entrapment, administration, and recovery enzymatic activity in autologous erythrocytes ried out as described in detail under Materials Methods.

hibiting tion.

M

0.09 M KC1 0.17 M NaCl 71,000

(major

HEPATIC,

Renal

4.6 8.1 X lo-5 98% bound 72% eluted

TABLE II COMPARISON OF HEPATIC RECOVERY OF ERYTHROCYTE-ENTRAPPED BOVINE HEPATIC AND SPLENIC P-GLUCURONIDASE ACTIVITIES ADMINISTERED TO P-GLUCURONIDASE-DEFICIENT MICE”

3 4 5 7 8

OF BOVINE Enzyme

Characteristic

Time postinjection (days)

403

P-GLUCURONIDASES

4.6 8.2 X lo-” 98% bound 70% eluted

M

0.09 M KC1 0.17 M NaCl 71,000

identical identical

or 12.5% of total activity in an equal mixture, would be expected to be observed; deviations from this value might provide a key to the source of the differential retention observed for the individual enzymes. Utilizing this bioassay system, the following mixing experiments were carried out as summarized in Table III. When equal activities of hepatic and splenic enzymes or renal and splenic enzymes were mixed and then administered intravenously to mice, average values of 29 and 23%, respectively, of total dose were recovered in murine liver at 24 h. These values also may be expressed as 58 and 46%, respectively, of the splenic enzyme’s contribution to the total dose. These recoveries were significantly greater than expected and were reproducible when postDEAE, post-CM-cellulose, or dialyzed enzyme preparations were used. When splenic enzyme was administered 30 min after the injection of hepatic or renal activity (all hepatic or renal activity was cleared from the circulation by 30 min; Fig. 1 and Ref. 7), the observed values for murine liver recovery were identical with that expected for the splenic contribution alone (Table III). These data suggested that the two enzymatic activities had to be in contact with each other prior to administration in order to observe the enhancement of recovery described above. Intriguingly, however, when hepatic or renal enzymes were administered 1.0 or

404

FIDDLER TABLE

In

III

Viuo

RENAL,

FATE OF MIXTURES OF BOVINE AND SPLENIC &GLUCURONIDASE IN P-GLUCURONIDASE-DEFICIENT

Enzyme

preparation

AND

Percentage covered Expected

HEPATIC, ACTIVITIES MICE

dose” rein liver Observed

(24 h postinjection) Hepatic Renal

+ splenic + splenic

Hepatic + renal Hepatic followed 30 min later by splenic Renal followed 30 min later by splenic Hepatic followed 30 min later by renal Hepatic followed 1 min later by splenic Renal followed 1 min later by splenic Hepatic followed 1 min later by renal Hepatic followed 3.5 min later by splenic Renal followed 3.5 min later by splenic Hepatic followed 3.5 min later by renal Hepatic + 60°C inactivated splenic Splenic + 60°C inactivated hepatic

12.5* 12.5* 0’ 12.5b 12.5 0’ 12.5b 12.5b 0’ 12.5” 12.5b 0 0’

29 W-45) 23 (19-28) 0 12 (11-14) 12 (11-13) 0 19 (14-21) 18 (14-20) 0 16 (13-19) 16 W-20) 0

Od

0

25d

24 (23-27)

LI Equal mixtures (1100-2400 U) of enzyme preparations were administered intravenously to 3 to 10 mice; mice were sacrificed at 24 h postinjection and liver was assayed for thermostable and thermolabile activities as described under Materials and Methods. Observed values are means and ranges. * Expected values calculated as (25% splenic dose)+(splenic dose + hepatic or renal dose) x 100. c Expected values determined from recovery of hepatic and renal activities when administered independently. d Incubation of bovine p-glucuronidase at 60°C for 1 h destroys all enzymatic activity; expected values determined by previous assessment of recovery of non-thermal-inactivated hepatic and splenic activities, respectively.

3.5 min prior to the splenic enzyme (time points at which approximately 85 and 50%, respectively, of the hepatic and renal activities remained in the circulation), average values of 18.5 and 16% of total dose

DESNICK

were recovered in murine liver at 24 h. These values were higher than the expected 12.5% recovery and suggested that an interaction between the hepatic or renal and splenic enzyme preparations may occur in vivo. As expected, undetectable levels at 24 h were observed following administration of hepatic and renal activities separated by 1.0 or 3.5 min. Finally, when the splenic enzymatic activity was destroyed by incubation at 60°C for 1 h prior to mixing with hepatic enzyme and administration, no activity was recovered in murine liver as expected for hepatic activity alone. In a reciprocal experiment, destruction of the hepatic activity by thermal inactivation prior to mixing with splenic activity resulted in an observed value for recovery of splenic activity identical to the expected splenic component alone (Table III). In Vitro Studies In order to control for possible nonadditivity or to identify possible enhancement TABLE

IV

In Vitro

MIXING OF BOVINE HEPATIC, RENAL, AND SPLENIC /?-GLUCURONIDASE ACTIVITIES AND MURINE (C3H/HeJ) LIVER HOMOGENATES

Enzyme

sources

EX-

petted”

Ob-

served

PC?l=

centage deviation from expected

(units)

Bovine hepatic +bovine splenic Bovine hepatic + bovine splenic +murine liver homogenate Bovine hepatic +bovine renal Bovine hepatic + bovine renal +murine liver homogenate Bovine renal +bovine splenic Bovine renal +bovine splenic +murine liver homogenate n Expected values assayed individually.

7.0

7.2

+3%

9.2

9.6

+4%

6.8

7.0

+3%

9.0

9.2

+2%

7.2

6.8

-5%

9.4

9.0

-4%

calculated

as sums

of activities

FATES

OF

BOVINE

TISSUE

resulting from enzyme interactions, mixing experiments were carried out for all combinations of the three enzymes, at various levels of activity, and in the presence or absence of murine liver homogenates. Table IV shows representative data indicating that no significant deviations from expected values were observed in these in vitro experiments demonstrating the additivity of the enzymatic activities. Experiments were conducted to determine potential differential in vitro characteristics of the three enzymes, including stability in murine homogenates, resistance to proteases, and affect of urea denaturation. Incubation of the three bovine enzymes with murine liver homogenate and a lysosomally enriched fraction, as well as incubation of a lysosomally enriched fraction obtained after enzyme injection, resulted in a loss of only ll-25% of activity by 4 h for all three enzymes. By 24 h, the incubations showed that more than 70% of activity remained. All three enzymes were remarkably resistant to proteolysis by trypsin, protease, or pepsin; 9095% of /3-glucuronidase activities were observed throughout the incubation period. Finally, only l&20% of each initial activity could be recovered following urea denaturation and dialysis. Thus, no in uiuo experiments were carried out with enzyme treated in this manner. DISCUSSION

The previous demonstration of a rapid liver uptake and clearance in the murine system of commercially prepared bovine hepatic P-glucuronidase was replicated by the bovine hepatic and renal activities isolated from fresh tissue. However, the surprising finding of an almost threefold greater murine liver retention of activity isolated identically from bovine splenic tissue stimulated the further characterization of these enzymes. The inability to demonstrate any physical or kinetic differences among the three enzyme preparations provided no insights into the nature of their differential in uiuo stabilities (see Table I). Nonetheless, the identical affinity and elution patterns on immobilized Con A indicates that this glyco-affinity

@GLUCURONIDASES

405

substrate may be utilized for the purification of all three enzymes. Utilization of Sepharose-Con A after the ammonium sulfate cut proves to be 10 times more efficient than Sephadex G-200 and may be readily substituted for that cumbersome gel filtration step for large-scale purification. Prolonged retention of splenic activity when administered in erythrocytes, however, does demonstrate that the differential stability is directly associated with an inherent feature of the enzyme or enzyme preparation and not the result of a differential cellular-lysosomal response stimulated by the interaction of the enzymes at the plasma membrane. Previous investigation of erythrocyte entrapment has demonstrated that little, if any, enzyme adheres or is exposed at the surface of the erythrocytes (10, 21), thus minimizing the possibility that uptake is mediated through a feature of the entrapped protein. The series of in uiuo mixing experiments provides the most intriguing data and an inferential basis for the existence of differences between the splenic and hepatic or renal enzymes. The enhanced recovery of administered activity when the splenic preparation was mixed with one or the other enzymes prior to injection suggests that differences may exist in the subunits or other associated molecule(s). In addition, the subunits, or other component(s), may exchange in such a manner as to confer upon the hepatic and renal enzymes the in uiuo stability of the splenic enzyme. Whatever the mechanism is that results in greater than expected recovery of activity at 24 h, it is subject to variability; this is attested to by the wide range of values observed, always significantly greater than expected, particularly for the hepatic plus splenic injections which represent a series of 10 experiments. When combinations of splenic with hepatic or renal enzymes were intravenously administered separated by 1.0 or 3.5 min, the greater than expected recovery of bovine activity suggests that these enzymes may be exchanging subunits, or other molecules, in the circulation. Indeed, because approximately 15 and 50%, respectively, of the activity injected first was already

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cleared when the splenic activity was administered, the hypothesized process of recognition and/or exchange may be very efficient. It also appears that in order for the hypothetical exchange process to occur, both enzymes must be in an undenatured state as evidenced by the reciprocal heat inactivation mixing experiments. Attempts to promote disassociation and reassociation of subunits by urea denaturation were hampered by the inability to recover significant activity upon removal of the urea. There is at least one alternative explanation which is also consistent with the in uiuo mixing experiment data. Simultaneous uptake of both the hepatic or renal and the splenic enzymes by murine liver cells, as may occur for all combinations (except the injections separated by 30 min), might result in a retarded degradation of the more stable splenic enzyme within digestive vacuoles containing both bovine enzymes. Proteolysis may be initiated first on the “less stable” hepatic or renal enzymes and thus a higher than expected recovery of enzymatic activity at 24 h could result from the delayed degradation of “more stable” splenic enzyme in deference to the hepatic or renal enzyme. Because the three enzymes, or any combination of their subunits, cannot be distinguished electrophoretically after uptake by murine tissue, this possibility cannot be directly tested against the “subunit (or factor) exchange” theory at this time. The results of the in vitro mixing experiments support the concept that the prolonged and enhanced retention of splenic activity, alone or in combination with the other enzymes, is the result of a biological phenomenon and not an artifact of the assay system due to kinetic differences or differential inhibition of the enzymes by components of murine liver tissue. It should be noted that the prolonged stability of the splenic enzyme could not be observed in several in vitro test systems (21). There was no difference in the rates of degradation of the three enzymes in mixtures of enzymes plus liver homogenate or enzymes plus a lysosomally enriched subcellular fraction incubated at 37°C. Similarly, the degradation rates of bovine ac-

DESNICK

tivities in a lysosomal fraction isolated 30 min after injection of the individual enzyme and then incubated at 37°C were identical; this is in contrast to the in uiuo rate of enzyme destruction. Finally, all three enzymes were equally resistant to pepsin, trypsin, and pronase at the appropriate pH values for these degradative enzymes. The nature of the differences between the three enzyme preparations cannot be assessed definitively from these experiments. The mechanisms which directly effect the turnover of proteins (221, and isozymes in particular, have not been described in any system although studies with catalase, lactate dehydrogenase, and aldolase demonstrate that isozymic forms of a protein may, in fact, show markedly different rates of turnover dependent upon the subunit composition of the isozyme and its tissue source (23, 24). Further, turnover studies of fatty acid synthetase (25) and myosin (26) have indicated that subunit exchange may occur in uiuo. Because p-glucuronidase purified from bovine liver has been demonstrated to be approximately 5% carbohydrate (20), it is tempting to speculate that the structural gene coding for the primary amino acid sequence and primarily responsible for catalytic activity is the same in hepatic, renal, and splenic tissue; presumably, therefore, tissue specific post-translational modifications, such as the differential addition of carbohydrates, may result in the differential in uiuo properties of the three tissue enzymes. This would be in contrast to the suggestion that subunit size is a primary factor in protein turnover (27). The post-translational addition of a specific “stability” protein to the splenic enzyme, analogous to egasyn which governs the subcellular localization of murine pglucuronidase (281, is not supported by any of the,electrophoretic results including the SDS-polyacrylamide studies. However, the possibility that a protein is associated with the hepatic and renal enzymes which promote degradation, relative to the splenic enzyme, cannot be ruled out. The somewhat elevated recovery of bovine renal activity in murine kidneys (Fig. 11, compared to the kidney uptake of bo-

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vine hepatic activity, and the transient, but unfortunately not reproducible, recovery of bovine splenic activity in murine spleen, may also reflect a tissue specific system of post-translational modification. Further suggestion of this is found in the somewhat greater liver uptake of the splenic enzyme reminiscent of a “high-uptake” enzyme (6). If, indeed, there are biochemical differences among tissue enzymes, the further use of in viuo systems, such as the murine system utilized here, may prove valuable in identifying these subtle differences (e.g., isoenzymes) by functional parameters even when electrophoretic or other chromatographic criteria fail to do so. It should be restated that the basis of the bovine ,&glucuronidase tissue differences may have to await complete purification and subsequent chemical analyses and the significance of these findings in nature should be assessedby differential turnover rates of tissue enzymes in situ. Nonetheless, these findings are applicable to the selection of specific enzyme sources for use in enzyme replacement endeavors. The need for examination of different tissues as the sources of enzyme for therapeutic administration was suggested by the results of early trials of enzyme therapy (29) and recent studies of the differential uptake efficiencies of enzymes isolated from multiple tissue sources by cultured fibroblasts (6-8, 30). The present studies provide another parameter which may be a function of enzyme source-in vivo retention. The administration of appropriate purified enzyme which is maximally stable either by its own inherent structure or by chemical modification (31) is among the requisites for effective enzyme therapy (20, 30). Thus, the examination and selection of specific tissue isozymes for prolonged substrate metabolism upon administration as a native protein or entrapped in vesicles such as autologous erythrocytes (10) or liposomes (33-35) to patients with inherited metabolic diseases is emphasized. ACKNOWLEDGMENTS The authors ciation to Dr.

wish to express their grateful appreS. R. Thorpe for her collaboration in

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the development of these studies, to Ms. Lynn D. Steger for her critical discussions and review of the manuscript, to Ms. Carolyn Dullum for her expert technical assistance, and to Ms. Ardys Ferman and Ms. Jackie Metelak for their expert clerical assistance. This work was supported in part by a grant (AM 15174)‘from the National Institutes of Health and a grant (l-273) from the National Foundation-March of Dimes. M.B.F. was a recipient of predoctoral fellowships from the National Science Foundation and the National Institutes of Mental Health (ITIMH 10679) and R.J.D. is the recipient of a National Institute of Health Research Career Development Award (K04 AM 00042). REFERENCES 1. RYSER, H. J.-P. (19681 Science 59, 390-396. 2. ASHWELL, G., AND MORELL, A. G. (1974)Aduan. Enzymol. 41, 99-128. 3. WINTERBURN, P. J., AND PHELPS, C. F. (1972) Nature (London) 236, 147-151. 4. FIDDLER, M. B., WOLD, F., AND DESNICK, F. J. (1975) Znt. J. Biochem. 6, 793-799. 5. GREGORIADIS, G. (1975) in Lysosomes in Biology and Pathology (Dingle, J. T., and Dean, R. T., eds.), Vol. 4, pp. 265-294, American Elsevier, New York. 6. BROT, F. E., GLASER, J. H., ROOZEN, K. J., AND SLY, W. S. (1974) Biochem. Biophys. Res. Commun. 59, 941-946. 7. NICOL, D. W., LAGUNOFF, D., AND PRITZL, P. (1974) Biochem. Biophys. Res. Commun. 59, 941-946. 8. GLASER, J. H., ROOZEN, K. J., BROT, F. E., AND SLY, W. S. (1975) Arch. Biochem. Biophys. 166, 536-542. 9. THORPE, S. R., FIDDLER, M. B., AND DESNICK, R. J. (1974)Biochem.Biophys. Res. Commun. 61, 1464-1470. 10. THORPE, S. R., FIDDLER, M. B., AND DESNICK, R. J. (1975) Pediat. Res. 9, 918-923. 11. PLAPP, B. Y., AND COLE, R. D. (1966) Arch. Biochem. Biophys. 116, 193-206. 12. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 13. CLARK, d. T. (1964) Ann. N. Y. Acad. Sci. 121, 428-436. 14. SWANK, R. T., AND BAILEY, D.W. (1973) Science 181, 1249-1251. 15. GANSCHOW, R., AND BUNKER, B. (1970)Biochem. Genet. 4, 127-133. 16. REISFELD, R. A., LEWIS, V. J., AND WILLIAMS, D. E. (1962) Nature (London) 195, 281-283. 17. IKONNE, J. U., AND ELLIS, R. B. (1973) Biochem. J. 135, 457-462. 18. BEUTLER, E., GUINTO, E., AND KUHL, W. (1975) J. Lab. Clin. Med. 85, 672-677.

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19. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4406-4412. 20. HIMENO, M., HASHIGUCHI, Y., AND KATO, K. (1974) J. Biochem. 76, 1245-1252. 21. FIDDLER, M. B. (1976) Ph.D. thesis. University of Minnesota, Minneapolis, MN. 22. SCHIMKE, R. T., AND KATUNUMA, N. (19’75) Intracellular Protein Turnover, Academic Press, New York. 23. MASTERS, C. J., AND HOLMES, R. J. (1975) in Haemoglobin, Isozymes, and Tissue Differentiation, Vol. 42, pp. 207-254, North Holland, Amsterdam. 24. HORECKER, B. L. (1975) International Conference on Isozymes, Academic Press, New York. 25. TWETO, J., DEHLINGER, P., AND LARRABEE, A. R. (1972) Biochem. Biophys. Res. Commun. 48, 1371-1373. 26. WI~KMAN-COFFELD, J., ZELIS, R., FENNER, C., AND MASON, D. T. (1973) J. Biol. Chem. 248, 5206-5207. 27. DICE, J. F., DEHLINGER, P. J., AND SCHIMKE, R. T. (1973) J. Biol. Chem. 248, 4220-4228. 28. SWANK, R. T., AND PAIGEN, K. (1973) J. Mol.

DESNICK Biol. 77, 371-389. 29. DESNICK, R. J., THORPE, S. R., AND FIDDLER, M. B. (1976) Physiol. Reu. 56, 57-99, 30. SHAPIRO, L. J., HICKMAN, S. G., HALL, C. W., AND NEUFELD, E. F. (1974) Amer. J. Hum. Genet. 26, 79A. 31. WOLD, F. (1973) in Enzyme Therapy in Genetic Diseases, Birth Defects: Original Article Series (Desnick, R. J., Bernlohor, R. W., and Krivit, W., eds.), Vol. IX, pp. 46-54, Williams and Wilkins, Baltimore, Md. 32. DESNICK, R. J., KRIVIT, W., AND FIDDLER, M. B. (19’75) in The Prevention of Mental Retardation and Genetic Disease (Milunsky, A., ed.), pp. 317-342, Saunders, Philadelphia, Pa. 33. GREGORIADIS, G., AND LEATHWOOD, R. D. (1971) FEBS Lett. 14, 95-99. 34. RYMAN, B. E. (1974) in Enzyme Therapy in Lysosomal Storage Diseases (Tager, J. M., Hooghwinkel, G. J. M., and Daems, W. Th., eds.), pp. 149-162, North-Holland, Amsterdam. 35. STEGER, L. D., AND DESNICK, R. J. (1977) Biochim. Biophys. Actu, in press.