Clearance of lysosomal hydrolases following intravenous infusion

ARCHIVES

OF

BIOCHEMISTRY

Clearance Kinetic

AND

BIOPHYSICS

of Lysosomal

and Competition

PHILIP

STAHL,2

Department

Hydrolases

(1976)

following

Intravenous

Experiments with @Glucuronidase Glucosaminidase’

JANE

of Physiology

117, 594-605

SOMSEL

RODMAN,

and Biophysics, St. Louis,

AND

Received

May

and N-Acetyi-p-D-

PAUL

Washington University Missouri 63110

Infusion

SCHLESINGER3

School

of Medicine,

3, 1976

Clearance experiments with highly purified lysosomal glycosidases, /3-glucuronidase and N-acetyl-p-n-glucosaminidase, following intravenous infusion revealed widely varying clearance profiles which depended on the tissue source of the enzyme. Normal rat serum P-glucuronidase and epididymal N-acetyl-/3-n-glucosaminidase were cleared slowly from the circulation when compared with rat preputial gland P-glucuronidase, liver lysosomal p-glucuronidase, and liver lysosomal N-acetyl-p-n-glucosaminidase, respectively, which were cleared rapidly. Experiments comparing the catalytic properties and molecular dimensions of the enzymes revealed no differences between rapid and slow clearance forms. Kinetic analysis using the rapid clearance forms of pglucuronidase has allowed the resolution of at least two components, rapid and slow. Clearance of the rapid component is saturable and appears to reflect binding or uptake by a limited number of sites. By contrast, the clearance rate of the slow component increased linearly with respect to dose and may be due to nonspecific or low-affinity binding. Competition experiments with pglucuronidase-free lysosomal extract and highly purified lysosomal enzymes, but not serum glycoproteins or colloidal silver, suggest that one lysosomal enzyme inhibits clearance of others and that a common mechanism may be involved in their binding.

plasma)

Lysosomal enzymes are for the most part glycoproteins having an intracellular localization (1, 2). A great deal is known about the structure and function of the lysosome as an organelle (3); however, the dynamics of the intracellular translocation and packaging of newly formed, or preexisting, hydrolases is obscure. For instance, it has been observed that many cells actively secrete lysosomal enzymes into the extracellular environment (4, 5). While the magnitude of this secretory element is unknown, the extracellular levels (i.e., ’ Supported by NIH Grants CA 12858 and GM 24096. a To whom all correspondence should be addressed. R Fellow in Medical Genetics, Division of Medical Genetics, Washington University Medical School (NIGMS Training Grant 1511). 594 Copyright All rights

0 1976 by Academic Press, of reproduction in any form

Inc. reserved.

of lysosomal

enq-ms

are very

low. The extracellular fate of lysosomal enzymes is of some current interest in relation to (i) the regulation of extracellular processes to which lysosomal enzymes contribute (e.g., the inflammatory response (4)) and (ii) the possible role of selective cellular uptake of lysosomal hydrolases in the biogenesis of lysosomes and its implication in the treatment of lysosomal enzyme deficiency storage diseases (6). An extracellular route for the packaging of lysosomal enzymes has been proposed by Neufeld and her colleagues (7, 8). Previous work from this laboratory has demonstrated the occurrence of different forms of rat pglucuronidase with “rapid” and “slow” clearance properties following intravenous injection (9). Moreover, in

CLEARANCE

OF

LYSOSOMAL

uiuo infusion experiments with several highly purified rat liver lysosomal glycosidases, including p-glucuronidase, a-mannosidase, a-fucosidase, and N-acetyl-/3-Dglucosaminidase, indicated that all of the enzymes exhibit “rapid” clearance kinetics (10). In the present report, the clearance properties of two lysosomal hydrolases, figlucuronidase and N-acetyl-p-nglucosaminidase, have been shown to vary with tissue of origin. Kinetic and competition experiments are reported with “rapid” clearance forms of p-glucuronidase and Nacetyl-B-D-glucosaminidase which suggest that rapid clearance of different lysosomal enzymes may be mediated by a common mechanism. EXPERIMENTAL

PROCEDURE

Materials Wistar rats were purchased from National Laboratory Animals, St. Louis, Missouri. Phenolphthalein glucuronic acid (PGA),4 4-methylumbelliferyl (MU)-p-glucuronic acid, p-nitrophenyl (PNP)-Nacetyl-p-glucosaminide, MU-N-acetyl-P-glucosaminide, PNP-a-mannoside, PNP-a-fucoside, PNPP-galactoside, Sephadex G-ZOO, Concanavalin A Sepharose, and Sepharose 6B were obtained from Sigma Chemical Co., St. Louis, Missouri. MU-ufucoside and MU-a-mannoside were obtained from Research Products Inc., Elk Grove, Illinois. Neosilvol was a gift from Parke Davis Co., Detroit, Michigan All other reagents were laboratory grade, obtained from local sources. Methods Enzyme assays. p-Glucuronidase was assayed using 0.001 M PGA as described by Stahl and Touster (11) or MU-glucuronic acid. The MU-glucuronide assay was performed in a total volume of 0.1 ml containing a final concentration of 0.05 M sodium acetate, pH 5.0, 0.0025 M substrate, and bovine serum albumin (1 @g/ml). Fluorescence was measured using an Aminco-Bowman fluorometer equipped with Corning filters (primary No. 5860; secondary, No. 3387 and No. 5543). Isoelectric focusing was 4 Abbreviations used: PGA, phenolphthalein glucuronic acid; MU, 4-methylumbelliferyl; PNP, pnitrophenyl; NAGA, N-acetyl-p-o-glucosaminidase; Con A, concanavalin A; GFLE, P-glucuronidase-free lysosomal extract; RSA, relative specific activity; DEAE, diethylaminoethyl; IgG, immunoglobulin G; CM, carboxymethyl.

ENZYMES

595

performed as described by Owens et al. (12). NAcetyl-p-n-glucosaminidase (NAGA) was assayed in 0.05 M sodium citrate, pH 4.5, containing 0.001 M PNP-N-acetyl-fl-glucosaminide or 0.0026 M MU-Nacetyl-Pglucosaminide in a final volume of 1.0 or 0.1 ml, respectively. a-Mannosidase was measured with 0.004 M PNP-a-mannoside or 0.0075 M MU-omannoside. The reaction mixture contained 0.01 M ZnSO, and 0.1 M sodium acetate, pH 4.3. a-Fucosidase was assayed with 0.004 M PNP-a-fucoside or MU-a-fucoside. The reaction mixture contained 0.2 M sodium acetate, pH 6.0. P-Galactosidase was assayed with 0.0125 M PNP-p-galactoside or 0.003 M MU-P-galactoside. All the assays for glycosidase activity employing calorimetric or fluorometric substrates were terminated by the addition of alkaline stopping reagent (0.133 M glycine, 0.067 M sodium chloride, and 0.083 M sodium carbonate, pH 10.7). Units of activity for all enzyme assays are micromoles per hour. The ratio of MU units/PGA units for /3-glucuronidase is approximately 2.0. Enzyme preparations. Rat preputial gland p-glucuronidase was isolated by antibody-Sepharose chromatography as described by Owens et al. (12) followed by DEAE-cellulose chromatography as described by Stahl and Touster (11) and gel filtration on Sepharose 6B. The latter was performed on a 2.5 x 80-cm column equilibrated with 0.1 M NaCl and 0.01 M Tris-Cl, pH 7.5. Except where otherwise noted, all manipulations in the preparation of enzyme were performed at 4°C. Briefly, antibodySepharose specific for P-glucuronidase was prepared with IgG fractions isolated from rabbits which had been immunized with highly purified rat preputial fi-glucuronidase. The antibody-Sepharose, prepared by the cyanogen bromide method (13), adsorbs pglucuronidase from all rat tissues tested. The enzyme was eluted from the resin with 68 M urea. Urea appears innocuous to /3-glucuronidase in that full enzymatic activity can be recovered by dialysis against urea-free buffers. Rat preputial gland p glucuronidase was also purified by a modification of the method of Ohtsuka and Wakabayashi (14). All of the rat preputial gland P-glucuronidase preparations employed in the animal experiments had a specific activity of >2000 PGA unitsimg. Liver lysosomal /%glucuronidase was isolated as described by Stahl and Touster (11) and modified by Owens et al.

WV. Rat serum pglucuronidase was isolated from large quantities (200-500 ml) of rat serum by antibody-Sepharose chromatography prepared as described above. Rat serum, which normally contains small amounts of P-glucuronidase (specific activity, 0.005 PGA unitsimg), was passed over antibodySepharose followed by elution with 8 M urea. Elution was routinely carried out at room temperature. The enzyme was subsequently chromatographed on

596

STAHL.

RODMAN

AND

Sepharose 6B equilibrated with 0.1 M NaCl and 0.01 M Tris-Cl, pH 7.5. The specific activity of the puritied enzyme was IO PGA units/mg. N-Acetyl-p-n-glucosaminidase was isolated from rat liver lysosomes. A mitochondrial-lysosomal fraction was prepared as described by Stahl and Touster (11). The fraction was suspended in 0.005 M sodium acetate, pH 5.0 (4 ml/g of liver). After standing for 20 min, the solution was sonicated for 30 s using a Bronwill sonicator (Model 11A) at setting 60. NaCl (1 M) was added to achieve a final concentration of 0.15 M. The solution was then centrifuged at 15 x lo4 g ‘min. The supernatant contained the bulk of the NAGA activity and was dialyzed against 0.05 M Na-acetate, pH 5.0. The dialyzed enzyme was chromatographed on CM-cellulose (Whatman CM52) equilibrated with 0.005 M Na-acetate, pH 5.0. Elution was achieved with a linear NaCl gradient (0 to 0.3 M). The enzyme activity, eluted as a single peak, was pooled, concentrated, and chromatographed on Sephadex G-200 equilibrated with 0.1 M NaCl and 0.05 M Na-acetate, pH 6.0. The specific activity of the enzyme at various stages of purification was as follows: homogenate, 2.8 PNP units/mg; mitochondrial-lysosomal fraction, 66 units/mg; CMcellulose, 260 units/mg; Sephadex G-200,2060 units/ w Rat epididymal NAGA was isolated from frozen tissue using the same chromatographic steps as described for liver lysosomal NAGA. After thawing, the epididymides were homogenized in 0.005 M Trisacetate, pH 6.0 using a Polytron homogenizer. The homogenate was centrifuged at 2 x 10” g.min in a Type 35 Beckman rotor. The pellet was resuspended in 0.1 M NaCl containing 0.005 M Tris-acetate, pH 6.0 and sonicated for 30 s. Following recentrifugation, the bulk of the enzyme activity was found in the supernatant fraction. The enzyme was then processed identically to lysosomal NAGA, i.e., CM-

cellulose and Sephadex G-200 chromatography. The following specific activities were found at each step of the isolation procedure: homogenate, 15 PNP units/mg; salt extract, 103 units/mg; CM-cellulose, 740 units/mg; Sephadex G-200, 2920 units/mg. p-Glucuronidase-free lysosomal extract (GFLE) was prepared from a rat liver mitochondrial-lysosoma1 fraction isolated by differential centrifugation in 0.25 M sucrose as described by Stahl and Touster (11). The isolated fraction was subjected to an osmotic shock by suspension in cold 0.005 M Tris-Cl, pH 7.5 (4 ml/g of liver). After 20 min, the solution was adjusted to 0.15 M in NaCl followed by centrifugation at 15 x 10“ g ‘min. The resulting supernatant (lysosomal extract) contained the bulk of the lysosoma1 hydrolases tested. These included Bglucuronidase, N-acetyl-p-n-glucosaminidase, a-fucosidase, cu-mannosidase, and pgalactosidase. The extract was then passed over a small pglucuronidase-specific antibody-Sepharose column as described for the purification of pglucuronidase. Other lysosomal enzyme activities such as NAGA and Pgalactosidase passed through the column unretarded. The p-glucuronidase-free extract was further purified on a column of Con A-Sepharose. All of the lysosomal enzymes tested were adsorbed to Con ASepharose. After washing with 0.15 M NaCl, the column (11 x 1.5 cm) was eluted with 0.70 M o-methyl mannoside containing 10 mM Tris-PO,, pH 7.8, and 0.05 M EDTA. The effluent was dialyzed against 0.005 M Tris-Cl, pH 7.5, containing 0.15 M NaCl followed by concentration. A summary of the copurification of several lysosomal enzyme activities appears in Table I. Animal preparations. The anesthetized female rat preparation was used for clearance experiments. Except where noted, 200-g animals were employed. The animals were anesthetized with sodium pentobarbital (30 mg/kg, ip). The femoral artery and con-

TABLE SUMMARY

OF LYSOSOMAL

ENZYME Lysosomal

U/ml /+Glucuronidase NAGA P-Galactosidase a-Mannosidase n-Fucosidase

U/mg

COPURIFICATION LYSOSOMAL

RSA 5.6 7.5

3.6

4,850

13.1 1.0

17,500 1,341

0.76

1,017

0.59

Concanavalin

Total

5.4

0.88

I IN THE PREPARATION EXTRACT”

supernatant

19.5 1.5 1.13

792

SCHLESINGER

U/ml (Not 146

U/w

OF ~GLUCURONIDASE-FREE A-Sepharose Total

detectable) 237

Overall recovery

(%)

RSA -

136 223

13.7 22

214 106

21.6 10.4

8,231

12.5

11.0

17.8

621

7.8 4.5

13.0 8.5

21.0 13.8

479

734

-

” A mitochondrial-lysosomal fraction was prepared and a solubilized extract was derived by suspending the fraction in hyoptonic buffer followed by high-speed centrifugation (see Methods). The extract was passed over a pglucuronidase-specific antibody-Sepharose column followed by concanavalin A-Sepharose chromatography. Units for Pglucuronidase are PGA units; for other enzyme activities, PNP units. Relative specific activity (RSA) is specific activity of the fraction divided by the specific activity of the original homogenate. Overall recovery refers to the amount of activity recovered, expressed as a ratio (x 100) to the activity in the original homogenate.

CLEARANCE

OF

LYSOSOMAL

tralateral vein were cannulated with PE 10 tubing filled with heparinized saline. Enzyme preparations, in 0.5 ml of 0.15 M NaC1, were infused using a Harvard constant infusion pump over a period of 70 s. Arterial blood samples were taken, starting 20 s after termination of the enzyme infusion, using standard heparinized capillary tubes. This method assured minimal blood loss to the animal during the experiment. Hematocrit values were also routinely determined. Plasma, taken from each capillary tube, was used for enzyme assays. RESULTS

Clearance of P-Glucuronidase Acetyl-b-~Glucosaminidase Intravenous Infusion

and Nfollowing

P-Glucuronidase and NAGA, from different rat tissues, were clearance following intravenous The basis for these experiments vided by earlier studies showing tain liver lysosomal glycosidases idly cleared from the circulation

TIME (hhtes)

isolated tested for infusion. was prothat cerare rapfollowing

ENZYMES

intravenous infusion (10, 15). The results summarized in Fig. 1 confirm the existante of “rapid” and %low” clearance forms of @-glucuronidase and NAGA. The enzyme preparations used were normal rat serum P-glucuronidase, rat preputial gland pglucuronidase, rat liver lysosomal NAGA, and rat epididymal NAGA. The enzyme preparations were administered to separate rats as described in Fig. 1. The clearance curves for the four enzyme preparations show that normal rat serum pglucuronidase (t 1,2 > 40 min) and epididyma1 NAGA (t,,, > 13 min) are %low” clearance forms when compared with rat preputial gland /3-glucuronidase and liver lysosomal NAGA (t,,, < 5 min), which are rapidly cleared. These conclusions are supported by the actual plasma enzyme levels before and after enzyme infusion (Table II). The “slow” clearance forms reach higher initial levels and return to control levels more slowly when compared with

TIME (Mmuter)

1. Clearance of &glucuronidase and N-acetyl-P-n-glucosaminidase. Preparations of pglucuronidase (left) and NAGA (right) isolated as described in Methods were diluted into 0.15 M NaCl and infused intravenously into the anesthetized rat. The infusate (0.5 ml), was administered in 70 s and the first blood sample was taken at 90 s with subsequent sampling as indicated. Hematocrit values were unchanged. The following enzymes were administered: preputial fi-glucuronidase, 10 PGA units; normal rat serum P-glucuronidase, 10 PGA units; liver lysosomal NAGA, 50 PNP units; epididymal NAGA, 50 PNP units. Each line represents the average of two or three animals. Enzyme assays were performed on plasma using fluorometric substrates and the enzyme levels at each point were expressed as a percentage of the first postinfusion time point. Preinfusion levels of enzyme were subtracted from postinfusion values. FIG.

597

598

STAHL, TABLE’

PRE-

AND

POSTINFUSION GLUCURONIDASE

AND

II PLASMA LEVELS AND NAGA”

Time

Enzyme 0 Normal serum pglucuronidase Preputial pglucuronidase Epididymal NAGA Liver lysosomal NAGA

RODMAN

OF p-

(min)

1.5

15

30

0.032

2.31

1.53

1.23

0.056

1.72

0.11

0.03

0.32 0.31

2.59 1.87

1.18 0.09

0.84 -b

(1 The anesthetized rat preparation was infused with various enzyme preparations as described in Fig. 1. Preinfusion plasma enzyme levels (zero time) were subtracted from postinfusion values. Data are expressed as MU units per milliliter of plasma. h Value below control level.

their rapidly cleared counterparts. Highly purified liver lysosomal p-glucuronidase, while not shown here, is cleared in a manner indistinguishable from rat preputial gland p-glucuronidase (10, 15). Since the clearance experiments revealed large differences between the same enzyme (i.e., enzyme activity), isolated from different tissues, experiments were undertaken to compare the catalytic and physical properties of the enzymes. It is conceivable, for instance, that enzyme activity, isolated from different tissues, represents the catalytic activity of different enzyme species. The results summarized in Table III show that normal serum p-glucuronidase and rat preputial gland p-glucuronidase display identical Km’s toward phenolphthalein glucuronide. A similar K, was found for liver lysosomal p-glucuronidase (9). Moreover, gel filtration chromatography experiments indicate that the molecular dimensions of the two pglucuronidases do not differ. However, isoelectric focusing analysis shows a more acid $ for the serum enzyme as compared with preputial pglucuronidase. Similarly, epidymal and liver lysosomal NAGA display identical K m’s using PNP-N-acetyl-@-glucosaminide. Chromatography on Sephadex G-200 does not distinguish the NAGA isolated from the two sources.

SCHLESINGER

Clearance of p-Glucuronidase: Relationships

Dose-Rate

The rapid clearance of p-glucuronidase and other lysosomal enzymes following intravenous injection appears to be mediated by specific recognition sites associated with the enzymes as well as specific tissue binding sites. Current experiments (16) indicate that liver is the major, if not exclusive, site of enzyme uptake. As shown in Fig. 1, the clearance of pglucuronidase is rapid and appears to be composed of several components. The presence of a slow component in the clearance curve for preputial Pglucuronidase or lysosomal NAGA is a consistent feature of the enzyme preparation, but its magnitude varies from one preparation to the next. For this reason, the experiments reported in this section were performed with selected preparations of enzyme. In addition, the kinetic experiments reported here, were made with 50-g rats in an effort to conserve enzyme. Figure 2 shows a typical clearance experiment with 50 PGA units of pglucuronidase. A semilog plot of plasma enzyme level versus time demonstrates at least two components: a rapid component (A) and a slow component @). The term component in the present context is used in a functional sense to denote enzyme clearance rates and does not necessarily refer to any one molecular species of pglucuronidase. The initial plasma level in MU units was estimated for each component by extrapolating back to zero time. While the PGA unit was used to determine the administered dose, the estimated initial plasma level in MU units was used for kinetic evaluations. Table IV summarizes the dose-rate relationships for a series of p-glucuronidase infusions. The initial rate of clearance of component A, comprising the bulk of the injected dose (see Fig. 11, was estimated from the earliest points on the clearance curve. The initial rate can be shown to be concentration dependent and saturable. This relationship is shown in Fig. 3 where rate of clearance is plotted as a function of dose. The change in initial rate with respect to dose (Fig. 3) clearly demonstrates

CLEARANCE

OF

LYSOSOMAL

TABLE SUMMARY

OF KINETIC Enzyme

AND CLEARANCE

preparation

PROPERTIES

III OF VARIOUS

Clearance

Preputial Pglucuronidase Normal serum P-glucuronidase Liver lysosomal NAGA Epididymal NAGA

599

ENZYMES

- 3 > 40 - 3 > 13

t,,, min min min min

LYSOSOMAL

ENZYME

PREPARATIONS”

Km (M) 7.7 7.4 3.1 4.2

x x x x

PI

lo-” 10-5 10-4 lo-”

6.0 5.6

’ Purified preparations of fl-glucuronidase and NAGA were tested for clearance (Fig. 1, Table I) in the anesthetized rat. The catalytic properties of each p-glucuronidase preparation were evaluated using phenolphthalein glucuronide, under standard conditions of buffer and pH (see Methods) with substrate varied from lo-’ to 2 x lo-” M. NAGA preparations were tested using lo-” to 2 x lo-” M PNP-N-acetyl-pglucosaminide. K, values are based upon double-reciprocal plots of the velocity versus substrate concentrations. Isoelectric focusing was performed as described in Methods. Clearance half-times were estimated from data described in Fig. 1 and are defined as the time required to clear 50% of t.he initial activity from the plasma.

100 y

CLEARANCE

TABLE IV RATES FOR VARYING GLUCURONIDASE”

Zero-time plasma pglucuronidase (units/ ml) “,:zpi4.7 6.7 9.8 16.2 22.0 51.0 200.0 440.0 1500.0 3010.0

TIME

(Minutes)

FIG. 2. Evaluation of a typical clearance curve for preputial pglucuronidase. Fifty PGA units of preputial p-glucuronidase were infused into the anesthetized rat as described in Fig. I. A semilog plot of plasma enzyme levels (minus preinfusion levels) vs time allows the resolution of at least two components, a rapid component (A) and a slow component (B). Initial or zero-time plasma enzyme levels (units/ml) for each component were estimated by extrapolation to zero time. The initial clearance rate (units/ml/min) for each component was calculated from their respective slopes. The estimated initial plasma levels and clearance rates are expressed in MU units.

Clearance

DOSES OF prate ml/min)

Component B

Component A

1.9 1.9 3.3 4.5 8.2 28.2 48.0 220.0 -

0.41 0.60 0.91 1.50 1.80 4.20 17.50 25.5 66.0 83.0

(units/

%Yi%0.11 0.08 0.26 0.33 0.47 2.10 2.4 10.0 -

a A series of doses of preputial p-glucuronidase, ranging from 5 to 5000 PGA units, was infused into the anesthetized rat preparation as described in Methods. Zero-time plasma levels (dose) were estimated by extrapolation for the slow (B) and rapid (A) clearance components as described in Fig. 2. The clearance rate at each dose and for each component was estimated from the appropriate slope as described in Fig. 2. Component A refers to the rapid clearance component; B refers to the slow component. Units of enzyme activity are MU units.

the saturability of component A clearance at high plasma concentrations of enzyme. The same data, expressed in a double-reciprocal plot (F’ig. 41, reveals a straight line and allows for the calculation of a K, and V, 865 units/ml and 76.9 MU unitsamin-‘, respectively. The estimation

600

STAHL.

RODMAN

AND

SCHLESINGER

of clearance rates and intercept values (dose) for component B corresponding to the same series of doses of pglucuronidase are also summarized in Table IV. The slow component B shows a rate of clearance which is linearly dependent upon dose (Fig. 5) over the dose range employed in these experiments. Evidence that Different Lysosomal Enzymes Utilize the Same Uptake Process IO DOSE

20 [Umtr)

30

x W2

FIG. 3. The variation of the clearance rate of component A of P-glucuronidase as a function of the estimated zero-time plasma level of enzyme. Clearance rates and initial plasma levels were estimated for component A using a series of p-glucuronidase injections, varying from 5 to 5000 PGA units, as described in Fig. 2 and summarized in Table IV. The curve shows the saturable nature of the rapid clearance component.

25 1

The saturation-type kinetics displayed by the clearance of P-glucuronidase (component A) suggest that a finite number of binding sites mediate the uptake of enzyme from extracellular space. Since several highly purified liver lysosomal enzymes were shown by earlier work to behave similarly to /3-glucuronidase (10, 15) (i.e., rapid clearance), a blocking reagent was prepared from liver lysosomal fractions for inhibition experiments to determine whether clearance of pglucuronidase could be impeded or retarded. P-Glucuronidase-free lysosomal extract (GFLE) was prepared as described in Methods using concanavalin A-Sepharose chromatography. As a control to lysosomal enzymes purified by Con A-Sepharose chromatography, serum glycoproteins were isolated

DOSE ~3-GLUCURONIDASE

4. Double-reciprocal plot of p-glucuronidase (component A) clearance rate and initial plasma level of enzyme. The data derived from the dose-rate experiment (Table IV) described in Fig. 3 are plotted by a double-reciprocal plot. AK, (865 MU units/ml) estimated from a slope of 11.25 and a V of 76.9 MU units.min-‘. The latter was estimated using an intercept value of 0.013. FIG.

(Units)

FIG. 5. The variation of the clearance rate of component B of p-glucuronidase as a function of the estimated zero-time plasma level of enzyme. Clearance rates and initial plasma levels were estimated for component B using a series of p-glucuronidase injections, varying from 5 to 700 PGA units, as described in Fig. 2 and summarized in Table IV. The curve shows the linear dependence of clearance rate on initial plasma enzyme levels.

CLEARANCE

OF

LYSOSOMAL

from fresh rat serum by the same method. Neosilvol (colloidal silver) was used as an agent for blocking colloidal nonspecific clearance by the reticuloendothelial system. The effect of varying doses of GFLE on the clearance of pglucuronidase is shown in Fig. 6. The extract was injected 2 min prior to a test dose (10 PGA units) of flglucuronidase. Using three doses of extract (0.31,0.72, and 3.3 mg), the clearance rate of p-glucuronidase was retarded in dose-related fashion (Fig. 6, Table V). The results are consistent with the notion that certain recognition signals mediate the binding and clearance of fi-glucuronidase. By presenting other lysosomal enzymes to the system, the clearance of fl-glucuronidase is effectively reduced. Other agents (e.g., serum glycoproteins, colloidal silver) administered in the same way as GFLE, had no effect on the clearance rate of /3glucuronidase (Table V). Inhibition ofNAGA Clearance Preputial p-Glucuronidase

with Rat

Rat liver lysosomal NAGA, like p-glucuronidase, is cleared very rapidly following intravenous injection (Fig. 1). To test whether one highly purified lysosomal enzyme (preputial pglucuronidase) will compete with another (NAGA) for clearance, experiments were undertaken using one enzyme as agonist and the second as antagonist. Using a constant dose (10 PNP units) of NAGA, varying doses of pglucuronidase were administered simultaneously to determine whether the clearance rate of the former would be impeded. The results shown in Fig. 7 show clearance of NAGA with the plasma levels of enzyme expressed as a percentage of the first postinfusion time point. PGlucuronidase administration produced a shift in the clearance curve for NAGA with a large increase in plasma half-life. Clearance rates, calculated as described in Fig. 2 and summarized in Fig. 8, show that the rate of NAGA clearance was reduced in dose-related fashion. The highest dose of pglucuronidase reduced the clearance rate fourfold. Control infusion of p-glucuronidase alone had no effect on plasma levels of NAGA over a 60-min period.

601

ENZYMES

10

lnflucn

5

10

TIME (M~wtes)

FIG. 6. Effect of /3-glucuronidase-free lysosomal extract on clearance of pglucuronidase. Semipuritied lysosomal enzymes, rendered free of p-glucuronidase by specific adsorption with antibody-sepharose, was used as an antagonist to the clearance of /3-glucuronidase. The extract was injected 2 min prior to the test dose of Pglucuronidase (10 PGA units). Clearance is expressed as a percentage of the first postinfusion time point with preinfusion plasma values subtracted as described in Fig. 1. The clearance rates for these and other antagonists are summarized in Table V.

DISCUSSION

The concept that lysosomal enzymes possess recognition sites which mediate their pinocytic uptake by cultured cells has been proposed and documented by Neufeld and her colleagues (7,8) and Brot et al. (17). We have focused on the in uiuo clearance of lysosomal enzymes from plasma. Previous work has demonstrated (i) that of the five purified liver lysosomal enzymes tested, all were cleared from the circulation with apparent “rapid” kinetics (10, 15) and (ii) that rapidly cleared enzymes like /$glucuronidase and NAGA can be converted into slow clearance forms by oxidation with sodium periodate (10, 15), suggesting that recognition is mediated by carbohydrate residues associated with the enzymes. Moreover, certain organophosphate compounds elevate a form of @-glucuronidase in plasma which, when isolated and tested for clearance, displays “slow” clearance kinetics (9). The mechanism of clearance in viuo involves the liver and possibly the spleen since evisceration substantially reduces clearance (9, 16). Whether specific uptake of enzymes by fi-

602

STAHL,

RODMAN

AND TABLE

EFFECT

OF VARIOUS

AGENTS

V

ON THE CLEARANCE

RATE

OF ~GLUCURONIDASE”

Inhibitor

Enzyme Preputial Preputial Preputial Preputial Preputial Preputial

SCHLESINGER

pglucuronidase pglucuronidase P-glucuronidase /3-glucuronidase Pglucuronidase Pglucuronidase

(10 (10 (10 (10 (10 (10

U) U) LJ) U) U) U)

GFLE GFLE GFLE Serum Neosilvol

Clearance Rate (units/ml/min)

(0.31 mg) (0.72 mg) (3.1 mg) glycoproteins (25 mg)

0.35 0.25 0.10 0.09 0.36 0.26

(3.3 mg)

n The clearance rate of preputial pglucuronidase (10 PGA units) was determined following the administration of various potential inhibitory agents. Two minutes prior to infusion of p-glucuronidase, varying doses of &glucuronidase-free lysosomal extract were injected intravenously. Following enzyme infusion, blood samples were taken as described in Fig. 1. Clearance rates were calculated from slopes as described for component A in Fig. 2. pGlucuronidase activity is expressed as MU units.

O-O NAGA -NAGA* u P-GLUC WNAGA.12OOv p-~tt~ -NAGA+2@3OuP-GLUC

L

I

Xc

1000

I

IS00

DOSE OF D-GLUCURONIDASE

200( IUml

I1

FIG. 8. Effect of p-glucuronidase on the clearance rate of NAGA. Clearance rates for liver lysosoma1 NAGA were estimated as described in Fig. 2 before and after addition of various amounts of purified -preputial pglucuronidase to the infusate as described in Fig. 7. The clearance rate (units/ml/ min) was reduced fourfold by the highest dose of pglucuronidase. Enzyme activity is expressed as MU units.

TIME (M(n)

FIG. 7. Effect of /%glucuronidase on the clearance of liver lysosomal NAGA. The clearance of NAGA (20 PNP units) was determined as described in Fig. 1 except that various amounts of preputial pglucuronidase (500, 1200, and 2000 PGA units) were included in the test injection. The t,,? for NAGA (3.0 min) was shifted to 5.5, 8.5, and 12.0 min, respectively, corresponding to the three levels of pglucuronidase blockade. Independent control experiments showed that injection of Pglucuronidase alone had no effect on plasma NAGA levels. Each line was drawn from the average of two animals.

broblasts in culture as opposed to clearance of enzyme in. vivo are comparable phenomena is uncertain. Preliminary experiments indicate that the two may be

dissimilar, e.g., rapidly cleared /?-glucuronidases (rat liver lysosomal, rat preputial) are not taken up by cultured fibroblasts (Sly, Touster, and Stahl, unpublished observations). The observation that purified normal serum pglucuronidase displays slow clearance properties is interesting and may account the natural occurrence of extracellular enzyme. Since normal serum p-glucuronidase is very similar to rat pglucuronidase from other sources (e.g., being isolable by antibody-Sepharose chromatography, having similar catalytic properties and physical dimensions), it is possible that minor differences, perhaps reflected in a different pI, are due to the absence of an exposed recognition site. On the other hand, the similarity between normal se-

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rum /3-glucuronidase and organophosphate-induced serum p-glucuronidase is striking. The latter displays slow clearance and has an acid isoelectric point (pH 5.6), but is in all other respects similar to rapid clearance forms of pglucuronidase. It is possible that trace amounts of lysosoma1 enzymes occur in plasma for obscure physiologic reasons, but these are questions which must await definitive biosynthetic and structural testing. Unfortunately, the quantities of p-glucuronidase normally found in plasma (co.1 PGA unit/ ml plasma) are very low. Rat liver and epididymis, on the other hand, are rich sources of NAGA and may provide suficient material for structural comparisons. The material used in the clearance experiments was highly purified and the two preparations, while having divergent clearance rates, had very similar specific activities. While the normal cellular disposition of epididymal NAGA has not been disclosed, one possibility is that epididyma1 NAGA is a form of the enzyme which is destined for secretion. The clearance profiles for preputial gland /3-glucuronidase and liver lysosomal /3-glucuronidase are very similar, if not identical. Since preputial gland pglucuronidase is readily available in highly purified form, it was used in the present study. The clearance curve for preputial pglucuronidase, as shown in Fig. 2, can be resolved into two components: a “rapid” component (A) comprising the bulk of the injected dose and a “slow” component (B). Similar observations have been made for liver lysosomal pglucuronidase, NAGA, and other liver lysosomal enzymes. The amount of slow component (B) varies from preparation to preparation. To analyze, in more quantitative terms, the clearance of the two components, a dose-rate experiment was made using doses which varied from 5 to 5000 PGA units of enzyme. For calculations, initial or zero-time plasma enzyme levels were estimated by extrapolation. This method protects against possible error deriving from incomplete delivery of the injected dose or inaccurate estimation of circulating volume. Using this convention, the clearance of component A is a saturable

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process and obeys Michaelis-Menten kinetics. The estimated K, would suggest that an injected dose sufficient to produce an initial plasma concentration of 865 MU units/ml would produce a clearance rate which is half-maximal. The slow component B, on the other hand, shows linear kinetics where the rate of clearance is directly proportional to the plasma enzyme concentration over the range of enzyme concentrations achieved. Evaluation of component B is difficult at the highest dose of enzyme since at that dose component A is nearly saturated and the change in slope of the semilog plot of clearance was not clearly defined over a 60-min experiment. From the data in hand, it is clear that the clearance of component B is distinct from that of component A. Several explanations for this distinction are possible: (i) Enzyme preparations are composed of multiple forms, some of which lack the specific recognition sites necessary for rapid clearance as component A. These enzyme forms would be cleared slowly or not at all by the receptors responsible for component A. (ii) Enzymes are also cleared by nonspecific or fluid pinocytosis that does not involve specific binding sites or cellular receptors. This system could have large capacity and would not necessarily be saturated by the doses employed in this study, but would display linear dose dependence. (iii) Alternative possibilities for the presence of more than one component include uptake by different liver cell types, uptake by other specific mechanisms having low-affinity binding, or complex variations in the populations of cell receptors or enzyme recognition sites responsible for binding. The current data do not allow one to construct an exclusive kinetic model since any of the above possibilities cannot be ruled out. The concept that a cell surface receptor or class of receptors mediates the clearance of lysosomal enzymes is supported by the observation that p-glucuronidase-free lysosomal extract was able to reduce, in dose-related fashion, the clearance of pglucuronidase. On the other hand, serum glycoproteins, isolated using the Con ASepharose chromatography method, were ineffective in combatting clearance. This

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control was done to demonstrate that glycoproteins which bind Con A-Sepharose are not necessarily successful inhibitors. Several lysosomal enzyme activities present in the GFLE, i.e., pgalactosidase, NAGA, a-fucosidase, and a-mannosidase, were followed for clearance in separate experiments. Interestingly, at low doses of GFLE, the respective activities were rapidly cleared from the circulation whereas at high doses of GFLE, their clearance was appreciably retarded. These observations, along with those where clearance of p glucuronidase was impeded, support the conclusion that at high doses of GFLE, the mechanism for rapid clearance (i.e., receptors) was saturated and further increases in enzyme concentration only increased clearance by slower, high capacity, nonspecific mechanisms. The experiments showing inhibition of NAGA clearance by fl-glucuronidase were designed as such because the p-glucuronidase employed as antagonist was known to be of near-maximal specific activity (>2000 PGA units/mg). While the NAGA preparations were highly purified, we were not able to show that they were homogeneous by polyacrylamide gel electrophoresis. The experiments show that /3glucuronidase effectively serves as an antagonist to the clearance of NAGA and reduces the clearance rate of the latter. The results are consistent with the proposal that a population of receptor(s) exist, tentatively assigned to liver, which mediate the rapid component (A) of lysosomal enzyme clearance. Colloidal silver (Neosilvol) was employed to rule out the possibility that enzyme clearance was being mediated by the mechanisms similar to those for nonspecific colloid uptake by Kupffer cells. Earlier experiments (9) had already ruled out the possibility that enzyme clearance is mediated by mechanisms similar to those described by Ashwell and Morel1 (18) for blood asialoglycoproteins. The latter are not competitive with preputial or lysosoma1 /3-glucuronidase for clearance. The recent reports of Lunney and Ashwell (19) and Stockett et al. (20) demonstrating the presence, in liver, of receptors which recognize terminal N-acetyl-glucosamine res-

AND

SCHLESINGER

idues on modified serum glycoproteins are of considerable interest and may be relevant to the findings reported in this paper. Recent experiments from our laboratory have demonstrated that agalacto-orosomucoid is an effective inhibitor of lysosoma1 enzyme clearance (Stahl, et al. (21), submitted for publication), suggesting that N-acetyl-glucosamine plays an important role in the clearance mechanism. These results, along with earlier studies showing that periodate oxidation of lysosomal enzymes abolishes their rapid clearance (10, 15), are consistent with the concept that recognition is mediated via specific sugar residues associated with lysosomal glycosidases. The rapid clearance of lysosomal enzymes from the plasma appears to involve two determinants, recognition sites associated with enzyme molecules and binding sites associated with cells. The physiological importance of such sites, aside from keeping extracellular levels of lysosomal enzymes very low, is unclear. They may play some role in the packaging of newly formed enzyme; however, there is no evidence of a biosynthetic or kinetic nature at the present time to support such a hypothesis. ACKNOWLEDGMENTS The authors are pleased to acknowledge the assistance of Dr. J. W. Owens in the preparation of enzyme. Special thanks are due to Dr. W. S. Sly, Dr. D. R. Geller, and Dr. 0. Touster for their critical review of the manuscript and to Ms. Jill Miller for technical assistance. REFERENCES 1. DE DUVE, C., PRESSMAN, B. C., GIANETTO, R., WATTIAUX, R., ANDAPPLEMANS, F. (1955)&othem. J. 60, 694617. 2. BISHAYEE, S., AND BACHHAWAT, B. K. (1974) Biochim. Biophys. Acta 334, 378388. 3. DE DUVE, C., AND WATTIAUX, R. (1966) Ann. Rev. Physiol. 28, 435-492. 4. HENSON, P. (1971)5. Exp. Med. 1347,114s-135s. 5. WEISMAN, U., DIDONATO, S., AND HERSCHKOWITZ, N. N. (1975)Biochem. Biophys. Res. Commun. 66, 1338-1343. 6. SLY, W. S., GLASER, J., ROOZEN, K., BROT, F., AND STAHL, P. (1974) in Enzyme Therapy in Lysosomal Storage Diseases (Tager, J., Hooghwinkel, G. J. M., and Deams, W. Th., eds.), pp. 228-291, North-Holland, Amster-

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dam. 7. NEUFELD, E. F. (1975) in Cell Communication (Cox, R., ed.), pp. 217-231, Wiley, New York. 8. HICKMAN, S., AND NEUFELD, E. (1972)Biochem. Biophys. Res. Commun. 49, 992-999. 9. STAHL, P., MANDELL, B., RODMAN, J. S., SCHLESINGER, P., AND LANG, S. (1975) Arch. Biochem. Biophys. 170, 536-546. 10. STAHL, P., SIX, H., RODMAN, J. S., SCHLESINGER, P., TULSIANI, D., AND TOUSTER, 0. (1976) Proc. Nat. Acad. Sci. USA, in press. 11. STAHL, P., AND TOUSTER, 0. (1971) J. Biol. Chem. 246, 5398-5406. 12. OWENS, J. W., GAMMON, K. L., AND STAHL, P. (1975) Arch. Biochem. Biophys. 166, 258-272. 13. CUATRECASAS, P. (1970) J. Biol. Chem. 245, 30593065. 14. OHTSUKA, K., AND WAKABAYASHI, M. (1970)Enzymologia 39, 109.

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15. STAHL, P., SCHLESINGER, P., SIX, H., AND TousTER, 0. (1975) Abstracts of the 29th Annual Meeting of the Society of General Physiologists, p. 9a. 16. SCHLESINGER, P., RODMAN, J. S., FREY, M., LANG, S., AND STAHL, P. (1976) Arch. Biothem. Biophys. 177, 606-614. 17. BROT, F. E., GLASER, J. H., ROOZEN, K., SLY, W., AND STAHL, P. (1974) Biochem. Biophys. Res. Commun. 57, 14. 18. ASHWELL, G., AND MORELL, A. (1975) Biochem. Sot. Symp. 404, 117-124. 19. LUNNEY, J., AND ASHWELL, G. (1976) Proc. Nat. Acad. Sci. USA 73,341343. 20. STOCKERT, R., MORELL, A., AND SCHIENBERG, I. (1976) Biochem. Biophys. Res. Commun. 68, 988-993, 21. STAHL, P., SCHLESINGER, P. RODMAN, J. S., AND DOEBBER, T. (1976) Nature, in press.