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Assay and purification of a solubilized membrane receptor that binds the lysosomal enzyme α-l -iduronidase
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 214, No. 2, April 1, pp. 681-687, 1982
Assay and Purification of a Solubilized Membrane Receptor the Lysosomal Enzyme cu-L-lduronidase’ ANTON W. STEINER
AND
That Binds
LEONARD H. ROME’
Department of Biological Chemistry and the Mental Retardation Research Center, UCLA School of Medicine, LOSAngeles, California 900.24 Received September
23, 1981, and in revised form November
7, 1981
A receptor that binds the lysosomal enzyme cu-L-iduronidase via phosphorylated mannose residues on the enzyme has been solubilized from Swarm rat chondrosarcoma membranes using a pH 9.5 buffer containing 0.1% Triton X-100. Detergent-solubilized receptor in crude and purified preparations was measured by assay of bound a-L-iduronidase after adsorbing the receptor-enzyme complex onto insoluble phospholipid vesicles (liposomes). Binding of a-L-iduronidase to the liposomes required receptor and was completely inhibited by mannose 6-phosphate but not glucose 6-phosphate, indicating that the receptor maintained specificity following solubilization. Receptors from rat chondrosarcoma and human diploid fibroblasts were purified to apparent homogeneity using a phosphomannan-Sepharose affinity column. Both had identical molecular weights in polyacrylamide gels containing sodium dodecyl sulfate (Mr = 215,000). Amino acid analysis and two-dimensional gel electrophoresis was carried out on the purified rat chondrosarcoma receptor. Two forms of the receptor with different PI’S were observed (~1 5.5 and 6.2). One form (~1 5.5) was made more basic (PI 5.8) by treatment with neuraminidase.
The existence of cell surface receptors for lysosomal enzymes was first proposed by Hickman and Neufeld (l), in order to explain the efficient uptake of certain hydrolytic enzymes by cultured fibroblasts. Evidence for these receptors came initially from kinetic studies of enzyme internalization (2-4) and later from direct binding experiments using intact fibroblasts (5, 6) and isolated fibroblast membranes (‘7, 8). Membranes of the Swarm rat chondrosar’ Research supported by USPHS Grant HD-06576, American Cancer Society Institutional Grant IN-131, and the NIH Cancer Center Core Support Grant USPHS CA 16042 (UCLA Jonsson Compreshensive Cancer Center); California Institute for Cancer Research. A.W.S. is an NICHD postdoctoral trainee (HD-07032). * To whom correspondence should be sent. 681
coma were found to be particularly rich in receptor (7). The membrane-associated receptors bind only to lysosomal enzymes that contain a specific recognition marker. A number of studies have demonstrated that the recognition marker is a terminal mannose 6phosphate moiety (9-11). Comparison of binding and internalization properties suggest that these receptors are identical to the cell surface proteins that mediate enzyme internalization and packaging of these enzymes into lysosomes. Recently a receptor that binds mannose 6-phosphate moieties of fl-galactosidase has been purified from bovine liver (12). In the present study we have solubilized and purified a receptor that binds a-L-iduronidase from rat chondrosarcoma and human fibroblasts. The purification was 0003-9861/82/040681-07$02,00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
682
STEINER
facilitated by an assay for receptor that is based upon the association of soluble receptor with insoluble phospholipid vesicles. A preliminary report of this work has been presented (13). EXPERIMENTAL
PROCEDURES
Materials. Swarm rat chondrosarcoma tissue, a rat cartilagenous tumor, was the gift of Dr. J. Kimura, NIH, Bethesda, Maryland. Phosphomannan from Hansenuh ho&ii (NRRL Y-2448) was a gift of Dr. M. Slodki, USDA Northern Regional Research Laboratory, Peoria, Illinois. Neuraminidase Type VI was purchased from Sigma. The barium salt of mannose 6-phosphate was from U. S. Biochemicals and was converted to the sodium salt by passage over Dowex 5OW-X8 (Na+ form). Sodium mannose 6-phosphate was decolorized with charcoal, lyophilized, and redissolved in water. Iodogen was obtained from Pierce Chemicals. Amerlite XAD-2 beads were purchased from Sigma and washed with methanol and water before use. Carrier free Na’? was from Amersham. oc-L-Iduronidase was purified from normal human urine as described (2, 5). of mewdrrane fra,&ms. One hundred Preparation grams of solid chondrosarcoma tissue, pressed through a stainless-steel sieve (14), was suspended in 300 ml of 2X concentrated buffer A (10 mM KH2POI, pH 6.0, 0.15 M NaCl, and 0.02% NaN3). The suspension was homogenized in a Polytron for 3 min and subsequently processed in a Gaulin Mill Model 15M (Gaulin Corp., Everett, Mass.) at 6000 psi. The suspension was recirculated through the press several times while the temperature was maintained at 20°C by the addition of crushed ice. The final solution (ea. 500 ml) was centrifuged for 40 min at 10,000 rpm in a Sorvall GSA rotor. The resulting supernatant was recentrifuged at 35,000 rpm for 1 h (100,OOOg) in a Beckman type 42.1 rotor. The high-speed membrane pellet was suspended in buffer A containing 10 mM mannose 6-phosphate and placed on ice for 1 h. This step was included to displace endogeneous lysosomal enzymes bound to the receptor. The membranes were recentrifuged, and the pellets washed with buffer A and centrifuged again. Final membrane pellets were resuspended in a small volume of buffer A and assayed for binding activity as described (7). Human diploid fibroblasts were grown and harvested as described (5). Cells obtained from 18 tissue culture flasks (150 cm’) were suspended in 0.25 M sucrose and disrupted by sonication for 10 min with a probe sonicator. The broken cells were centrifuged at 12,000 rpm in a Sorvall SM-24 rotor for 20 min and the resulting supernatant centrifuged for 1.5 h at 35,000 rpm in a Beckman type 40 rotor. Final membrane pellets were washed once with buffer A containing 10 mM mannose 6-phosphate and once with buffer A alone as described above. Membrane preparations were routinely stored at -20°C.
AND ROME Receptor solubilization. Approximately 20 mg of rat chondrosarcoma 100,OOOg membrane protein was stirred overnight at 4°C with 3 ml of extraction buffer which contained: 0.1 M sucrose, 0.05 M KCl, 0.04 M KH2P04, pH 9.5, 0.03 M EDTA, 0.1% Triton X100, and 13% glycerol. Insoluble material was removed by centrifugation at 120,OOOgfor 2 h, and the extract was dialyzed against 1 liter of 20 mM TrisHCl, pH 7.5. Following dialysis, the solution was again clarified by centrifugation and the supernatant used as the source of soluble receptor. Fibroblast membranes were solubilized3 with 2.5 ml of extraction buffer and processed in the same manner. Detergent-solubilized rat chondrosarcoma membrane protein (2.1 mg) was iodinated with 2 mCi of Na’%I using the iodogen procedure (15). Fibroblastsoluble membrane protein was labeled witch 750 &i Na’%I. Unreacted iodide was removed by dialysis against buffer A containing0.05% Triton X-100. Typically 30% of the added radioactivity was incorporated into nondialyzable material. Phosphmnnan-Sepharose af$nity column. Phosphomannan was acid hydrolyzed to core and pentasaccharide fragments (16), both presumably terminating in mannose 6-phosphate moieties (17,18). The core was precipitated with 1 vol 95% ethanol, centrifuged, and resuspended in 0.1 M sodium bicarbonate, pH 8.6. Approximately 5 g of core was mixed with 125 ml of Sepharose 4B activated with 27 g of cyanogen bromide. Coupling and removal of reactive groups was as described (19). The phosphomannanSepharose was washed and equilibrated before use with buffer B (50 mM citrate-phosphate buffer, pH 6.0, 0.15 M NaCI, 0.1% Triton X-100, and 0.1% NaN,) containing 1 mg/ml BSA.4 Soluble receptor assay. Prior to assay for receptor binding activity, Triton X-100 was removed from the solubilized membrane preparations by addition of Amberlite XAD-2 beads (1.2 g/ml of detergent solution). After 1 h of gentle shaking at 4”C, the beads were removed by a brief centrifugation. Binding mixtures contained in a final volume of 150 ~1; 1.25 units of a-L-iduronidase (2.5 X lo-’ M final concentration), 50 mM 2-(N-morpholino)ethanesulfonic acid (Na+), pH 6.4, and detergent-free membrane protein. Mannose 6-phosphate or glucose 6-phosphate was included in some assays at a final concentration of 10 mM. Binding mixtures were incubated for 1.5 h at 0°C. Twenty-five microliters of a lipsome mixture of egg lecithin and stearylamine, prepared according to Eimerl et al. (20), was then added and the solution maintained at 0°C for 15 min. Liposomes were aggregated by the addition of 5 ~1 of 1 M MgC12. The
3The term “solubilized” is used through out this manuscript to refer to material that does not sediment when centrifuged at 120,OOOgfor 2 h. 4 Abbreviations used: BSA, bovine serum albumin; SDS, sodium dodecyl sulfate.
ASSAY
AND PURIFICATION
OF A RECEPTOR
mixture was placed on ice for 30 min, and the liposomes were collected by centrifugation at 13,OOOgfor 15 min. Liposome pellets were resuspended in 50 ~1 buffer A containing 0.1% Triton X-100 and 100 rg/ ml BSA and assayed for cY-L-iduronidase activity as described (5), where one unit of enzyme activity represents the conversion of 1 nmol of substrate to product per hour at 37°C.
RESULTS
Receptor solubilixation and assay. Detergent extraction of the 100,OOOgchondrosarcoma membrane fraction released about 60% of the total membrane protein into solution. Approximately 37% of the original binding activity remained membrane associated following detergent solubilization and recentrifugation. An assay method was developed to detect and quantitate the amount of soluble receptor which remained active following solubilization. This method was based on a technique developed by Eimerl et al+ (20) for insertion of the p-adrenergic receptor into intact cells. A rapid assay was desired to
IL
&-oy? 10
20
30
Protein
ENZYME
Mw Buffer Buffer STDS Wash +G6P
683
Buffer +M6P
200K116K94K68K-
AND DISCUSSION
I
FOR A LYSOSOMAL
I
I
I
I
40
50
60
(gg9)
FIG. 1. Soluble receptor assay. Detergent-free soluble membrane protein at the concentrations indicated, was incubated with ol-L-iduronidase, adsorbed onto liposomes and bound enzyme quantitated as described under Experimental Procedures. Crude soluble membrane protein (0); crude soluble membrane protein not retained by the phosphomannan-sepharose affinity column (0). The values shown have been corrected for nonspecific binding (i.e., enzyme bound in the presence of 10 mM mannose 6-phosphate). This binding did not exceed 12 munit/assay.
FIG. 2. Purification of ‘%I-labeled rat chondrosarcoma receptor. Rat chondrosarcoma membranes were solubilized, iodinated, and passed over a l-ml phosphomannan-Sepharose affinity column. The column was washed with 100 ml of buffer B containing 1 mg/ ml BSA, followed by 10 ml of 10 m!d glucose B-phosphate in buffer B containing 1 mg/ml BSA, and linally eluted with 10 ml of 10 mM mannose 6-phosphate in buffer B containing 1 mg/ml BSA. Selected fractions from each wash (containing 15,000-30,000 cpm) were pooled and protein was precipitated by addition of 10 vol of cold 95% ethanol. Precipitates were resuspended in sample buffer containing SDS and subjected to electrophoresis in an 8% acrylamide gel (23). Autoradiography was carried out on the dried gel. The molecular weights indicated represent the following standards (Bio-Rad Laboratories): myosin, 200,000; fl-galactosidase, 116,250; phosphorylase B, 92,500; bovine serum albumin, 66,200; ovalbumin, 45,000.
facilitate the comparison of various procedures used to extract active receptor from membranes. The liposome assay method used relies on the immobilization of receptor by adsorption onto insoluble phospholipid vesicles. The receptor is quantitated by assay of a bound ligand, the enzyme a-L-iduronidase. Figure 1 shows that the amount of a-~iduronidase which is specifically bound to the insoluble lipid vesicles is dependent on the amount of crude solubilized membrane protein present. The solubilization treatment used did not significantly destroy receptor binding activity since up to 70% of the binding capacity which was lost from the membranes after solubilization could be accounted for with the liposome assay. That the binding of enzyme to liposomes was mediated through a specific
684
STEINER PROTEIN
STAIN
warm 200K-
-
116K94K-
^___ m
68K-
xI_
43 K-
c%- -FIG. 3. Comparison of rat chondrosarcoma and human diploid fibroblast receptors. Rat chondrosarcoma and human diploid fibroblast (HDF) membranes were solubilized as described under Experimental Procedures. Affinity-purified ‘%I-receptor was added as tracer to the crude chondrosarcoma-soluble protein and the mixture passed over a 16-ml phosphomannan-Sepharose affinity column. After extensive washing, receptor was eluted with 10 mM mannose 6-phosphate in buffer B (75 ml), and aliquots were analyzed as described above. Fibroblast membranes were extracted, iodinated, and purified on a l-ml phosphomannon-Sepharose affinity column. RCl and RC2 represent the material obtained from two separate purifications of rat chondrosarcoma tissue.
receptor was shown by testing receptorfree membrane protein in the liposome assay. This material was not retained by the phosphomannan-Sepharose affinity column used for receptor purification. As expected, practically no cY-L-iduronidase was bound when this fraction was used as the source of protein in the assay. Saturable binding of cY-L-iduronidase to the soluble receptor was observed using the liposome assay. A dissociation constant of 1 X lo-‘M, calculated by the method of Scatchard (21), was similar to that found for intact chondrosarcoma membranes (7). The soluble receptor assay should be useful for detection of a wide variety of membrane-derived receptors by quantitation of bound ligand. Pur$cation of receptor. Detergent-solubilized membrane proteins were iodinated and passed over the phosphomannan-Sepharose affinity column. Extensive washing with buffer B (usually 100 column ~01s) was necessary to reduce the back-
AND ROME
ground radioactivity. Figure 2 shows that when mannose 6-phosphate (but not glucose 6-phosphate) was included in the wash buffer, a radioactive protein of molecular weight 215,000 was eluted. We presumed that this labeled protein was the receptor for a-L-iduronidase and that iodination of the impure mixture of membrane proteins did not destroy the mannose 6-phosphate specific binding properties of the receptor. The amount of rat chondrosarcoma tissue used for purification was increased to 750 g in order to have quantities sufficient for further characterization. The material obtained was analyzed by gel electrophoresis in the presence of SDS. As shown in Fig. 3, two separate receptor preparations were found to be homogeneous with respect to protein staining by Coomassie blue. Purified ‘%I-receptor was added as tracer during affinity purification to facilitate its location in column fractions. The purified unlabeled receptor comigrated with the labeled tracer receptor. Approximately 700 pg of purified receptor (determined by the method of Lowry et al, (26)) was obtained from 1 g of high-speed membrane protein (from ca. 750 g tumor). Furthermore, iodination of the purified receptor protein and subsequent electrophoresis did not reveal even minor contaminating proteins. High-speed membranes from human diploid fibroblasts were solubilized, labeled with Nan9 and passed through the phosphomannan-Sepharose affinity column. A radioactive protein of molecular weight 215,000was specifically eluted with 10 mM mannose 6-phosphate indicating that at least with respect to molecular weight on polyacrylamide gels in the presence of SDS, the receptor from rat chondrosarcoma and from human fibroblasts are identical. Recently an ‘%I-labeled receptor of 215,000 molecular weight that binds mannose 6-phosphate residues of bovine testicular B-galactosidase was isolated from bovine liver, human diploid fibroblasts, and Chinese hamster ovary cells by Sahagian et al. (12). In addition we have found that a-mannosidase purified from Lktyostelium discoideum and shown to
ASSAY
AND PURIFICATION
OF A RECEPTOR TABLE
FOR A LYSOSOMAL
685
ENZYME
I
SOLUBLE RECEPTOR ASSAY WITH PURIFIED ?-RECEPTOR’ Pellet Incubation ‘“I-receptor
“Mock”
+ enzyme
receptor
‘z51-Receptor only
+ enzyme
SN (wm)
Enzyme bound (munit)
Addition
wm
sb
None Man 6-P” Glc 6-P
1736 1559 1567
21.0 20.0 19.6
6522 6215 6414
91.1 7.7 83.9
None Man 6-P
-
-
-
14.8 14.6
None Man 6-P
1795 1792
22.1 21.1
6319 6679
-
a Affinity-purified ‘%I-receptor from rat chondrosarcoma was mixed with the unbound protein fraction from the affinity column. “Mock” receptor was prepared by substituting buffer B for the labeled receptor. After removal of detergent the sample was assayed for soluble receptor. Liposome pellets were assayed for both ol-L-iduronidase activity and bound radioactivity. b Percentage of total radioactivity in pellet + supernatant (SN). ’ Man 6-P, mannose 6-phosphate (10 mM); Glc 6-P, glucose 6-phosphate (10 mM).
bind to human diploid fibroblasts via a phosphate specificity in the soluble recepmannose 6-phosphate recognition marker tor assay. Mannose 6-phosphate inhibited (22), also specifically bound rat chondro92% of the a-L-iduronidase binding activsarcoma membranes (data not shown). Thus it appears that a common 215,000 TABLE II molecular weight protein is present on a AMINO ACID ANALYSIS OF RAT variety of different cell types that can CHONDROSARCOMA RECEPTOR bind a number of hydrolytic enzymes bearAmino acid Residues/m01 ing a mannose 6-phosphate recognition marker. This binding protein is most likely Asx 184 involved in the internalization and packThreonine 110 aging of lysosomal enzymes. Serine 142 Soluble receptor assay with puri&ed 1251Glx 178 receptor. In order to further validate the Proline 110 Glycine 165 soluble receptor assay and demonstrate Alanine 127 directly that the 215,000 molecular weight Valine 129 protein isolated from the affinity column Methionine 17 was the receptor for a-L-iduronidase, we Isoleucine 52 used affinity-purified ‘%I-receptor in the Leucine 96 soluble receptor assay. Prior to assay, the Tyrosine 75 purified receptor was first mixed with the Phenylalanine 75 protein fraction not retained on the affinHistidine 44 ity column. This step was necessary to Lysine 100 prevent nonspecific adsorption of the reArginine 84 ceptor to the Amberlite XAD-2 beads used Note. Forty-eight micrograms of affinity-purified to remove detergent. receptor was hydrolyzed with 6 M HCl overnight at Table I shows that approximately 20% 110°C under vacuum and amino acid analysis perof the total soluble receptor was adsorbed formed on a Beckman Model 120C amino acid anaonto liposomes independent of the pres- lyzer. Values represent the averages of two separate ence or absence of enzyme. As observed analyses. Calculations are based on a molecular with crude receptor preparations, purified weight of 215,000 and excluded analysis of half-cyslabeled receptor displayed mannose 6- tine, tryptophan, and ammonia.
686
STEINER PROTEIN
AND ROME AUTORADIOGRAPH
STAIN Conlrol
Treated 8.0
7.0
6.0
5.0
4.0
MW
STDS #
FIG. 4. Neuraminidase treatment of purified ?-receptor. Affinity purified ‘l-receptor was treated with neuraminidase for 5 h at 3’7°C. Reaction mixtures contained in a final volume of 250 ~1: 50 mM citrate-phosphate buffer, pH 5.0,0.15 M NaCI, 150 pg BSA, 0.05 unit neuraminidase, and 15,000 cpm of receptor. In control incubations, neuraminidase was omitted. Reactions were terminated by addition of trichloroacetic acid to 15%. Precipitated proteins were collected by centrifugation and resuspended in the isoelectric focusing sample buffer (24). The sample was subjected to two-dimensional gel electrophoresis (25) as modified by Breithaupt et al. (24). Autoradiography was performed on the dried gels. The positions of BSA and major BSA contaminants have been drawn onto the autoradiographs to serve as reference points. The arrows indicate the position of the ~15.5 form of the receptor.
ity whereas glucose 6-phosphate inhibited only 8%. The adsorption of receptor onto liposomes in the presence or absence of enzyme was unaffected by mannose 6phosphate, thus this phosphorylated sugar specifically inhibited the enzyme-receptor binding. In the absence of receptor, a very small amount of a+iduronidase was nonspecifically bound to liposomes. The above results strongly suggest that the pure 215,000 molecular weight protein isolated by affinity purification on phosphomannan-Sepharose is indeed a receptor for the lysosomal enzyme a-L-iduronidase. It is possible that the membrane proteins that are added to the liposome assay contain a factor that is necessary for a-L-iduronidase binding. This is unlikely since we have recently been able to couple the purified 215,000 molecular weight protein to diazotized paper discs. After coupling, the
paper discs displayed saturable a-L-iduronidase binding that could be inhibited by mannose 6-phosphate but not by glucose 6-phosphate. This binding activity was not dependent on the addition of any other soluble membrane proteins (data not shown). As stated previously, up to 70% of crude solubilized receptor was bound in the liposome assay (calculated as a percentage of the original membrane binding activity that had been solubilized). We do not know why association of the purified lz51-receptor with liposomes was limited to only 20% in the soluble receptor assay. Increasing the quantity of liposomes did not increase the amount of receptor adsorbed. It is possible that during removal of detergent with the XAD-2 beads, receptor molecules formed dimers or small aggregates through their hydrophobic regions
ASSAY AND PURIFICATION
OF A RECEPTOR
which were previously occupied by detergent. This self-association could prevent a hydrophobic receptor-liposome interaction. This explanation seems likely since attempts to remove detergent from the pure receptor without first adding soluble membrane proteins resulted in adsorption of receptor to the hydrophobic XAD-2 beads. We feel that the membrane proteins function in a stabilizing capacity rather than participate directly in the binding to mannose 6-phosphate. Alternately, the limited association of the purified receptor with liposomes could result from an inactivation of some liposome binding sites on the receptor during the purification. Receptor characterization. Because of the availability of the chondrosarcoma receptor we attempted to further characterize it with respect to amino acid composition and charge heterogeneity. The receptor is somewhat enriched in acidic amino acids (Table II) although ammonia analysis was not performed. This acidic nature of the receptor was further indicated by two-dimensional gel electrophoresis employing isoelectric focusing and polyacrylamide gel electrophoresis in the presence of SDS (Fig. 4). The rat chondrosarcoma receptor was comprised of two forms differing in isoelectric point (PI 6.2 and 5.5). The pI 5.5 form became more basic (PI 5.8) following treatment with neuraminidase, presumably due to removal of sialic acid residues. Separation and purification of the individual charged species should allow us to determine the role of sialic acid moieties in receptor function. ACKNOWLEDGMENTS We thank Dr. Audrbe Fowler for carring out the amino acid analysis, Drs. A. L. Miller and H. H. Freeze for their generous gift of a-mannosidase, and Dr.G. Lawrence and M. Mehrahian for reading the manuscript. REFERENCES 1. HICKMAN, S., AND NEUFELD, E. F. (1972) ~&hem Biophys. Res. Cmnmun 49,992-999. 2. SANDO, G. N., AND NEUFELD, E. F. (1977) Cell 12, 619-627.
FOR A LYSOSOMAL
ENZYME
68’7
3. KAPLAN, A., ACHORD, D. T., AND SLY, W. S. (1977) Proc. Nat. Acad Sci. USA 69,2026-2030. 4. ULLRICH, K., MERSMANN, G., WEBER, E., AND VON FIGURA, K. (1978) Biochem J. 170, 643-650. 5. ROME, L. H., WEISSMANN, B., AND NEUFELD, E. F. (1979) Proc. Nat Acad Sci. USA 76,23312334. 6. GONZALEZ-NORIEGA, A., GRUBB, J. H., TALKAD, v., AND SLY, W. S. (1980) J. Cell BioL 85, 839852. 7. ROME, L. H., AND MILLER, J. (1980) B&hem. Bicphys. Res. Commun. 92, 986-993. 8. FISHER, H. D., GONZALEZ-NORIEGA, A., AND SLY, W. S. (1980) J. BioL Chem. 255, 5069-5074. 9. NATOWICZ, M. R., CHI, M.-M. Y., LOWRY, 0. H., AND SLY, W. S. (1979) Proc. Nat. Acad Sci. USA 76.4322-4326. 10. VON FIGURA, K., AND KLEIN, U. (1979) Eur. J. B&hem. 94, 347-354. 11. SAHAGIAN, G., DISTLER, J., HIEBER, V., SCHMICKEL, R., AND JOIJRDIAN, G. W. (1979) Fed. Proc. 38,467. 12. SAHAGIAN, G., DISTLER, J., AND JOURDIAN, G. W. (1981) Proc. Nat. Acad Sci. USA 78,4289-4293. 13. STEINER, A. W. AND ROME, L. H. (1981) Fed Proc 40, 1820. 14. FALTZ, L. L., REDDI, A. H., HASCALL, G. K., MARTIN, D., PITA, J. C., AND HASCALL, V. C. (1979) J. BioL Chem 254, 1375-1380. 15. MARKWELL, M. A. K., AND Fox, C. F. (1978) Birr chemistry 17, 4807-4817. 16. SLODKI, M. E., WARD, R. M., AND BOUNDY, J. A. (1973) Biochim Biqvhya Acta 304, 449-456. 17. BRE~HAUER, R. K., KAC~OROWSKI, G. J., AND WEISE, M. J. (1973) Biochemistry 12, 12511256. 18. KAPLAN, A., FISCHER, D., AND SLY, W. S. (1978) J. BioL Chem. 253, 647-650. 19. CUATRECASAS, P. (1970) J BioL Chem. 245,30593065. 20. EIMERL, S., NEUFELD, G., KORNER, M., AND SCHRAMM, M. (1980) Proc. Nat. Acd Sci USA 77,760-764. 21. SCATCHARD, G. (1949) Ann N. Y. Acad Sci. 51, 670-672. 22. FREEZE, H. H., MILLER, A. L., AND KAPLAN, A. (1980) J. BioL Chem. 255, 11081-11084. 23. LAEMMLI, U. K. (1970) Nature (Londmz) 227,680685. 24. BREITHAUPT, T. B., NYSTROM, I. E., HODGES, D. H., JR., AND BABITCH, J. A. (1978) AnaL Biochem. 84,579-582. 25. O’FARRELL, P. H. (1975) J. BioL Chem 250,40074021. 26. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. BioL Chem. 193, 265-275.
Assay and Purification of a Solubilized Membrane Receptor the Lysosomal Enzyme cu-L-lduronidase’ ANTON W. STEINER
AND
That Binds
LEONARD H. ROME’
Department of Biological Chemistry and the Mental Retardation Research Center, UCLA School of Medicine, LOSAngeles, California 900.24 Received September
23, 1981, and in revised form November
7, 1981
A receptor that binds the lysosomal enzyme cu-L-iduronidase via phosphorylated mannose residues on the enzyme has been solubilized from Swarm rat chondrosarcoma membranes using a pH 9.5 buffer containing 0.1% Triton X-100. Detergent-solubilized receptor in crude and purified preparations was measured by assay of bound a-L-iduronidase after adsorbing the receptor-enzyme complex onto insoluble phospholipid vesicles (liposomes). Binding of a-L-iduronidase to the liposomes required receptor and was completely inhibited by mannose 6-phosphate but not glucose 6-phosphate, indicating that the receptor maintained specificity following solubilization. Receptors from rat chondrosarcoma and human diploid fibroblasts were purified to apparent homogeneity using a phosphomannan-Sepharose affinity column. Both had identical molecular weights in polyacrylamide gels containing sodium dodecyl sulfate (Mr = 215,000). Amino acid analysis and two-dimensional gel electrophoresis was carried out on the purified rat chondrosarcoma receptor. Two forms of the receptor with different PI’S were observed (~1 5.5 and 6.2). One form (~1 5.5) was made more basic (PI 5.8) by treatment with neuraminidase.
The existence of cell surface receptors for lysosomal enzymes was first proposed by Hickman and Neufeld (l), in order to explain the efficient uptake of certain hydrolytic enzymes by cultured fibroblasts. Evidence for these receptors came initially from kinetic studies of enzyme internalization (2-4) and later from direct binding experiments using intact fibroblasts (5, 6) and isolated fibroblast membranes (‘7, 8). Membranes of the Swarm rat chondrosar’ Research supported by USPHS Grant HD-06576, American Cancer Society Institutional Grant IN-131, and the NIH Cancer Center Core Support Grant USPHS CA 16042 (UCLA Jonsson Compreshensive Cancer Center); California Institute for Cancer Research. A.W.S. is an NICHD postdoctoral trainee (HD-07032). * To whom correspondence should be sent. 681
coma were found to be particularly rich in receptor (7). The membrane-associated receptors bind only to lysosomal enzymes that contain a specific recognition marker. A number of studies have demonstrated that the recognition marker is a terminal mannose 6phosphate moiety (9-11). Comparison of binding and internalization properties suggest that these receptors are identical to the cell surface proteins that mediate enzyme internalization and packaging of these enzymes into lysosomes. Recently a receptor that binds mannose 6-phosphate moieties of fl-galactosidase has been purified from bovine liver (12). In the present study we have solubilized and purified a receptor that binds a-L-iduronidase from rat chondrosarcoma and human fibroblasts. The purification was 0003-9861/82/040681-07$02,00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
682
STEINER
facilitated by an assay for receptor that is based upon the association of soluble receptor with insoluble phospholipid vesicles. A preliminary report of this work has been presented (13). EXPERIMENTAL
PROCEDURES
Materials. Swarm rat chondrosarcoma tissue, a rat cartilagenous tumor, was the gift of Dr. J. Kimura, NIH, Bethesda, Maryland. Phosphomannan from Hansenuh ho&ii (NRRL Y-2448) was a gift of Dr. M. Slodki, USDA Northern Regional Research Laboratory, Peoria, Illinois. Neuraminidase Type VI was purchased from Sigma. The barium salt of mannose 6-phosphate was from U. S. Biochemicals and was converted to the sodium salt by passage over Dowex 5OW-X8 (Na+ form). Sodium mannose 6-phosphate was decolorized with charcoal, lyophilized, and redissolved in water. Iodogen was obtained from Pierce Chemicals. Amerlite XAD-2 beads were purchased from Sigma and washed with methanol and water before use. Carrier free Na’? was from Amersham. oc-L-Iduronidase was purified from normal human urine as described (2, 5). of mewdrrane fra,&ms. One hundred Preparation grams of solid chondrosarcoma tissue, pressed through a stainless-steel sieve (14), was suspended in 300 ml of 2X concentrated buffer A (10 mM KH2POI, pH 6.0, 0.15 M NaCl, and 0.02% NaN3). The suspension was homogenized in a Polytron for 3 min and subsequently processed in a Gaulin Mill Model 15M (Gaulin Corp., Everett, Mass.) at 6000 psi. The suspension was recirculated through the press several times while the temperature was maintained at 20°C by the addition of crushed ice. The final solution (ea. 500 ml) was centrifuged for 40 min at 10,000 rpm in a Sorvall GSA rotor. The resulting supernatant was recentrifuged at 35,000 rpm for 1 h (100,OOOg) in a Beckman type 42.1 rotor. The high-speed membrane pellet was suspended in buffer A containing 10 mM mannose 6-phosphate and placed on ice for 1 h. This step was included to displace endogeneous lysosomal enzymes bound to the receptor. The membranes were recentrifuged, and the pellets washed with buffer A and centrifuged again. Final membrane pellets were resuspended in a small volume of buffer A and assayed for binding activity as described (7). Human diploid fibroblasts were grown and harvested as described (5). Cells obtained from 18 tissue culture flasks (150 cm’) were suspended in 0.25 M sucrose and disrupted by sonication for 10 min with a probe sonicator. The broken cells were centrifuged at 12,000 rpm in a Sorvall SM-24 rotor for 20 min and the resulting supernatant centrifuged for 1.5 h at 35,000 rpm in a Beckman type 40 rotor. Final membrane pellets were washed once with buffer A containing 10 mM mannose 6-phosphate and once with buffer A alone as described above. Membrane preparations were routinely stored at -20°C.
AND ROME Receptor solubilization. Approximately 20 mg of rat chondrosarcoma 100,OOOg membrane protein was stirred overnight at 4°C with 3 ml of extraction buffer which contained: 0.1 M sucrose, 0.05 M KCl, 0.04 M KH2P04, pH 9.5, 0.03 M EDTA, 0.1% Triton X100, and 13% glycerol. Insoluble material was removed by centrifugation at 120,OOOgfor 2 h, and the extract was dialyzed against 1 liter of 20 mM TrisHCl, pH 7.5. Following dialysis, the solution was again clarified by centrifugation and the supernatant used as the source of soluble receptor. Fibroblast membranes were solubilized3 with 2.5 ml of extraction buffer and processed in the same manner. Detergent-solubilized rat chondrosarcoma membrane protein (2.1 mg) was iodinated with 2 mCi of Na’%I using the iodogen procedure (15). Fibroblastsoluble membrane protein was labeled witch 750 &i Na’%I. Unreacted iodide was removed by dialysis against buffer A containing0.05% Triton X-100. Typically 30% of the added radioactivity was incorporated into nondialyzable material. Phosphmnnan-Sepharose af$nity column. Phosphomannan was acid hydrolyzed to core and pentasaccharide fragments (16), both presumably terminating in mannose 6-phosphate moieties (17,18). The core was precipitated with 1 vol 95% ethanol, centrifuged, and resuspended in 0.1 M sodium bicarbonate, pH 8.6. Approximately 5 g of core was mixed with 125 ml of Sepharose 4B activated with 27 g of cyanogen bromide. Coupling and removal of reactive groups was as described (19). The phosphomannanSepharose was washed and equilibrated before use with buffer B (50 mM citrate-phosphate buffer, pH 6.0, 0.15 M NaCI, 0.1% Triton X-100, and 0.1% NaN,) containing 1 mg/ml BSA.4 Soluble receptor assay. Prior to assay for receptor binding activity, Triton X-100 was removed from the solubilized membrane preparations by addition of Amberlite XAD-2 beads (1.2 g/ml of detergent solution). After 1 h of gentle shaking at 4”C, the beads were removed by a brief centrifugation. Binding mixtures contained in a final volume of 150 ~1; 1.25 units of a-L-iduronidase (2.5 X lo-’ M final concentration), 50 mM 2-(N-morpholino)ethanesulfonic acid (Na+), pH 6.4, and detergent-free membrane protein. Mannose 6-phosphate or glucose 6-phosphate was included in some assays at a final concentration of 10 mM. Binding mixtures were incubated for 1.5 h at 0°C. Twenty-five microliters of a lipsome mixture of egg lecithin and stearylamine, prepared according to Eimerl et al. (20), was then added and the solution maintained at 0°C for 15 min. Liposomes were aggregated by the addition of 5 ~1 of 1 M MgC12. The
3The term “solubilized” is used through out this manuscript to refer to material that does not sediment when centrifuged at 120,OOOgfor 2 h. 4 Abbreviations used: BSA, bovine serum albumin; SDS, sodium dodecyl sulfate.
ASSAY
AND PURIFICATION
OF A RECEPTOR
mixture was placed on ice for 30 min, and the liposomes were collected by centrifugation at 13,OOOgfor 15 min. Liposome pellets were resuspended in 50 ~1 buffer A containing 0.1% Triton X-100 and 100 rg/ ml BSA and assayed for cY-L-iduronidase activity as described (5), where one unit of enzyme activity represents the conversion of 1 nmol of substrate to product per hour at 37°C.
RESULTS
Receptor solubilixation and assay. Detergent extraction of the 100,OOOgchondrosarcoma membrane fraction released about 60% of the total membrane protein into solution. Approximately 37% of the original binding activity remained membrane associated following detergent solubilization and recentrifugation. An assay method was developed to detect and quantitate the amount of soluble receptor which remained active following solubilization. This method was based on a technique developed by Eimerl et al+ (20) for insertion of the p-adrenergic receptor into intact cells. A rapid assay was desired to
IL
&-oy? 10
20
30
Protein
ENZYME
Mw Buffer Buffer STDS Wash +G6P
683
Buffer +M6P
200K116K94K68K-
AND DISCUSSION
I
FOR A LYSOSOMAL
I
I
I
I
40
50
60
(gg9)
FIG. 1. Soluble receptor assay. Detergent-free soluble membrane protein at the concentrations indicated, was incubated with ol-L-iduronidase, adsorbed onto liposomes and bound enzyme quantitated as described under Experimental Procedures. Crude soluble membrane protein (0); crude soluble membrane protein not retained by the phosphomannan-sepharose affinity column (0). The values shown have been corrected for nonspecific binding (i.e., enzyme bound in the presence of 10 mM mannose 6-phosphate). This binding did not exceed 12 munit/assay.
FIG. 2. Purification of ‘%I-labeled rat chondrosarcoma receptor. Rat chondrosarcoma membranes were solubilized, iodinated, and passed over a l-ml phosphomannan-Sepharose affinity column. The column was washed with 100 ml of buffer B containing 1 mg/ ml BSA, followed by 10 ml of 10 m!d glucose B-phosphate in buffer B containing 1 mg/ml BSA, and linally eluted with 10 ml of 10 mM mannose 6-phosphate in buffer B containing 1 mg/ml BSA. Selected fractions from each wash (containing 15,000-30,000 cpm) were pooled and protein was precipitated by addition of 10 vol of cold 95% ethanol. Precipitates were resuspended in sample buffer containing SDS and subjected to electrophoresis in an 8% acrylamide gel (23). Autoradiography was carried out on the dried gel. The molecular weights indicated represent the following standards (Bio-Rad Laboratories): myosin, 200,000; fl-galactosidase, 116,250; phosphorylase B, 92,500; bovine serum albumin, 66,200; ovalbumin, 45,000.
facilitate the comparison of various procedures used to extract active receptor from membranes. The liposome assay method used relies on the immobilization of receptor by adsorption onto insoluble phospholipid vesicles. The receptor is quantitated by assay of a bound ligand, the enzyme a-L-iduronidase. Figure 1 shows that the amount of a-~iduronidase which is specifically bound to the insoluble lipid vesicles is dependent on the amount of crude solubilized membrane protein present. The solubilization treatment used did not significantly destroy receptor binding activity since up to 70% of the binding capacity which was lost from the membranes after solubilization could be accounted for with the liposome assay. That the binding of enzyme to liposomes was mediated through a specific
684
STEINER PROTEIN
STAIN
warm 200K-
-
116K94K-
^___ m
68K-
xI_
43 K-
c%- -FIG. 3. Comparison of rat chondrosarcoma and human diploid fibroblast receptors. Rat chondrosarcoma and human diploid fibroblast (HDF) membranes were solubilized as described under Experimental Procedures. Affinity-purified ‘%I-receptor was added as tracer to the crude chondrosarcoma-soluble protein and the mixture passed over a 16-ml phosphomannan-Sepharose affinity column. After extensive washing, receptor was eluted with 10 mM mannose 6-phosphate in buffer B (75 ml), and aliquots were analyzed as described above. Fibroblast membranes were extracted, iodinated, and purified on a l-ml phosphomannon-Sepharose affinity column. RCl and RC2 represent the material obtained from two separate purifications of rat chondrosarcoma tissue.
receptor was shown by testing receptorfree membrane protein in the liposome assay. This material was not retained by the phosphomannan-Sepharose affinity column used for receptor purification. As expected, practically no cY-L-iduronidase was bound when this fraction was used as the source of protein in the assay. Saturable binding of cY-L-iduronidase to the soluble receptor was observed using the liposome assay. A dissociation constant of 1 X lo-‘M, calculated by the method of Scatchard (21), was similar to that found for intact chondrosarcoma membranes (7). The soluble receptor assay should be useful for detection of a wide variety of membrane-derived receptors by quantitation of bound ligand. Pur$cation of receptor. Detergent-solubilized membrane proteins were iodinated and passed over the phosphomannan-Sepharose affinity column. Extensive washing with buffer B (usually 100 column ~01s) was necessary to reduce the back-
AND ROME
ground radioactivity. Figure 2 shows that when mannose 6-phosphate (but not glucose 6-phosphate) was included in the wash buffer, a radioactive protein of molecular weight 215,000 was eluted. We presumed that this labeled protein was the receptor for a-L-iduronidase and that iodination of the impure mixture of membrane proteins did not destroy the mannose 6-phosphate specific binding properties of the receptor. The amount of rat chondrosarcoma tissue used for purification was increased to 750 g in order to have quantities sufficient for further characterization. The material obtained was analyzed by gel electrophoresis in the presence of SDS. As shown in Fig. 3, two separate receptor preparations were found to be homogeneous with respect to protein staining by Coomassie blue. Purified ‘%I-receptor was added as tracer during affinity purification to facilitate its location in column fractions. The purified unlabeled receptor comigrated with the labeled tracer receptor. Approximately 700 pg of purified receptor (determined by the method of Lowry et al, (26)) was obtained from 1 g of high-speed membrane protein (from ca. 750 g tumor). Furthermore, iodination of the purified receptor protein and subsequent electrophoresis did not reveal even minor contaminating proteins. High-speed membranes from human diploid fibroblasts were solubilized, labeled with Nan9 and passed through the phosphomannan-Sepharose affinity column. A radioactive protein of molecular weight 215,000was specifically eluted with 10 mM mannose 6-phosphate indicating that at least with respect to molecular weight on polyacrylamide gels in the presence of SDS, the receptor from rat chondrosarcoma and from human fibroblasts are identical. Recently an ‘%I-labeled receptor of 215,000 molecular weight that binds mannose 6-phosphate residues of bovine testicular B-galactosidase was isolated from bovine liver, human diploid fibroblasts, and Chinese hamster ovary cells by Sahagian et al. (12). In addition we have found that a-mannosidase purified from Lktyostelium discoideum and shown to
ASSAY
AND PURIFICATION
OF A RECEPTOR TABLE
FOR A LYSOSOMAL
685
ENZYME
I
SOLUBLE RECEPTOR ASSAY WITH PURIFIED ?-RECEPTOR’ Pellet Incubation ‘“I-receptor
“Mock”
+ enzyme
receptor
‘z51-Receptor only
+ enzyme
SN (wm)
Enzyme bound (munit)
Addition
wm
sb
None Man 6-P” Glc 6-P
1736 1559 1567
21.0 20.0 19.6
6522 6215 6414
91.1 7.7 83.9
None Man 6-P
-
-
-
14.8 14.6
None Man 6-P
1795 1792
22.1 21.1
6319 6679
-
a Affinity-purified ‘%I-receptor from rat chondrosarcoma was mixed with the unbound protein fraction from the affinity column. “Mock” receptor was prepared by substituting buffer B for the labeled receptor. After removal of detergent the sample was assayed for soluble receptor. Liposome pellets were assayed for both ol-L-iduronidase activity and bound radioactivity. b Percentage of total radioactivity in pellet + supernatant (SN). ’ Man 6-P, mannose 6-phosphate (10 mM); Glc 6-P, glucose 6-phosphate (10 mM).
bind to human diploid fibroblasts via a phosphate specificity in the soluble recepmannose 6-phosphate recognition marker tor assay. Mannose 6-phosphate inhibited (22), also specifically bound rat chondro92% of the a-L-iduronidase binding activsarcoma membranes (data not shown). Thus it appears that a common 215,000 TABLE II molecular weight protein is present on a AMINO ACID ANALYSIS OF RAT variety of different cell types that can CHONDROSARCOMA RECEPTOR bind a number of hydrolytic enzymes bearAmino acid Residues/m01 ing a mannose 6-phosphate recognition marker. This binding protein is most likely Asx 184 involved in the internalization and packThreonine 110 aging of lysosomal enzymes. Serine 142 Soluble receptor assay with puri&ed 1251Glx 178 receptor. In order to further validate the Proline 110 Glycine 165 soluble receptor assay and demonstrate Alanine 127 directly that the 215,000 molecular weight Valine 129 protein isolated from the affinity column Methionine 17 was the receptor for a-L-iduronidase, we Isoleucine 52 used affinity-purified ‘%I-receptor in the Leucine 96 soluble receptor assay. Prior to assay, the Tyrosine 75 purified receptor was first mixed with the Phenylalanine 75 protein fraction not retained on the affinHistidine 44 ity column. This step was necessary to Lysine 100 prevent nonspecific adsorption of the reArginine 84 ceptor to the Amberlite XAD-2 beads used Note. Forty-eight micrograms of affinity-purified to remove detergent. receptor was hydrolyzed with 6 M HCl overnight at Table I shows that approximately 20% 110°C under vacuum and amino acid analysis perof the total soluble receptor was adsorbed formed on a Beckman Model 120C amino acid anaonto liposomes independent of the pres- lyzer. Values represent the averages of two separate ence or absence of enzyme. As observed analyses. Calculations are based on a molecular with crude receptor preparations, purified weight of 215,000 and excluded analysis of half-cyslabeled receptor displayed mannose 6- tine, tryptophan, and ammonia.
686
STEINER PROTEIN
AND ROME AUTORADIOGRAPH
STAIN Conlrol
Treated 8.0
7.0
6.0
5.0
4.0
MW
STDS #
FIG. 4. Neuraminidase treatment of purified ?-receptor. Affinity purified ‘l-receptor was treated with neuraminidase for 5 h at 3’7°C. Reaction mixtures contained in a final volume of 250 ~1: 50 mM citrate-phosphate buffer, pH 5.0,0.15 M NaCI, 150 pg BSA, 0.05 unit neuraminidase, and 15,000 cpm of receptor. In control incubations, neuraminidase was omitted. Reactions were terminated by addition of trichloroacetic acid to 15%. Precipitated proteins were collected by centrifugation and resuspended in the isoelectric focusing sample buffer (24). The sample was subjected to two-dimensional gel electrophoresis (25) as modified by Breithaupt et al. (24). Autoradiography was performed on the dried gels. The positions of BSA and major BSA contaminants have been drawn onto the autoradiographs to serve as reference points. The arrows indicate the position of the ~15.5 form of the receptor.
ity whereas glucose 6-phosphate inhibited only 8%. The adsorption of receptor onto liposomes in the presence or absence of enzyme was unaffected by mannose 6phosphate, thus this phosphorylated sugar specifically inhibited the enzyme-receptor binding. In the absence of receptor, a very small amount of a+iduronidase was nonspecifically bound to liposomes. The above results strongly suggest that the pure 215,000 molecular weight protein isolated by affinity purification on phosphomannan-Sepharose is indeed a receptor for the lysosomal enzyme a-L-iduronidase. It is possible that the membrane proteins that are added to the liposome assay contain a factor that is necessary for a-L-iduronidase binding. This is unlikely since we have recently been able to couple the purified 215,000 molecular weight protein to diazotized paper discs. After coupling, the
paper discs displayed saturable a-L-iduronidase binding that could be inhibited by mannose 6-phosphate but not by glucose 6-phosphate. This binding activity was not dependent on the addition of any other soluble membrane proteins (data not shown). As stated previously, up to 70% of crude solubilized receptor was bound in the liposome assay (calculated as a percentage of the original membrane binding activity that had been solubilized). We do not know why association of the purified lz51-receptor with liposomes was limited to only 20% in the soluble receptor assay. Increasing the quantity of liposomes did not increase the amount of receptor adsorbed. It is possible that during removal of detergent with the XAD-2 beads, receptor molecules formed dimers or small aggregates through their hydrophobic regions
ASSAY AND PURIFICATION
OF A RECEPTOR
which were previously occupied by detergent. This self-association could prevent a hydrophobic receptor-liposome interaction. This explanation seems likely since attempts to remove detergent from the pure receptor without first adding soluble membrane proteins resulted in adsorption of receptor to the hydrophobic XAD-2 beads. We feel that the membrane proteins function in a stabilizing capacity rather than participate directly in the binding to mannose 6-phosphate. Alternately, the limited association of the purified receptor with liposomes could result from an inactivation of some liposome binding sites on the receptor during the purification. Receptor characterization. Because of the availability of the chondrosarcoma receptor we attempted to further characterize it with respect to amino acid composition and charge heterogeneity. The receptor is somewhat enriched in acidic amino acids (Table II) although ammonia analysis was not performed. This acidic nature of the receptor was further indicated by two-dimensional gel electrophoresis employing isoelectric focusing and polyacrylamide gel electrophoresis in the presence of SDS (Fig. 4). The rat chondrosarcoma receptor was comprised of two forms differing in isoelectric point (PI 6.2 and 5.5). The pI 5.5 form became more basic (PI 5.8) following treatment with neuraminidase, presumably due to removal of sialic acid residues. Separation and purification of the individual charged species should allow us to determine the role of sialic acid moieties in receptor function. ACKNOWLEDGMENTS We thank Dr. Audrbe Fowler for carring out the amino acid analysis, Drs. A. L. Miller and H. H. Freeze for their generous gift of a-mannosidase, and Dr.G. Lawrence and M. Mehrahian for reading the manuscript. REFERENCES 1. HICKMAN, S., AND NEUFELD, E. F. (1972) ~&hem Biophys. Res. Cmnmun 49,992-999. 2. SANDO, G. N., AND NEUFELD, E. F. (1977) Cell 12, 619-627.
FOR A LYSOSOMAL
ENZYME
68’7
3. KAPLAN, A., ACHORD, D. T., AND SLY, W. S. (1977) Proc. Nat. Acad Sci. USA 69,2026-2030. 4. ULLRICH, K., MERSMANN, G., WEBER, E., AND VON FIGURA, K. (1978) Biochem J. 170, 643-650. 5. ROME, L. H., WEISSMANN, B., AND NEUFELD, E. F. (1979) Proc. Nat Acad Sci. USA 76,23312334. 6. GONZALEZ-NORIEGA, A., GRUBB, J. H., TALKAD, v., AND SLY, W. S. (1980) J. Cell BioL 85, 839852. 7. ROME, L. H., AND MILLER, J. (1980) B&hem. Bicphys. Res. Commun. 92, 986-993. 8. FISHER, H. D., GONZALEZ-NORIEGA, A., AND SLY, W. S. (1980) J. BioL Chem. 255, 5069-5074. 9. NATOWICZ, M. R., CHI, M.-M. Y., LOWRY, 0. H., AND SLY, W. S. (1979) Proc. Nat. Acad Sci. USA 76.4322-4326. 10. VON FIGURA, K., AND KLEIN, U. (1979) Eur. J. B&hem. 94, 347-354. 11. SAHAGIAN, G., DISTLER, J., HIEBER, V., SCHMICKEL, R., AND JOIJRDIAN, G. W. (1979) Fed. Proc. 38,467. 12. SAHAGIAN, G., DISTLER, J., AND JOURDIAN, G. W. (1981) Proc. Nat. Acad Sci. USA 78,4289-4293. 13. STEINER, A. W. AND ROME, L. H. (1981) Fed Proc 40, 1820. 14. FALTZ, L. L., REDDI, A. H., HASCALL, G. K., MARTIN, D., PITA, J. C., AND HASCALL, V. C. (1979) J. BioL Chem 254, 1375-1380. 15. MARKWELL, M. A. K., AND Fox, C. F. (1978) Birr chemistry 17, 4807-4817. 16. SLODKI, M. E., WARD, R. M., AND BOUNDY, J. A. (1973) Biochim Biqvhya Acta 304, 449-456. 17. BRE~HAUER, R. K., KAC~OROWSKI, G. J., AND WEISE, M. J. (1973) Biochemistry 12, 12511256. 18. KAPLAN, A., FISCHER, D., AND SLY, W. S. (1978) J. BioL Chem. 253, 647-650. 19. CUATRECASAS, P. (1970) J BioL Chem. 245,30593065. 20. EIMERL, S., NEUFELD, G., KORNER, M., AND SCHRAMM, M. (1980) Proc. Nat. Acd Sci USA 77,760-764. 21. SCATCHARD, G. (1949) Ann N. Y. Acad Sci. 51, 670-672. 22. FREEZE, H. H., MILLER, A. L., AND KAPLAN, A. (1980) J. BioL Chem. 255, 11081-11084. 23. LAEMMLI, U. K. (1970) Nature (Londmz) 227,680685. 24. BREITHAUPT, T. B., NYSTROM, I. E., HODGES, D. H., JR., AND BABITCH, J. A. (1978) AnaL Biochem. 84,579-582. 25. O’FARRELL, P. H. (1975) J. BioL Chem 250,40074021. 26. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. BioL Chem. 193, 265-275.