Purification and partial characterization of the carbohydrate structure of lysosomal N-acetyl-β-d-hexosaminidases from bovine brain

0020-71 IX x2 010025-07303.00 0 Cnpynght 0 1982 Pergamon Prer\ Ltd

PURIFICATION AND PARTIAL CHARACTERIZATION OF THE CARBOHYDRATE STRUCTURE OF LYSOSOMAL N-ACETYL-P-D-HEXOSAMINIDASES FROM BOVINE BRAIN B. OVERDIJK. G. VAN STEIJN. J. H. WOLF and J. J. W. LISMAN Department

of Medical Chemistry, Vrije Universiteit. van der Boechorststraat NL-1007 MC Amsterdam. The Netherlands (Recc~irrd 8 Junr

7,

19X1)

Abstract--l. The lysosomal forms A and B. and an Intermediate form I of N-acetyl-ii-o-hcxosaminidase (EC 3.2.1.30) were isolated from bovine brain. resulting in the following purification factors and specific activities: hexosaminidase A 20255. 103 U mg- ’ . hexosaminidase B 34715. I34 U rng- ’ : hexosaminidase I 15241. 7X U mgg’. 2. The molecular weights of the polypeptide chains were identical for each isoenzyme: two bands of 50 and 53 kdaltons were found. 3. Carbohydrate analysis showed the presence of mannose. galactose. N-acetylglucosamine and sialic acid. This composition. and the absence of N-acetylgalactosamme, indicated that only iV-glycosidically linked oligosaccharide chains are present. 4. The amino-acid composition showed no substantial differences for the three isoenrymes.

enzymes from neuronal tissues, compared with other mammalian tissues. In this manuscript we describe the purification of the lysosomal 1V-acetyl-[j-o-hexosaminidases (EC 3.2.1.30) from bovine brain tissue, the subunit composition and the carbohydrate and amino-acid analysis.

INTRODUCTION

In 1972. Hickman & Neufeld proposed their excretionrecapture hypothesis for lysosomal enzymes. to explain the abnormal distribution of a variety of these enzymes between cultured skin flbroblasts from patients with I-cell disease and the medium in which these cells were grown. The authors predicted that “recognition sites on lysosomal hydrolases and the complementary sites on cell surfaces should prove a fertile field of study” (Hickman & Neufeld. 1972). Since then it has become evident that the recognition marker of the Iysosomal enzymes is situated on the carbohydrate moiety of these molecules. Analogously, the blood-group specificity and the specific recognition between molecules during processes like celll cell interactions and lectin interactions. is mediated by the carbohydrate part of glycoproteins and glycolipids (Aminoff er nl., 1977, 1979a,b; Childs et al., 1979; Critchley et al., 1979; Monsigny YCal., 1980; Yamakawa & Nagai. 1978; Williams, 1978). Two types of lysosomal recognition markers are known. Uptake of lysosomal enzymes by fibroblasts appears to be dependent on the presence of mannose6-phosphate residues on carbohydrate chains of the enzyme molecules (Kaplan er ((I., 1977: Natowicz rr al., 1979). For the cells of the reticula-endothelial system mannose or N-acetylglucosamine functions as recognition marker (Stahl rr (I/.. 1978. 1980). It has been postulated that carbohydrate chains also play a role in the intracellular transport of lysosomal enzymes (for a review. see Hasilik. 1980). In order to get more insight into the biosynthetic pathway of these enzymes and their final packaging in lysosomes, it is necessary to determine the structure of the carbohydrate chain(s). There is relatively little known about the carbohydrate composition and structure of lysosomal

MATERIALS AYD METHODS

The follovvmg chemicals were supplied by the companres Indicated. DEAE-Sephacel. SP Sephadex C-50. CHSepharose 48, ConA -Sepharose [Pharmacia Fine Chemtcals. Uppsala. Sweden): z-methylmannoside. Wacetylglucosamine. naphthol AS-BI-2-acetamido-2-deoxy-P-D_elucopyranoside. 4-methylumbelliferyl-2-acetamtdo-2-deoxy-/G o-glucopyranostde. p-nitrophenyl-2-acetamido-2-deoxy-/Ir)-galactopyranoside (Koch-Light Laboratories Ltd. Colnbrook. Bucks.. U.K.): Fast Black (Serva. Hetdelberg. F.R.G.I.

All procedures were performed at O-4 C unless stated otherwtse. Bovine brain tissue (600 g) was homogenized in 0.02 M Tris HCI:0.05 M KCI solutton. pH 7.0. which contained 0.2”,, (v;v) Triton X-100. The homogenate (2.4 I.) was centrifuged in a 6 x 300 ml rotor of MSE (2 hr at 75.UOOy). The pellet was discarded. The supernatant (1675 ml). readjusted to pH 7.0 after addition of NaCl to I M. was applied -to a column of ConA~-Sepharose (20 x 2.5 cm diam:).’ The column material had been preequtlibrated wtth 0.02 M TrissHCl solution. pH 7.0. containing 1.0 M NaCI. 0.02 M CaCl, and 0.02 M MnCI,. The unretained material was washed off with 600 ml of 0.02 M TrissHCI. pH 7.0. containmg 1.0 M NaCI. Elution was continued with IOOml 0.5 M r-methylmannoside in the above buffer. The column was then brought to room temperature. and after I hr of equilibration the elution was continued with 500 ml 0.5 M r-methylmannoside in the buffer. The fractions which contained /Ghexosaminidase activity were dialyzed against a 25

B.

26 Table

1. Purification Total

Homogenate 75,COO 9 supernatant ConA-Sepharose GalNAc-Sepharosc DEAE-Sephacel Hexosaminidase A Hexosaminidase I Hexosaminidase B SP-Sephadex Hexosaminidase B”

activity (U)

435 336 230 127

OVERDIJK

et al.

of p-hexosaminidase

isoenzymes

Total protein (mg)

Specific activity (U mg-‘)

Yield (“;,)

Purification (fold)

100 77 53 29

1.0 2.5 48 268 1

65.0 17.1 25.9

0.63 0.22 1.36

103 78 19

I5 3.9 6.0

20255 15241 3730

60.3

0.45

134

5.5

34715

0.05 M sodium phosphate buffer at pH 7.4, containing 0.15 M NaCl (PBS). The enzyme sample was applied to a GalNAc-Sepharose affinity column @-aminophenyl-2-acetamido-2-deoxy-b-D-galactopyranoside coupled to CHSepharose; 6 x 5 cm diam.). which has been described earlier (Lisman & Overdijk, 1978). The retained enzyme activity was eluted with 0.2 M N-acetylglucosamine in PBS. It was then dialyzed against 0.05 M Tris-HCl/0.05 M KCI, pH 7.0, and concentrated with the aid of an Amicon concentrator, fitted with a PM-10 filter, to 25 ml. This was brought to a column of DEAE-Sephacel (19 x 2.5cm diam.), equilibrated in the latter buffer. Prior to the elution with a 300ml gradient of 0.05-0.55 M KC1 in that buffer, 5OOml of the starting buffer was pumped through. The unretained activity peak (hexosaminidase B) was dialyzed against a 0.02 M imidazole-HCI buffer, pH 6.2, and applied to a 1Oml column of SP-Sephadex C-50, equilibrated in the above buffer. After elution with 50ml of the starting buffer, a 50 ml gradient of O-O.3 M NaCl in the buffer was applied. The enzyme activity was eluted in the gradient. analysis

Cellogel electrophoresis was done following Poenaru & Dreyfus (1973). Polyacrylamide gel electrophoresis was performed on slab gels (1 mm thick, 11 cm long and 15 cm wide). Electrophoresis on 15% gels containing SDS was done as described by Lugtenburg et al. (1975). The molecular weight of the polypeptides was determined following Weber & Osborn (1969). Electrophoresis of the native enzymes in polyacrylamide gels was done according to Maurer (1968) in 7.5”/, gels. For enzymatic activity these gels were stained in a 0.1 M sodium citrate buffer, pH 4.5, containing 0.5 mM naphtol AS-BI-2-acetamido-2-deoxy+ D-glucopyranoside and 1.0 mg ml- ’ Fast Black. Protein bands were visualized in a solution of Coomassie Brilliant Blue R-250 (0.25:,,, w/v) in methanol-acetic acid-water (4: 1: 5, by vol).

The carbohydrate composition of the purified enzymes was determined according to Reinhold (1972). The gas chromatograph (Packard Becker model 421) was equipped with a glass column (1.8 mm x 2 m) containing 3.8% SE-30 CC grade on Chromosorb WHP, lo&200 mesh. The programming rate was l’/min. Mannitol was used as internal standard. Amino-acid

brain

5.1 X 1o-3 12.8 X 1om3 0.25 13.7

85400 26215 931 9.3

’ The results of the final SP-Sephadex step for fi-hexosaminidase can not be related to the above homogenate value. One unit of enzyme activity liberates 1 pmol 4-methylumbelliferone Lowry C? ul. (1951).

Electrophoretic

from bovine

analysis

The samples were hydrolyzed under N, at 105°C in 6 M HCI for 24 hr. The amino-acid composition was determined with the aid of a Biotronic LC-6000 chromatograph. equipped with a column (31 x 0.6cm diam.). filled with

B have been obtained

per min at 37’C. Protein

Durrum DC-6-A fornia, U.S.A.). Enzyme

in a separate

(Durrum

experiment

was determined

Instruments,

and

following

Sunnyvale,

Cali-

ussu!~

The /?-hexosaminidase activity was determined as described earlier (Overdijk et al.. 1975) with 4-mkthylumbelliferyi-2-acetamido-2-deoxy-/l-D-glucopyranoside as substrate.

All procedures were done at least 3 times. with the determination of the enzymatic activity in duplicate. RESULTS

AND

DISCUSSION

The results of the purification of the various lysosoma1 forms of bovine brain /I-hexosaminidase have been summarized in Table 1. The enzyme was separated into three fractions. Hexosaminidase B was not retained by the DEAE-Sephacel column. The intermediate fraction I was eluted after the peak of the B-form, without raising the salt concentration of the eluting buffer. Hexosaminidase A was eluted in a salt gradient at about 0.25 M KCI (Fig. 1). We did not investigate the possible identity of the above I-fraction with the intermediate /I-hexosaminidase forms in serum, described by Price & Dance (1972). With the aid of Cellogel electrophoresis the identity of the enzyme forms A and B. eluted from the DEAESephacel, was confirmed. The electrophoretic analysis of the purified enzymes in 7.5% polyacrylamide gels showed that all bands which could be made visible by protein staining were enzymatically active. Hexosaminidase I comigrated with the A enzyme. Samples which had been reduced and denatured in the presence of /?-mercaptoethanol and SDS, showed on SDS-electrophoresis in 1.50;,gels an identical pattern for each isoenzyme. Two bands were visible with molecular weight of 53 and 50 kdaltons, respectively (Fig. 2). In a recent paper by Mahuran & Lowden (1980) a survey of literature data is presented of molecular weights of polypeptide chains of human placental hexosaminidases. Values of 5@60 kdaltons and of 25-30 kdaltons are reported for the enzymes of this tissue. The 60 kdalton a-chain which has been found by Mahuran & Lowden (1980) could not be dissociated with those means that normally disrupt disulfide bonds, thus leaving the possibility that the cc-unit is composed of polypeptide chains linked via bonds other than disulfide bridges. In human liver

Carbohydrate

composition

27

of hexosaminidases

Fig. 1. DEAE-Sephacel chromatography of P-hexosaminidases from bovine brain. Experimental details of the ion exchange chromatography are described in Materials and Methods. The horizontal bars indicate the fractions containing hexosaminidase A, B and 1. respectively.

molecular weights of 50. 37 and 25 kdaltons were found for the A enzyme, and 50 and 25 kdalton chains for the B enzyme (Wolf er ul., 1980). With the same electrophoretic procedure only chains at about 50 kdaltons were found in the present study. The cause of the splitting up found for all three isoenzymes. is unknown. It is also seen for cc-glucosidase and the marker protein phosphorylase B (Hasilik & Neufeld. 1980). The latter authors speculated that small changes could be due to modification of the carbohydrate side chains or of the polypeptides. For some P-hexosaminidases the reported purification factors and specific activities have been summarized (Table 2). There appears to be hardly any reTable 2. Purification

Tissue

Human placenta

Hexosaminidase A Specific Purification factor activity

4300h 5100

Human kidney Human brain Monkey brain

6500” 7450h 966’ 1015h 3300” 2.560”

Bovine brain

20255b

Human

liver

results

I50 23 I80 70 251 90 85 3.1 5.6 103

of /?-hexosaminidase

lationship between the obtained specific activities and the corresponding purification factors, even when one kind of tissue is considered (e.g. human placenta). Taking into account that different methods have been used by the various authors for the calculation of the purification factors, it can be concluded that our purification procedure gives better results. The amino-acid composition of the three preparations is given in Table 3. The compositions of the three forms are comparable with each other. The results of the glc-analysis of the carbohydrates present in the enzyme preparations have been summarized in Table 4. In Table 5 the carbohydrate compositions of hexosaminidases from various sources arc A and B from various

Hexosaminidase B Purification Specific factor activity

Substrate usedd

18000” 16080h 3860’ 1107h

207 23 240 130 546 180 93

MU MU MU MU MU MU PNP

2239” 34715b

5.0 134

PNP PNP MU

6000” 5800”

mammalian

tissues

Reference Lee & Yoshida (1976) Geiger & Arnon (1976.1978) Freeze cr rrl. (1979) Hasilik & Neufeld (1980) Wolf r~
,’ The calculation of the purification factor was based on the individual contribution to the total enzyme activity in the homogenate of hexosaminidase A and B. h The calculation of the purification factor was based on the total hexosaminidase activity in the homogenate. activity being hexosaminidase LThe calculation of the purification factor was based on 65”,, of the total hexosaminidase A and 35”,, hexosaminidase B in the tissue extract. d MU = 4-methylumbelliferyl-2-acetamido-2-deoxy-~-o-glucopyranoside: PNP = p-nitrophenyl-2-acetamido-2-deoxy/J’-o-glucopyranoside.

B. OVERDIJK

28 Table 3. Amino

acid composition

of bovine

Hexosaminidase Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine + half-cystine” Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysme Histidine Arginine

5.66 5.08 15.48 12.63 4.78 12.48 6.52 2.20 4.28 0.60 2.70 5.22 2.07 2.94 9.04 4.4X 3.34

i_ * + i f f ~

A

of ul

brain /I-hexosaminidase

Hexosammidase

0.02 0.32 2.48 0.12 0.27 1.15 0.10

7.51 5.56 15.08 12.45 4.22 12.49 6.79

f 0.79 _+ 1.02 k 0.22 _t 0.43 k 0.70 + 0.13 + 0.38 _t 0.75 f 0.26 + 0.15

2.36 4.31 0.32 2.82 5.36 2.12 3.12 8.40 4.36 3.55

B

i * + + f * + + + +

I

Hexosaminidase

If- 0.36 + 0.04 & 1.34 & 0.55 k 0.50 & 0.95 f 0.33

I

A. B and

4.99 & 0.16 5.39 * 0.20 14.71 * 1.89

12.03* 0.56 5.14 + 0.40 12.16 i 0.9’ 7.07 * 0.30

0.73 1.32 0.05 0.61 0.42 0.20 0.20 0.53 0.60 0.04

7.05 4.71 0.30 2.76 5.94 2.21 3.48 8.82 4.09 4.1 I

* 0.72 _+ 1.09 + 0.05 + 0.40 i 0.63 * 0.15 * 0.40 * 0.59 * 0.39

_t 0.13

,’ Determined as cystine. Values given represent means + SEM. The data are expressed as mol of amino acidilOOmol of total residues in the hydrolysate. Tryptophan was not determined.

present. but only N-glycosidically linked carbohydrate chains, either of :he high-mannose or of the complex type (Montreuil, 1980). The elucidation of the structure of these carbohydrate chains will be the subject of future research.

The glucose found in our preparations must be ascribed to contamination by column materials used. as was shown by glc-analysis of eluates of blank runs. The absence of N-acetylgalactosamine indicates that no mucin-type carbohydrate chains are

compared.

Table 4. Carbohydrate

composition

of purified

Hexosaminidase A ng sugar;:

brain

[j-hexosammidase

Hexosaminidase

carbohydrate residues; mol enzyme

100 1’8 protein

bovine

tsoenrymes

B

Hexoaaminidase I

ng sugar! tOOPg protein

carbohydrate residues/ mol enzyme

ng sugar. 100 1’9 protein

carbohydrate residues, mol en7yme

Mannose

2.70 i 0.06

Galactose NV-acetylglucosamine”

1.24 * 0.02 2.06 f 0.06

21.19 * 0.47 9.73 + 0.16 13.17 + 0.38

1.72 + 0.04 0.82 + 0.02 1.33 + 0.03

13.78 + 0.32 6.57 + 0.16 8.68 * 0.20

3.41 + 0.03 1.25 * 0.01 2.27 * 0.05

26.50 + 0.35 9.71 4 0.15 IS.63 _t 0.40

N-acetylneuraminic acid

0.18 & 0.01

0.82 _t 0.05

0.18 f 0.01

0.84 + 0.08

0.11 + 0.01

0.50 f 0.04

I’N-acetylglucosamine residues attached to the peptide A. Cheron. personal communication). Values given represent means + SEM. The molecular 1972: Overdijk. 1975). Protein was determmed according

Table 5. Carbohydrate

Enzyme

Tissue Human

placenta

Monkey

brain

Bovine bram

A B Converted A B A B

I

form

B

Mannose 2.17 2.37 3.48 27,’ 10.7 2.70 1.72 3.41

composition

Galactose 0.52 0.35 0.66 6.7

1.24 0.82 1.25

” Total amount of hexoses. h Total amount of hexosamines. y The following sugars were reported to be present: The data are expressed as 1lg sugar:lOOng protein.

chains

are not determined

with the used method

weight of the enzymes was taken to Lowry et trl. (1951).

of /I-hexosaminidases

N-acetylglucosamine 1.46 1.36 2.00 2.6h 5.3h 2.06 I .33 2.41

as 150.(x)0 (Robmson

from mammalian

0.34 0. IX 0.18 0.11

&

(‘1 trl..

tissues

X-acetvlneurammic . actd 0.60 0.09 0.04

IG. Streckcr

Reference Free/e

(‘I [I/. (1979)

Aruna & Basu (1Y75)’ Aruna & Basu (1976) Pre\cnt publicatron

mannose. galactose. glucose, hexosamine and stalic actd. Protein was determined according to Lcwry 1’1~1. II951 1

I Fig. 2. Polyacrylamide Experimental details of the following samples: hexosaminidase 1. 10 pg

2

3

4

567

8

gel electrophoresis in 15”<, SDS-gels of /Chexosaminidases from hovme brain. the electrophoresis are described in Materials and Methods. The lanes contain hexosaminidase A, 10/q protein (1). hexosaminidase B. IO/cg protein (2). protein (3) cytochrome c (4). aldolase (5). bovine serum albumin (6). catalase (7). ovalbumin (8).

29

Carbohydrate

composil tlon of hexosaminidases

ilc~,lo~~lpdgrn~r,lls~ We thank Mr L. A. W. Trippelvitz and Mr C. J. van der Wal for performing the carbohydrate and amino-acid analysis. REFERENCES AMINOFF D.. V~RDER BRUEC~GEW. F., BELL W. C., SARPOLE K. & WILLIAMS R. (1977) Role of sialic acid in survival of erythrocytes In the circulation: interaction of neuraminidase-treated and untreated erythrocytes with spleen and liver at cellular level. Proc. nutn. Acad. Sci. L’.S..4. 74, 1521 ~1524.

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31

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OVERDIJK B. (1975) Properties of /I-N-acetylhexosaminidases in human and bovine brain tissue. M.D. Thesis. Amsterdam. OVERDIJK B.. VAN DER KROEF W. M. J.. VEL~KAMP W. A. & HOOGHWINKEL G. J. M. (1975) The separation of bovine brain p-N-acetyi-D-hexosaminidases. Abnormal gel filtration behaviour of p-N-acetyl-D-glucosaminidase C. Biochem. J. 151, 257-261. POENARU L. & DREYFUS J.-C. (1973) Electrophoretic studies of hexosaminidases. Hexosaminidase C. Clinicu chim. Acru 43, 439-442. PRICE R. G. & DANCE N. (1972) The demonstration of multiple heat stable forms of ,Y-acetyl-/I-glucosaminidase in normal human serum. Biochim. hiopl1J.y. Acttr 271, 145-153. REINHOLD V. N. (1972) Gas- liquid chromatographic analysis of constituent carbohydrates in glycoproteins. In Methods in Enz!,moloyy. Vol. 25 (Edlted by HIRS C. H. W. & TIWASHEFF S. N.). pp. 244249. Academic Press. New York. ROBINSON D., JORDAN T. W. & HORSRURGH T. (1972) The N-acetyl-fi-hexosaminidases of calf and human brain. J. Nrurochem. 19, 1975- 1985. SANDHOFF K.. CONZELMAV~ E. & NEHRKORN H. (1977) Specificity of human liver hexosaminidases A and B against glycosphingolipids GU2 and G,>, Purification of the enzymes by affinity chromatography employing specific elutlon. Hoppr-Sry/rr’.\ Z. pltj%~/. C/wm 358, 779-787. STAHL P.. RODMAS J.. MILLER J. & SCHILSINC~ERP. (1978) Evidence for receptor-mediated binding of glycoprotelns. glycoconjugates. and lysosomal glycosidases by alveolar macrophages. Pro<,. nutn. Acud. .Ci. U.S.A. 75, 13991403. STAHL P.. SCHLESINGER P. H.. SIC;ARDSOKE.. RODMAS J. S. & LEF: Y. C. (1980) Receptor-mediated pinocytoais of mannose glycoconjugates by macrophages: characterization and evidence for receptor recycling. Cell 19, 207-215. WEBEKK. & OSBORX M. (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. hiol. Chrm. 244, 4406~~4412. WILLIAMS A. F. (1978) Membrane glycoproteins in recognition. Bloc,hr~~. Sot. Trutu 6, 490 494. WOLF J. H.. LISMAN J. J. W.. OVERDIJK B. & HOO(~HWINKEL G. J. M. (1980) On the structure of N-acetyl-B-ohexosaminidase from human liver. Arch int. PI~~wol. Biochim. 88, B252-B253. YAMAKAWA T. & NAGAI Y. (1978) Glycolipids at the cell surface and their biological functions. Trend\ hioclwm. SC;. 3, 128-131.