Galactosylceramide β-galactosidase in krabbe disease: Partial purification and characterization of the mutant enzyme

ARCHIVES

OF BIOCHEMISTRY

AND BIOPHYSICS

Vol. 196, No. 1, August, pp. 93-101, 1979

Galactosylceramidep-Galactosidase and Characterization YOAV BEN-YOSEPH, Departmnt

MELINDA

in Krabbe Disease: Partial Purification of the Mutant Enzyme’ HUNGERFORD,

AND

HENRY L. NADLER

of Pediatrics, Division of Genetics, Northwestern University Medical Children’s Memorial Hospital, Chicago, Illinois 60614

School,

Received January 29, 1979; revised March 26, 1979 Galactosylceramide /3-galactosidase (EC 3.2.1.46) has been partially purified from liver of a patient who died of Krabbe disease. Approximately 700-fold purification was achieved by solubilization, adsorption with immobilized concanavalin A, gel filtration through Bio-Gel A-1.5m and chromatography on immobilized sphingosine. The relative increase in crossreacting material and residual galactosylceramidase and lactosylceramidase I activities of the mutant enzyme was essentially identical to that obtained for the enzyme partially purified by the same procedure from normal liver control. An apparent molecular weight of about 750,000 and similar electrophoretic mobilities were observed for both enzymes. In contrast, catalytic properties and stability of the enzyme protein were severely affected in the mutant as compared to the normal enzyme. The apparent K, values of the mutant enzyme for P-galactosidase activities toward galactosylceramide and lactosylceramide in the presence of pure sodium taurocholate were 14 and 4 times, respectively, higher than the normal values. Incubation for 4 min at 52°C or dialysis against 1.3 M urea caused a 50% loss of residual enzymatic activity of the mutant enzyme, whereas a 35min incubation or dialysis against 5.6 M urea was required for 50% inactivation of the normal enzyme. These findings indicate that the mutation in Krabbe disease leads to synthesis of normal quantities of catalytically and structurally altered protein.

Globoid cell leukodystrophy or Krabbe are found to be normal in tissues from padisease is an inherited disorder of galacto- tients with another genetic disorder of galipid metabolism. Onset in early infancy is lactolipid metabolism, GM, gangliosidosis followed by rapidly progressive degenera- (7). Hydrolysis of lactosylceramide is also tion of the central nervous system ending in deficient in GM, gangliosidosis when assayed death generally before 2 years of age (1). in the presence of taurodeoxycholate, glycoDeficiency in activity of the enzyme galacto- deoxycholate, or taurochenodeoxycholate sylceramide /3-galactosidase (EC 3.2.1.46) (lactosylceramidase II) (6,8). However, the is the basic defect in this disease (2). In lactosylceramidase I assay is very specific addition to the diminished activity toward and determines almost exclusively the acgalactosylceramide in tissues of patients tivity of galactosylceramide P-galactosidase with Krabbe disease, deficient P-galacto- even in the presence of high levels of sidase activity has also been reported lactosylceramidase II activity (9). In a pretoward galactosylsphingosine (3), monoga- vious study, we used antibodies evoked lactosyldiglyceride (4), and lactosylcera- against galactosylceramide p-galactosidase mide when assayed in the presence of pure purified 3900-fold from normal placenta to sodium taurocholate (lactosylceramidase I) demonstrate approximately normal quanti(5,6). These four hydrolase activities appear ties of antigenically cross-reacting material to represent the same enzyme protein and in brain, liver, and skin fibroblasts of patients with Krabbe disease (10). In the present 1 This study was supported by grants from the Na- report, we describe biochemical and imtional Foundation-March of Dimes and the Kroc munochemical studies which were undertaken in order to characterize this catalytiFoundation. 93

0003-9861/79/090093-09$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

94

BEN-YOSEPH,

HUNGERFORD,

tally deficient galactosylceramide p-galactosidase partially purified from liver of a patient who died of Krabbe disease.

AND NADLER

radial immunodiffusion assay was performed in 0.8% agarose containing 0.44 mg/ml y globulin as described previously (16). Purified placenta enzyme (10) was used for calibration and goat antiserum to rabbit y globulin was used as a second antibody for visualization of the MATERIALS AND METHODS precipitin rings obtained with the first antibody. Enzyme m&cation. Liver tissue specimens were Sepharose 4B was from Pharmacia (Piscataway, N. J.); Bio-Beads SM-2 and Bio-Gel A-1.5m were from obtained at autopsy from patients with Krabbe disease Bio-Rad Laboratories (Richmond, Calif.) and agarose and normal controls. The specimens were frozen imwas from Aldrich Chemical (Milwaukee, Wise.). Acryl- mediately and kept at-20°C until used. Fifty grams amide, N,N’-methylenebisacrylamide, TEMED,2 am- of tissue was homogenized in 250 ml of 0.1% Cutscum, monium persulfate, Coomassie brilliant blue G-250 and 10 mM sodium phosphate buffer, pH 6.0, in a Waring cyanogen bromide were from Eastman-Kodak (Ro- Blendor for three 1-min periods at top speed. Lectins chester, N. Y.). Concanavalin A, wheat germ agglu- were immobilized on cyanogen bromide-activated tinin, blue Dextran, bovine serum albumin, 4-methyl- Sepharose 4B (17). The Sepharose-bound preparations umbelliferyl fl-n-galactopyranoside, and N-carbobenz- 50 ml each, contained 0.86 mgiml concanavalin A and oxy e-aminocaproic acid were from Sigma (St. Louis, 0.72 mg/ml wheat germ agglutinin. Adsorption of 200 ml liver supernatant (12,500g) was carried out in 0.1% MO.). Bovine y globulin, ovalbumin, dihydrosphingosine, galactosyl-N-stearoyl-dihydrosphingosine, lac- Cutscum, 10 mM sodium phosphate buffer, pH 6.0, and tosyl-N-stearoyl-dihydrosphingosine, and goat anti- elution of the enzyme fraction with the same buffer serum to rabbit y globulin were from Miles containing 0.5 M NaCl and 1 M of either methyl-a-nLaboratories (Elkhart, Ind.). Oleic acid, sodium tauro- mannoside or N-acetyl-n-glucosamine. The sugar comcholate, and sodium taurodeoxycholate were from Cal- petitor was removed by dialysis against 0.14 M NaCl, 10 mM sodium phosphate buffer, pH 6.0. The enzyme biochem (La Jolla, C&f.). Cutscum was from Fisher (Pittsburgh, Pa.) and Silica Gel G thin-layer plates fraction was concentrated by negative pressure to 5 ml were from Brinkman Instruments (Westbury, N. Y.). and applied on Bio-Gel A-1.5m column, 2 x 110 cm. Galactose oxidase was from Worthington Biochemical The gel filtration column was calibrated with known (Freehold, N. J.) and sodium boro[3H]hydride (270 molecular-weight markers: blue Dextran, 2,000,OOO; mCi/mmol) from New England Nuclear(Boston, Mass.). human plasma a,-macroglobulin &M), 720,000;bovine y globulin (IgG), 150,000; bovine serum albumin, Preparation of radiolabeled substrates. Galactosylceramide and lactosylceramide were labeled with 68,000; and ovalbumin, 45,000. The major enzyme fractritium at carbon 6 of the terminal galactose residue tion was concentrated and then chromatographed on a spningosine column. Dihydrosphingosine was coupled by the galactose oxidase-sodium boro[3H]hydride method (11). The radiolabeled products were purified with l -aminocaproic acid (18) and immobilized on by Silica Gel thin-layer chromatography in solvent Sepharose 4B (17). The Sepharose-bound N-e-aminosystem of chloroform/methanol/water (110/40/6). The caproyl-dihydrosphingosine (10 ml) contained 0.12 labeled substrates were diluted prior to use with the mglml l&and. The enzyme fraction was eluted by 50150 respective nonradioactive compounds to give specific ml linear gradient of O-50% ethylene glycol in 0.14 M NaCl, 10mM sodium phosphate buffer, pH 6.0, dialyzed activity of 400-600 cpminmol. Tritium radioactivity against 0.14 M NaCl, 10 mM sodium phosphate buffer, was measured in a Tri-Carb liquid scintillation pH 7.0 (PBS), to remove the ethylene glycol, and used spectrometer. for subsequent studies. Enzyme assays. Hydrolysis of I-methylumbelliferyl Immunochemical studies. Quantitation immuno/3-Dgalactoside was determined as previously described (10) and galactosylceramidase and lactosylceramidases precipitation was carried out in finai volume of 1.5 ml I and II activities were assayed according to Wenger by adding increasing amounts of the enzyme preparaet al. (8, 12). After removal of Cutscum using SM-2 tion to a constant amount of y globulin fraction of the beads (13), protein was determined by the Folin method antiserum (0.5 ml). The mixtures were incubated 1 h (14) with bovine serum albumin as a standard. Absor- at 37°C and 16 h at 4°C. The immune precipitates were bance measurements were made in a Beckman double- spun down, washed twice in cold PBS, redissolved in beam spectrophotometer. 0.1 M NaOH, and the protein content was determined Quantitation of antigenically cross-reacting ma- by the absorbance at 280 nm. Immunoelectrophoresis terial. y globulin fraction was prepared from rabbit was performed by electrophoresing the enzyme prepantiserum to placenta galactosylceramide /3-galacto- aration on 6% polyacrylamide gel (38/l, acrylamidei sidase (10) according to Levy and Sober (15). Single bisacrylamide) (19) following in which the gels were placed on 0.8% agarose plate containing a trough filled ’ Abbreviations used: TEMED, N,N,N’,N’-tetrawith the antibody preparation. After diffusion for 48 h methylene diamine; a,M, a*-macroglobulin; IgG, y at 4°C the agarose plate was washed for 72 h in 0.14 M globulin; PBS, phosphate buffered saline. NaCl, 10 mhf sodium phosphate buffer, pH 7.0, and

GALACTOSYLCERAMIDE

pGALACTOSIDASE

95

IN KRABBE DISEASE

stained for protein with Coomassie brilliant blue G-250, 0.25% in methanol/acetic acid/water (5/l/5). Kinetic studies. Galactosylceramidase and lactosylaceramidase I activities of partially purified galactosylceramide /3-galactosidase were assayed at various substrate concentrations. The apparent MichaelisMenten constants (K, values) were determined graphically according to Lineweaver and Burk (20). Znactivutiwn studies. Thermostability of the partially purified enzyme preparations was examined at a temperature of 52°C. Residual galactosylceramidase and lactosylceramidase I activities were determined after incubation for various periods of time. Susceptibility of the partially purified galactosylceramide P-galactosidase to urea denaturation was examined by dialyzing the enzyme preparations against solutions of various urea concentrations and determinations of residual enzymic activities after redialysis against 0.14 M NaCl, 10 mM sodium phosphate buffer, pH 7.0.

liver fractions but content of antigenically cross-reacting material was very similar. The relative increase in concentration of cross-reacting material within the protein was consistent with the increase in specific enzymic activities for both the mutant and the normal enzyme purifications. Replacement of the concanavalin A step by adsorption of the liver preparations with Sepharose-bound wheat germ agglutinin resulted in a poor yield of about 8%. Elution of the enzyme fraction from this lectin conjugate was not as efficient as that obtained by methyl-a-D-glucoside in the case of concanavalin A since only the free sugar N-acetylD-ghcOSa?IIine was employed as competitor. However, the purification-fold as calculated according to antigenically cross-reacting material and enzymatic activities was esRESULTS sentially the same for both the mutant and the normal galactosylceramide p-galactoPur@ication of Enzyme sidases and very similar to the fraction obThe purification of galactosylceramide tained from concanavalin A adsorption. P-galactosidase from liver of a patient with Liver samples from two additional patients Krabbe disease and a normal control is de- with Krabbe disease and three controls were scribed under Materials and Methods and subjected after solubilization to either consummarized in Table I. Purification-fold and canavalin A adsorption or sphingosine chroyield at each step were calculated according matography. Residual enzymic activities in to the quantity of antigenically cross-react- the patients liver preparations were about ing material and both galactosylceramidase 13 and 16%of normal liver controls and found and lactosylceramidase I activities. Residual to copurify with antigenically cross-reacting enzymic activities of fractions obtained from material in each of the purification steps. the patient liver were about 8% of control Purification-fold and yield in these two steps TABLE I PARTIAL PURIFICATION OF GALACTOSYLCERAMIDE P-GALACTOSIDASEFROM 50~ OF “Krabbe” ANDNORMALLIVERSPECIMENS~ Galactosylceramidase specific activity (nmol/h/mg protein) Step Liver homogenate Concanavalin A adsorption Bio-Gel A-1.5m gel filtration Sphingosine chromatography Overall yield (%) Overall purification

Cross-reacting material (pg/mg protein)

Krabbe

Normal

Krabbe

Normal

0.11 6.13 22.9 76.5

1.31 69.9 287 958

0.23 11.8 55.2 178

0.21 10.3 49.7 171

20.4 731

19.8 774

22.7 814

17.8 695

a The overall yield and purification are calculated for Krabbe and normal enzyme preparations according to galactosylceramidase activity and content of antigenically cross-reacting material.

BEN-YOSEPH,

HUNGERFORD, TABLE

AND NADLER

II

~GALACTOSIDASE ACTIVITIES TOWARD GALACTOSYLCERAMIDE (GalCer), LACTOSYLCERAMIDE (LacCer AND II), AND 4-METHYLUMBELLIFERYL P-D-GALACTOSIDE (MU-@-Gal) IN GALACTOSYLCERAMIDE ~GALACTOSIDASE PARTIALLY PURIFIED FROM “Krabbe” AND NORMAL LIVER P-Galactosidase

activity

(nmol/h/mg

protein)

Enzyme preparation

GalCer

Krabbe Liver homogenate Partially purified

0.11 76.5

0.34 217

20.8 28.2

293 64

Normal Liver homogenate Partially purified

1.31 958

3.67 2620

22.9 185

388 501

were similar for all mutant and normal liver enzymes as calculated according to crossreacting material and both galactosylceramidase and lactosylceramidase activities. Galactosylceramidase, lactosylceramidases I and II, and 4-methylumbelliferyl p-galactosidase activities in the partially purified preparations were compared to the respective activities in the liver homogenates. The results are shown in Table II. Purification of about 700-fold was found for p-galactosidase activities toward galacto-

LacCer I

LacCer II

I

MU-&Gal

sylceramide and lactosylceramide I in both the “Krabbe” and the normal preparations. In contrast, the purification factors for lactosylceramidase II and 4-methylumbelliferyl p-galactosidase activities were only 1.4 and 0.2 for the “Krabbe” preparations and 8.1 and 1.3 for the normal preparation, respectively. Molecular

Weight

The elution patterns of the mutant and normal galactosylceramide /3-galactosidases

FIG. 1. Gel filtration of galactosylceramidase activity of normal (0) and Krabbe (0) liver preparations on Bio-Gel A-1.5m. Five-milliliter fractions (180 mg protein) of Sepharose-concanavalin A eluate were filtered through 2 x llO-cm column in 0.14 M NaCl, 10 mM sodium phosphate buffer, pH 6.0, at 4°C at flow rate of 22 ml/h. Molecular weight markers: blue Dextran, 2 x 106; human plasma a,-macroglobulin (a,M), 7.2 x 105; bovine liver catalase, 2.4 x 105; bovine y globulin (IgG), 1.5 x 105; bovine serum albumin, 6.8 x 104; ovalbumin, 4.5 x lo* (m).

GALACTOSYLCERAMIDE

&GALACTOSIDASE

IN KRABBE DISEASE

97

from Bio-Gel A-1.5m column are shown in Fig. 1. The major peak of each column corresponded to a molecular weight of around 750,000. This enzyme fraction was pooled and further purified by column chromatography on Sepharose-bound N-c-aminocaproyl-dihydrosphingosine. Minor peaks of low molecular weight forms of galactosylceramide @galactosidase were also observed in the patient and normal liver preparations with similar proportion between the various enzyme forms. No attempts .were made to purify the low-molecular-weight forms. It FIG. 3. Immunoprecipitation of normal (0) and Krabbe (0) partially purified liver galactoslyceramide p-galactosidases with y globulin fractions of antiserum to the purified placenta enzyme. Antigenically crossreacting material in the enzyme preparations was determined by single radial immunodiffusion assay as described under Materials and Methods. Samples containing increasing amount of cross-reacting material were incubated with 0.5 ml of antiserum preparation in a final volume of 1.5 ml in 0.14 M NaCI, 10 mM sodium phosphate buffer, pH 7.0. Following 1 h at 37°C and 16 h at 4°C the immunoprecipitates were washed twice, dissolved in 0.1 M NaOH, and quantitated for protein by absorbance at 280 nm.

should be noted, however, that these minor enzyme forms demonstrated antigenic identity with the major high-molecularweight form when compared by double immunodiffusion against antiserum to the purified placenta galactosylceramide pgalactosidase. Electrophretic

Mobility

The partially purified enzyme preparations were subjected to polyacrylamide gel FIG. 2. Polyacrylamide gel electrophoresis of normal electrophoresis in order to compare the (N) and Krabbe (K) partially purified liver galactosylelectrophoretic mobility of the mutant enceramide &galactosidases followed by immunodiffusion in agarose gel against -y globulin fraction of anti- zyme with that of the normal. Since there serum to the purified placenta enzyme (ab). Enzyme is no activity staining available, the electrosamples containing 200 pg protein were applied to 10 phoresed enzyme was located by immunox O&cm disks of 6% polyacrylamide gel (38/l, acryl- diffusion against the anti-enzyme antiserum amide/bisacrylamide) and electrophoresed for 6 h at and similar mobilities were demonstrated 4°C at constant current of 2 mA per gel. Immunodiffor these mutant and normal enzymes (Fig. fusion was carried out by placing the polyacrylamide disks on agarose-gel plate (0.8% in 0.14 M NaCl, 10 2). Enzyme preparations from liver samples of two additional patients with Krabbe dismM sodium phosphate buffer, pH 7.0) with trough tilled with the antiserum preparation. Diffusion allowed ease and three controls had also similar electo proceed for 48 h at 4°C and the precipitin arcs were trophoretic mobilities when examined after chromatography on immobilized sphingosine. stained with Coomassie blue.

98

BEN-YOSEPH,

HUNGERFORD,

P

ZOO-

-

I’ I’ I/V hv# h/pm011

tk 0,’ ,’ ,’ ,’

loo-

AND NADLER

higher than the normal (Fig. 4) and about four times increase in the apparent K, was found for its lactosylceramidase I activity (Fig. 5). Similar differences in the apparent K, values of these enzymatic activities were also observed between sphingosine-chromatographed fractions of several other patients and controls liver samples.

” /

Heat Znactivation

,I0

The partially purified mutant enzyme was found to be more thermolabile than the % KRABBE / normal enzyme when incubated at 52°C NORMAL Km.16sg,~ . K..10.8pM (Fig. 6). Temperature of 56°C was required -jr-to demonstrate thermolability of liver prep-100 50 -50 0 arations from other patients with Krabbe I/S hedl-’ ) disease which were purified by either conFIG. 4. Double reciprocal plots of galactosylceramicanavalin A adsorption or sphingosine chrodase activity of normal (0) and Krabbe (0) partially matography. In all mutant and normal enpurified liver enzymes versus galactosylceramide concentration. Enzyme preparations containing 10 pg zyme preparations, lactosylceramidase I activity was more stable than galactosylprotein were added to a sonicated mixture of varying amounts of [3H]galactosylceramide (560 cpmnmol), ceramidase activity. However, both activi200 pg sodium taurocholate, and 20 pg oleic acid in 0.2 ties were inactivated much faster in prep,

,’

ml of 0.1 M sodium citrate/sodium phosphate buffer, pH 4.3. Following a 30-min incubation at 37°C the reaction was terminated and processed according to Wenger et al. (12).

Zmmunoprecipitation Quantitative precipitin tests were carried out employing the antigalactosylceramide P-galactosidase antiserum and the partially purified mutant and normal enzymes. Essentially identical precipitin curves were found for both enzyme preparations (Fig. 3). Less than 7% of the initial galactosylceramidase and lactosylceramidase I activities could be recovered in supernatants of the equivalence zone implying that the antiserum is monospecific and the antigenitally cross-reacting material represent the galactosylceramide pgalactosidase enzymes. Michaelis -Menten Constants A study of the apparent K, values of the mutant and normal partially purified enzymes toward galactosylceramide P-galactosidase is severely defective in respect to it’s substrate affinity. The apparent K, of the mutant enzyme galactosylceramidase activity was found to be about 14 times

I/S

(mM-‘I

FIG. 5. Double reciprocal plots of lactosylceramidase I activity of normal (0) and Krabbe (0) partially purified liver enzymes versus lactosylceramide concentration. Enzyme preparations containing 5 pg protein were added to a sonicated mixture of varying amounts of [3H]lactosylceramide (440 cpm/nmol), 500 fig sodium taurocholate, and 50 pg oleic acid in 0.2 ml of 0.1 M sodium citrate/sodium phosphate buffer, pH 4.3. Following a 20-min incubation at 37°C the reaction was terminated and processed according to Wenger et al. (12).

GALACTOSYLCERAMIDE

/3-GALACTOSIDASE

IN KRABBE

DISEASE

arations from Krabbe disease liver compared to controls.

99

as patients with Krabbe disease has been reported to be less than 5% of that of normal controls (1). In a previous study, we used Urea Denaturation antibodies evoked against the placenta enzyme to demonstrate the presence of antiThe susceptibility of the mutant enzyme genically cross-reacting material in brain, to urea was greater than that of the normal liver, and skin fibroblasts of patients with enzyme (Fig. ‘7). In contrast to heat inacKrabbe disease (10). Moreover, antigenic tivation, galactosylceramidase and lactosyl- identity was documented between galactoceramidase I activities were inactivated by sylceramide P-galactosidases from different urea to the same extent and the same urea organs as well as between the mutant and concentrations were required to cause loss of the normal enzymes. In the present study, activity in enzyme preparation purified by we elected to purify the mutant enzyme from either one or four step procedures. liver since the highest residual activity was measured in this tissue. In addition to deDISCUSSION termination of enzymatic activity, the antiDeficiency of certain enzymatic activity enzyme antibodies were also used in order is the basic defect in many diseases. Such to follow the catalytically deficient enzyme deficiency can be as severe as total absence protein throughout the purification proceor milder when high levels or residual ac- dure. The purification pattern was very tivity are found. In many cases, the reduced similar for the mutant and the normal liver enzymatic activity is barely detectable in enzymes indicating that both enzymes reveal preparations of patient tissues and thus affinity to the lectins concanavalin A and purification of a mutant enzyme should not wheat germ agglutinin and to the sphingobe solely based on its catalytic activity. The lipid N-•-aminocaproyl-dihydrosphingosine, average residual galactosylceramide p-ga- although differences in the degree of aflactosidase activity in various organs of finity may still exist. The enzyme prepara-

---A-

--_____

-. ---_

KRABBE -~------,-o

-LaCCer

KRABBE-

GolCw

1

------m----Q --------o 1

I

20

60

40 Incubation

Time

,

60

(min)

FIG. 6. Thermostability of galactosylceramidase (0, 0) and lactosylceramidaae I (A, A) activities of normal (-) and Krabbe (- - - -) partially purified liver galactosylceramide &@actosidases at 52°C. Enzyme preparations with protein concentration of 100 pg/ml were incubated in 0.14 M NaCl, 10 mM sodium phosphate, pH 7.0, for various periods of time as indicated. Residual enzymatic activities were assayed according to Wenger et al. (12).

100

BEN-YOSEPH,

HUNGERFORD,

L

AND NADLER

.

Urea

(Ml

FIG. 7. Susceptibility of galactosylceramidase (0, 0) and lactosylceramidase I (A, A) activities of normal (-) and Krabbe (- - - -) partially purified liver galactosylceramide P-galactosidases to urea denaturation. Enzyme preparations with protein concentration of 100pg/ml were dialyzed against 10 mM sodium phosphate buffer, pH 7.0, containing varying concentrations of urea as indicated. After redialysis against 0.14 M NaCl, 10 mM sodium phosphate buffer, pH 7.0, the residual enzymatic activities were assayed according to Wenger et al. (12).

tions were almost free of GM, ganglioside p-galactosidase as indicated by much greater lactosylceramidase I over lactosylceramidase II activity and galactosylceramidase over 4-methylumbelliferyl p-galactosidase activity. Both enzyme preparations filtered through Bio-Gel A-1.5m with a major activity peak in a molecular-weight range of 750,000. Similar proportions were demonstrated between the high- and low-molecular-weight forms. The smallest size form had a molecular weight of around 120,000 and it should be noted that sodium dodecyl sulfate-gel electrophoresis of the purified placenta enzyme revealed a similar molecular weight (10). The various molecular-weight forms of the mutant as well as the normal enzyme demonstrated complete antigenic identity suggesting that the higher molecular-weight forms are possible oligomers of the 120,000one. The antigenic determinants appear to be the same in both enzyme, as shown by identical precipitin curves and by inhibition of immunotitration of the normal enzyme by crude preparations obtained from three unrelated patients with Krabbe dis-

ease. Striking differences were observed in the apparent K, values of the mutant and the normal enzymes as well as in their thermostability and susceptibility to urea denaturation. Although complete study was carried out with only one partially purified liver preparation of each, these findings were also confirmed in less purified preparations from two additional patients with Krabbe disease and three liver controls. In conclusion, the present report strongly suggests that normal quantities of enzyme protein are synthesized in Krabbe disease but this mutant galactosylceramide P-galactosidase is severely defective catalytically and structurally. REFERENCES 1.

K., AND SUZUKI, Y. (1978) in The Metabolic Basis of Inherited Disease (Stanbury, J. B., Wyngaarden, J. B., and Fredrickson, D. S., eds.), pp. 747-769, McGraw-Hill, New York. 2. SUZUKI, K., AND SUZUKI, Y. (1970) Proc. Nat. SUZUKI,

Acad. Sci. USA 66, 302-309.

GALACTOSYLCERAMIDE

&GALACTOSIDASE

3. MIYATAKE, T., AND SUZUKI, K. (1972) Biochm. Biophys. Res. Common. 48, 538-543. 4. WENGER, D. A., SATTLER, M., AND MARKEY, S. P. (1973) Biochem. Biophys. Res. Commun. 53, 680-685. 5. WENGER, D. A., SATTLER, M., AND HIATT, W. (1974) Proc. Nat. Acad. Sci. USA 71,854-857. 6. TANAKA, H., AND SUZUKI, K. (1975) J. Biol. Chem. 250, 2323-2330. 7. O’BRIEN, J. S. (1975) Clin. Genet. 8, 303-313. 8. WENGER, D. A., SATTLER, M., AND CLARK, C. (1975) Biochim. Biophys. Acta 409, 297-303. 9. TANAKA, H., AND SUZUKI, K. (1977) Clin. Chim. Acta 75, 267-274. 10. BEN-Y• SEPH, Y., HUNGERFORD, M., AND NADLER, H. L. (1978) Amer. J. Hum. Genet. 30, 644-652. 11. RADIN, N. S., HOF, L., BRADELY, R. M., AND BRADY, R. 0. (1969) Brain Res. 14, 497-505. 12. WENGER, D. A., SATTLER, M., CLARK, C., AND

13. 14.

15. 16.

17. 18. 19. 20.

IN KRABBE

DISEASE

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MCKELVEY, H. (1974) Clin. Chim. Acta. 56, 199-206. HOLLOWAY, P. W. (1973) Anal. Biochem. 53, 304-308. LOWRY, D. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. LEVY, H. B., AND SOBER, H. A. (1960) Proc. SOC. Exp. Biol. Med. 103, 250-252. BEN-Y• SEPH, Y., BURTON, B. K., AND NADLER, H. L. (1977) Amer. J. Hum. Genet. 29, 575-580. AXEN, R., PORATH, J., AND ERNBACK, S. (1967) Nature (London) 214, 1302- 1304. GORDON, J. A., BLUMBERG, S., LIS, H., AND SHARON, N. (1972) FEBS Lett. 24, 193-196. DAVIS, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404-427. LINEWEAVER, H., AND BURK, D. (1934) J. Amer. Chem. Sot. 56, 658-666.