J. Biol. Macromol.3 (1) 15 – 22 (2003)

ArticlesStudies on Glucocerebrosidase from Bovine Brain
II: Molecular and kinetic characterization of the solubilized and immobilized
enzymes
Saori Yasui, Mutsumi Shimada, Azusa Hotta, Sumio Ishijima and Masatake Ohnishi
Graduate School of Agriculture, The Prefecture University of Kyoto(Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan)
Received September 20, 2003，Accepted February 10, 2003
Glucocerebrosidase
β-glucosidase.
Sepharose.

(EC

3.2.1.45)

is

a

and

membrane-bound

Bovine brain glucocerebrosidase was immobilized on decyl-

Immobilization did not change temperature and pH dependence ofthe enzyme activity.

The solubilized enzyme was a little more stable to heat

inactivation than the immobilized enzyme.
changelysosomal

kinetic

parameters,

Km

and

Immobilization did not or little
Vmax,

and

the

inhibitionbyconduritol-B-epoxide, but the immobilized enzyme was less susceptible to ceramide
inhibition.

Thus, bovine brain glucocerebrosidase can be immobilized on

decyl-Sepharose with little changing molecular and kinetic properties.
Keywords:

Glucocerebrosidase;

Immobilization;

Decyl-Sepharose;

Ceramide;

Conduritol-B-epoxide; Bovine brain

Glucocerebrosidase(glucosyl-

brosidase has been purified from a variety

ceramide-β-glucosidase, EC 3.2.1.45) is aof mammalian tissues2,3).

membrane-bound lysosomal hydrolase

purifiedthat participates in the degradation ofbrosidase using hydrophobic chromato-

glycosphingolipids by cleaving glucosyl-

graphy2).

In the preceding paper4), we

ceramide (glucocerebroside) to yieldreported

inhibition of bovine brain

glucose and ceramide1).

glucocerebrosidase by Dextran sulfate.

Glucocere-human

Furbish et al.

placental

glucocere-

16

Glucocerebrosidase, immobilized to decyl-Sepharose

We here reported immobilization of thesuspended in 0.1 M sodium carbonate

bovine brain enzyme to acyl-Sepharose,buffer (pH 9.5).

and indicated some of the properties of

For preparation of decyl-Sepharose,

the solubilized and immobilized enzyme.

25 ml of N-decylamine was mixed with
100 ml of N,N’-dimethylformamide andMaterials and methods

100 ml of 0.1 M NaHCO3 and pH wasMaterials.

4-Methylumbeliferyl-β-D-

adjusted to 9.5 by addition of citric acid.

glucopyranoside (MUG), and cyanogen

Activated Sepharose 4B was added to thebromide were obtained from Nacalaidecylamine solution and stirred for 20 h.

Tesque Inc. Sepharose 4B was obtained

Decylated Sepharose 4B was washedfrom Amersham Biosciences Corp.

with 1 l of distilled water and suspended

Preparation of bovine brain enzyme.

in 200 ml of 1 M 2-aminoethanol (pHGlucocerebrosidase was partially purified

9.5).

from bovine brain by ammonium sulfate

and washed with 500 ml of distilled water,

fractionation, acid precipitation at pH

and then 0.2 M acetic acid, distilled water,

5.35 and butanol extraction as described

50 mM NaOH, distilled water, 50% (v/v)

previously4).

1,4-dioxane, 0.2 M acetic acid, andPreparation of acyl-Sepharose.
steps were carried out at 4˚C.

All

Sepharose

The suspension was stirred for 4 h

finally washed with 1 l of distilled water,
respectively.

4B (80 ml) was washed with distilled

Other acyl-Sepharose was preparedwater and suspended in 200 ml of 2 M

by the same method as decyl-Sepharose.

Na2CO3 (pH 12.1).

Immobilization of glucocerebrosidase

Cyanogen bromide

solution (25 g/500 ml in distilled water)

to decyl-Sepharose.

that was made just before using was

glucocerebrosidase was dialyzed against

added to Sepharose gel suspension slowly

20 mM sodium phosphate buffer (pH 5.5)

with stirring.

containing 5 mM EDTA and 1 mM

During the addition ofPartially-purified

cyanogen bromide solution, the pH of the

2-mercaptoethanol.

gel

at

(final 30% (v/v)) was added to the11.0-12.0 by addition of NaOH solution.

dialysate and the sample was mixed with

The gel suspension was stirred gently fordecyl-Sepharose.

30 min.

Activated Sepharose 4B was

was washed with distilled water, and thenwashed with 2 l of cold water and 2 l ofsuspended in the same buffer that wascold 0.1 M NaHCO3 (pH 9.5), and thenused for dialysis of the enzyme.

suspension

wasmaintained

EthyleneglycolThe gel suspension

The gelS. Yasui et al.

suspension was stirred for 4 h andcentrifuged at 15,000xg for 15 min.

The

17

Results
Glucocerebrosidase is a membrane-pellet was washed with sodium phosphatebound enzyme.

buffer (pH 5.5) containing 5 mM EDTA

cerebrosidase has been purified usingandhydrophobic chromatography, and it was

1mM

2-mercaptoethanol,andHuman placental gluco-

suspended in the same buffer.

shown that the placental enzyme was

Enzyme assays.

eluted at 60% ethylene glycol during

The standard assay

chromatography

5 mM MUG in 0.1 M sodium phosphate

Reddy et al. 3) indicated that bovine brain

buffer (pH 5.5), 0.15% Triton X-100 andglucocerebrosidase was more hydropho-

0.125% sodium taurocholate4).

bic than the human placental enzyme.

courses

of

the

Time

enzyme-catalyzed

onoctyl-Sepharose2).

mixture with artificial substrate contained

Adsorptionofbovinebrain

variousacyl-hydrolysis of MUG were measured at

glucocerebrosidase

30˚C.

Sepharose was examined by batch

The

amountofproducedto4-methylumbeliferon was determined byfluorescence intensity exited at 340 nm.

120

The progress curves of the enzyme

Proteinconcentration was determined by the

60
40
20

albumin as standard.

0

Kinetic studies.

The apparent Km of

enzyme for MUG was determined bystandard assay with varying substrateconcentrations.

Kmvaluesdetermined by s/v~s plots.
constants,

Ki,

were

were

Inhibitor

determinedbystandard assay using MUG as substrate in
the presence of various concentrations ofinhibitor. Ki values were determined by
Dixon plots.

Decyl-

method of Bradford5) using bovine serum

Butyl-

determination.

Hexyl-

Protein80

Octyl-

over 8 to 16 min.

100
Adsorption(%)

reaction were practically straight lines

Acyl-Sepharose

Fig. 1.
Adsorption of glucocerebrosidase to acyl-Sepharose.
The enzyme was mixed with the indicatedacyl-Sepharose in the absence (open bar)and presence (closed bar) of 30% ethylene
glycol, and was allowed to stand for 15
min at 4˚C. After centrifugation, theenzyme activity of the supernatant was
assayed.

Glucocerebrosidase, immobilized to decyl-Sepharose

18

method in the absence and presence ofOptimum temperature.

30% ethylene glycol (Fig. 1).

In thetemperature on the activities of theabsence of ethylene glycol, 80% or more

glucocerebrosidase were examined withof the enzyme was adsorbed to decyl- and

MUG in 50 mM sodium phosphate buffer,octyl-Sepharose, and addition of 30%

pH 5.5, containing 0.15% Triton X-100,

ethylene glycol increased the amount of

0.125% sodium taurocholate.

the adsorbed enzyme.

highest activity of both enzyme was

The results

The effects ofThesuggested that bovine brain enzyme
cannot be eluted from octyl-Sepharose（ A)

column by addition of ethylene glycol.

120

We here adsorbed the bovine brain
terized the immobilized enzyme.

Activity(%)

enzyme to decyl-Sepharose, and charac-

100
80
60
40
20
0

Molecular characterization of solubilized

andimmobilized

0

40

50

60

70

120
100
Activity(%)

The property of immobilized enzyme

80
60
40

hydrolysis of MUG as the guidance.

20

Immobilized enzyme retained the activity0

in 1% Triton X-100, 2% sodium cholate,

30

(B )

brosidase

enzyme, using the enzyme-catalyzed

20

Te mp.(℃ ）glucocere-

was compared with the solubilized

10

4

5

6

7

8

pH

1% sodium taurocholate, and 1% CHAPS. Fig. 2. Temperature (A) and pH (B)In 80% ethylene glycol, 10% of enzyme dependence of solubilized ( ● ) andimmobilized ( ○ ) glucocerebrosidase
was eluted from the gel. By addition ofactivity.
100 mM sodium thiocyanate, about 20% The enzyme activity was assayed in 50
of glucocerebrosidase was eluted. mM sodium phosphate buffer (pH 5.5)
containing 0.15% Triton X-100 and
These results suggested that decyl- 0.125% sodium taurocholate (A) or incitrate/phosphatebufferSepharose-immobilized glucocerebrosi- wide-range
containing 0.15% Triton X-100 at 30˚C.
dase had enough stability to make The amount of produced 4-methylcomparison with solubilized glucocere- umbeliferon was determined at 8 to 15
min. The maximal activity was taken as
brosidase.
100%.

S. Yasui et al.

19

obtained at 50˚C (Fig. 2A) under theestimated to be pH 5.5 (Fig. 2B). The

conditions. At the higher temperatures,pHthe heat inactivation was observed during

immobilized glucocerebrosidase activity

the enzyme assay as shown below.

The

were not different.

temperature dependence of activity was

Thermal stability.

not so much changed between solubilized

immobilized

and immobilized glucocerebrosidase.

compared

Optimum pH.

The effects of pH on

dependenceofsolubilized

andThermal stability of

glucocerebrosidase

with

that

of

was

solubilized

glucocerebrosidase. When enzymes weresolubilized and immobilized glucocere-

incubated

at

5˚C,

solubilizedandbrosidase activity were examined withimmobilized

glucocerebrosidase

kept

MUG as the substrate in wide-range

almost all the activities at least for 150

buffer, containing 0.15% Triton X-100.

min.

The optimum pH of both enzymes werethan 90% activity after incubation at

Both enzymes also retained more

(A )
120

100

(B )

80

80

Activity (%)

Activity (%)

100

60
40
20

60
40
20

0
20

30

40

50

60

0

70

0

T e m pe r at u r e (℃ )

120

180

240

T im e (m in )

(C )
100

(D )

80
80

60

Activity (%)

Activity (%)

100

60

40
20
0
0

5

10

15

20

T im e (m in )

25

30

60
40
20
0
0

5

10

15

20

T im e (m in )Fig. 3. Thermal stability of solubilized and immobilized glucocerebrosidase.
(A) The enzyme was incubated at various temperature for 30 min in sodium phosphatebuffer (pH 5.5) and the enzyme activity was assayed. (B)―(D) The enzyme wasincubated at 50˚C (B), 60˚C (C) and 65˚C (D). At various intervals samples were
removed and assayed for glucocerebrosidase at 30˚C. The initial activity without
incubation was taken as 100%.

Glucocerebrosidase, immobilized to decyl-Sepharose

20

30˚C for 30 min (Fig. 3), and more than
70% activity for 120 min.

Thermalstability of both enzymes incubated at 50,
60 and 65˚C were examined.

Table 1.
Kinetic parameters of thesolubilized and immobilized glucocerebrosidase-catalyzed hydrolysis of MUG.
Glucocerebrosidase

The

Solubilized

Immobilized

1.1

1.3

0.55

0.54

solubilized enzyme kept the activity withKm

incubation at 50˚C at least for 240 min,Vmax (µM/min)(mM)

while the immobilized enzyme remained
about 60% activity at 240 min. The

with incubation for 90 min at 50˚C.

results indicated that the solubilized

Kinetic characterization of solubilized

enzyme was a little more stable withand immobilized glucocerebrosidase

incubation at 50˚C than the immobilized

Kinetic parameters.

enzyme.

While both enzymes wereconstant, Km, and maximum velocity,

relatively stable at 50˚C, the enzymes

Vmax, were obtained with MUG as a

incubated at 60˚C were lost most activity

substrate. The plot of the initial velocity,

until 30 min, and at 65˚C the activitiesv, against [MUG]0 apparently fitted awere not detected at 5 min.

Michaelis-Menten fashion. The Km andThese results suggested a possibility

The Michaelis

Vmax values evaluated with MUG arethat the immobilized enzyme was eluted

summarized in Table 1.

from the gel with incubation at highlized glucocerebrosidase showed almost

temperature.

Therefore, the extent of

the same values as solubilized one. The

the enzyme immobilized at 50˚C was

results suggested that immobilization didevaluated from the remaining protein:

not affect the activity of glucocere-

About 71% of the enzymes was yet

brosidase-catalyzed hydrolysis of MUG.

immobilized on the decyl-Sepharose gel

Inhibition by ceramide.

The immobi-

Effects ofTable 2.
Effects of ceramide on the solubilized and immobilized
glucocerebrosidase-catalyzed hydrolysis of MUG.
Solubilized

Ｉmmobilized
Ceramide (3 µM)

app

Km (mM)
Vapp (µmol/min)
Ki for ceramide (mM)

＋
4.7
6.6

−
1.0
3.8
0.055

＋
4.7
6.0

−
0.86
4.0
0.22

S. Yasui et al.

21

Table 3. Effects of conduritol-B-epoxide on the solubilized and immobilized
glucocerebrosidase-catalyzed hydrolysis of MUG.
Solubilized
Ｉmmobilized
Conduritol-B-epoxide (3 µM)
＋
−
＋
−
5.3
0.98
4.8
1.0
6.6
3.3
6.0
3.0

Kmapp (mM)
Vapp (µmol/min)ceramideonthesolubilized

andDiscussion

glucocerebrosidase-cata-

In the preceding paper4), we partiallylyzed hydrolysis of MUG were examined.

purified glucocerebrosidase from bovine

To obtain inhibitor constant, Ki, Dixon

brain.

plots of this reaction were figured, andglucocerebrosidase, reported here for theindicated that ceramide acted as a“solubilized” enzyme, were similar to

mixed-type inhibitor of both solubilized

thoseimmobilized

andimmobilized

Inhibitor

constant,

The properties of bovine brain

reportedfor3)

glucocerebrosidase.

glucocerebrosidase

values2)

bovine

brain

and

human

6)

were

placental and brain glucocerebrosidase,

estimated to be about 0.055 mM for thebut pH optimum for these enzymes,

solubilized glucocerebrosidase and 0.22

reported to be around 6.2, was a little

mM for the immobilized glucocere-higher than that for the bovine brain

brosidase (Table. 2).

Thus, the immobi-

enzyme characterized here (Fig. 2B).

lized enzyme was less susceptible toThis difference (6.2 vs 5.5) may be due toceramide inhibition

the difference of substrate used for theKi,conduritol-B-epoxide.

enzyme assay; the natural substrate was

Conduritol-B-epoxide (CBE) is a specificused for the reported enzymes, while theactive-site inhibitor of glucocerebrosidase.

artificial compound, MUG, was used

Effects of CBE on the glucocere-

here.

Inhibitionbybrosidase-catalyzed hydrolysis of MUG
were examined (Table. 3).

Bovine brain glucocerebrosidase was

Immobili-

adsorbed to decyl-Sepharose, and thezation of the enzyme little affected theimmobilization was practically stable.

inhibition of CBE.

The molecular and kinetic properties ofthesolubilized

andimmobilized

glucocerebrosidase were similar, such astemperature and pH dependence ofGlucocerebrosidase, immobilized to decyl-Sepharose

22

activity,

thermalparameters,

andstability,

kinetic

inhibitionbyconduritol-B-epoxide.
to

the

binds

activeglucocerebrosidase,irreversible

151-170 (2000).
2) F.S. Furbish, H.E. Blair, J. Shiloach,Conduritol-B-epoxide
covalently

and S. Karger AG, Tokyo, pp.

siteresulting7)

inhibition .

Since

P.G. Pentchev and R.O. Brady:ofProc. Natl. Acad. Sci. USA, 74,

in3560-3563 (1977).

no3) P.U.M. Reddy, G.J. Murray and J.A.

equilibrium exists to permit dissociation

Barranger:of the enzyme-inhibition complex, any Ki

200-210 (1985).

Biochem. Med., 33,

value calculated will be meaningless, but

4) M. Shimada, S. Yasui, A. Hotta, S.

Table 3 indicated that conduritol-B-

Ishijima and M. Ohnishi: J. Biol.

epoxideMacromol., 3, 7-14 (2003).

similarly

solubilized

inhibited

and

the

immobilized

glucocerebrosidase.
Thus,
brosidase248-254 (1976).

bovinebrain

canimmobilizedbe5) M.M. Bradford: Anal. Biochem., 72,

glucocereon

decyl-Sepharose with little changing
molecular and kinetic properties.

6) L.B. Daniels, P.J. Coyle, R.H. Glew,
N.S. Radin and R.S. Labow: Arch.
Neurol., 39, 550-556 (1982).
7) M.C. Stephens, A. Bernatsky, V.
Burachinski, G. Legler and J.N.
Kanfer:Neurochem.,

1023-1027 (1978).

References
1) S. Ishijima and M. Ohnishi:
Glycoenzymes,

J.

M.

Ohnishi,inT.

Hayashi, S. Ishijima and T. Kuriki,
eds., Japan Scientific Societies Press

Communicated by Ueno Hiroshi

30,