Activation of residual acidic α-mannosidase activity in mannosidosis tissues by metal ions

BIOCHEMICAL

Vol. 67, No. 4, 1975

ACTIVATION

AND BIOPHYSICAL

OF RESIDUAL

ACIDIC

IN MANNOSIDOSIS

Department

and

of Clinical

October

BY METAL

P.K.

85

ACTIVITY IONS

Masson

Chemistry,

S-221 Received

a-MANNOSIDASE

TISSUES

B. Hultberg

RESEARCH COMMUNICATIONS

University

Lund,

Hospital,

Sweden

30,1975

The residual acidic a-mannosidase activity from mannosidosis Summary: representing between 1 and 8 % of he activity found in normal tissues, tissues, these metal ions was significantly activated by Zn2+ and Co J+ p whereas respectively activated or inhibited the acidic enzyme activity from normal The defective enzyme from mannosidos,js liver bound most effectively tissues. . This metal ion also improved to the synthetic substrate in the presence of Co the hydrolysis of a natural substrate by the acidic enzyme from mannosidosis liver. The results indicate that the defective enzyme in the disease has an The demonstration that this defective altered capacity to bind metal ions. enzyme can be activated may have an important bearing on the therapy of the disease.

INTRODUCTION Human

mannosidosis

activities

of all

hydrolase,

type

affected Zn2+,

the molecular

E.C.

A neutral

3.2.1.24)

inhibited

and EDTA,

activity,

bovine

and rat tissues

enzyme

(1).

labile

of these

serum,

metal

was

The acidic

ions

shown

was

have

(4,5).

between

forms

hand,

stable

to be reduced in heart

been

it was

found

between 6.0

observed

(6). that

manno-

4.0

and 4.5

and 6.5,

is not

are activated

(2,3).

intestine,

this

activity

(2, 3).

Similar from in Zn 2+ -

of a-mannosidase

In those

by

by Zn 2+

on a-mannosidases

lung,

(1).

sialylated

by Co 2+ , inhibited

the activity

and brain

(a-g-mannoside

to be sialylated

in liver,

in the

and probably

is activated

not appear also

reduction

of the enzyme

are heat

Recently,

affected,

a pH optimum

a pH optimum

and does

but unaffected

activity

with

of a-mannosidase

have

on the other

is heat

rats

forms

by Co 2+ and EDTA,

effects

depleted

by the simultaneous

which

of enzyme,

in the disease

The neutral

and

is caused

kidney,

tissues

where

increased

spleen, the by

Vol. 67, No. 4, 1975

BIOCHEMICAL

approximately

60% in the presence

a 5% increase

in enzyme

of exogeneously

Zn 2+ , compared

added

from Zn 2+ -supplemented

activity

the aim of the present

investigation

on the residual

a-mannosidase

acidic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was

to study

activity

rats

the effects

(6).

to

Consequently

of Zn 2+ and Co 2+

in mannosidosis

tissues.

MATERIALS Samples

of liver,

sidosis

patient

Normal

liver,

brain,

(7).

These

brain,

and

at -2O’C

were

used

a patient

with

mannosidosis

were

cultured

patient

skin

with

monolayer, physiological

spleen

samples spleen

as controls.

(9) as described

confluent twice

from

and

were

had been

obtained

before

Serum

samples

centrifuged

obtained (10).

stored

at about

(8) had been biopsies

obtained

stored

from a manno-

subsequently

at -20’~.

the same time from a normal similarly

from a normal

The cells

at 1,500

at autopsy

were

and

stored

individual

for a year.

and Fibroblasts

and a mannosidosis

harvested

at the stage

g for 5 min and the cell

pellet

of washed

saline.

METHODS 10% (w/v) homogenates of liver, brain, and spleen were prepared in NaH2P04-NaOH buffer (10 mM with respect to phosphate), pH 6.0, using a Dounce tissue homogeniser. The homogenates were centrifuged at 43,000 g for 20 min at 4OC and the supernatants used for the analytical work. The fibroblast pellet was homogenised in 3.5 ml of the same buffer and centrifuged at Serum was diluted five-fold in the same 1,500 g for 10 min at room temperature. buffer. For the analysis, 100 ~1 of the enzyme were incubated at 37’C for various times with 100 J of a 1.6 mM solution of 4-methylumbelliferyl-a-~-mannoColnbrook, TJ’. K.) in citrate-Fhosphate pyranoside (Koch-Light Laboratories, buffers (100 mM citric acid and 200 mM Na2HP04) at the pH values indicated in Fig. 1 and Table I, and 20 ~1 of 27.5 or 55 mM solutions of ZnS04 or of 2.5 or 5 mM metal ion. The reaction CO(NO~)~ , giving final concentrations was stopped by adding 3 ml of 200 mM glycine-NaOH buffer, pH 10.4, and the fluorescence of the released 4-methylumbelliferone measured in an AmincoBowman spectrofluorimeter (excitation wavelength 348 nm, emission wavelength 450 nm). This procedure was repeated on the supernatants from liver using dip+rent su,J+trate concentrations in the presence and absence of 2.5 mM of or Co . Zn The liver supernatants were also incubated for 30 h with the natural substrate M2G (a-B-mannopyranoside-(1 -3)-P-D-mannopyranoside-(1-4)-2-acetamido-2-deoxy=lJ-glucose) which was isolasd from the urine of patients with mannosidosis (13. The incubation mixture contained 400 ~1 enzyme (200 Wl/15 h),

1474

Vol. 67, No. 4, 1975

50 ~1 of a 10 pH 4.5, and before (12). of unhydrolysed the substrate

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

mM solution of M G, 250 ~1 of 100 mM sodium acetate buffer, 2.5 mM Zn2+ or &+. The rest of the procedure was as described The degree of hydrolysis was correlated by quantitating the amount substrate at the end of the incubation. Each supernatant and were also incubated separately as controls.

RESULTS The effect is shown

in Fig.

pH range

la.

3.0 to 5.5

pH values. over

of Zn2+

Co2+

on the a-mannosidase

An increase

in the normal

in the presence

In the presence

the pH range

trast,

and

of Co

of Zn 2+

3.0 to 4.5 p and higher

the metal

ions

activated

mannosidosis

spleen

from

pH 3.0 to 4.5.

were

similar

Table tissues tissues,

ions

I shows studied,

in the presence

the enzyme

400

j

30

pH,

tissue

of the metal

spleen

in the

presence

over

than

(Fig.

In con-

activity exerted lb).

in the various ions.

of Co

2+

In normal and increased

co

Fig. 1. Effect of metal ions on the activity of a-mannosidase at various pH values. The enzyme activity was assayed as described in “Methods” with citrate-phosphate buffers ranging from pH 3.0 to 7.0 and a final concentration of 2.5 mM metal ion. The effects of the metal ions in normal spleen are shown in (a), and those in mannosidosis spleen in (b). a-Mannosi ase activity in the absence of metal ions (u), in the presence of and in the presence of Co2+ (e -.a). Zn %+ (C-----A),

1475

the

normal

to 7.0.

the effects

and absence

(181

50

in normal

lower

a-mannosidase

a-mannosidases

decreased

40

this

was

spleen

at higher

from pH 5.0

acidic

Above

observed

activity

normal

occurred

decreasing

of the acidic

activity

Normal

than

from normal

activity

, the activity

the residual

to those

the activities

enzyme

p the enzyme

both

the metal

2+

activity

in by

2

=:

and nmoles

and spleen,

377

are expressed

2+

390 15.3

14.3

3.2 3.2

1.0

4-methylumbelliferone/ml/min

4-methylumbelliferone

65

72

85.4 93

81

Mannosidosis

41

39

7.5 7.5

2.3

wet for serum

released/g

202

222

338 338

300

and fibroblasts.

weight/min

6.8

7.3

13.7 12.9

8.0

at pH 4.0

2.6

3.2

5.6 5.3

0.7

Serum Normal Mannosidosis

of a-mannosidase

Spleen Normal Mannosidosis

activities

I

at pH 4.5

on the acidic

Brain

ions

Normal

of metal

as nmoles

69

66.3

50 52.2

7.1

452

493 493

Mannosidosis

Liver

Normal

The results

co

co2+

2.5

5

Zn2+ Zn2+

Metal ion

2.5 5

0

Concentration hM)

Effect

Table

for liver,

4.0

4.8

8.9 9.5

6.2

c

1.2

brain,

1.5

ifI 9 s

F

I z

1.7 2.3

0.2

Fibroblasts Normal Mannosidosis

BIOCHEMICAL

Vol. 67, No. 4, 1975

in the presence tissues and

was

spleen

of Zn2+.

activated were

either

Co2+

Fig. acidic

Zn2+

metal

more

tissues

acidic ions.

enzyme

activity

The acidic

in the presence

behaved

RESEARCH COMMUNICATIONS

similarly

in mannosidosis

activities

of Co

at pH 6.0

2+

in liver,

than

Zn2+.

under

brain,

Both normal

the influence

of

or Zn’+. that

a-mannosidase was

a rectangular activity

by both

2 illustrates

hyperbola

The residual

increased

and mannosidosis

AND BIOPHYSICAL

observed

25% of M2G

was

2+

decreased case.

higher the Vmax

On the other

Vmax slightly. hand,

for the residual

only

with

A rectangular

this

tendency

acidic

in the presence

of Co

the normal

towards

a-mannosidase 2+

I even

though

activity

in the absence

from

of metal

of Co

2+

ions,

. On the other

normal

liver

was

found

hand,

to hydrolyse

of Zn 2+ , and

60% in the presence the residual

Normal

acidic

activity

Mannosidosis

,c-I’ ,

* __-- _--- : :’

//

_.’

100

0

both

the enzyme.

a-mannosidase

16% in the presence

a slightly

observed liver

activated

The acidic

gave

in each

mannosidosis

and Co’+

2+

and Co

hyperbola

from

Zn

I

I

I

I

1

1

2

3

4

5

Final

I

0 0

substrate

concentration

1

2

, 3

I

I

4

5

( mM )

Effect of substrate concentration on the activity of a-mannosidase. Fig. 2. The enzyme activity from liver was assayed as described in “Methods” in the presence of different substrate concentrations and 2.5 mM metal ion. a-Mannosidase activity in the absence of metal ions ( t-----m), in the presence of Zn2+ (L-----A), and in the presence of Co2+ (e.-.-.-+.

1477

Vol. 67, No. 4, 1975

from

BIOCHEMICAL

mannosidosis

absence

liver,

of metal

presence

which

ions,

of Zn2+

was

hydrolysed

found

and Co’+,

AND BIOPHYSICAL

only

RESEARCH COMMUNICATIONS

4% of the substrate

to hydrolyse

7.5%

in the

and 16% of M2G

in the

respectively.

DISCUSSION Lysosomal Zn2+

a-mannosidase

and inhibited

which

slightly

is cytoplasmic, by Co2+

on the acidic

enzyme

activity

from normal

findings,

indicating

verified

by kinetic

co

from

2+

results

altered bility

studies

which

this

metal

some extent The effect bovine presence

defective by Zn

kidney.

Whereas

of this from in this

The total

metal

substrate

was

the activity ion,

mannosidosis

case

may be more activity,

acidic

tested

kidney

that

for the was

a-mannosi-

of the

has an

the hydrolytic 2+

and to

of mannosidos

a-mannosidase was

in

increased

in the residual

capa-

in the acidic

that the resultant

severe.

known

to be labile

1478

(2,3),

was

of

considerably.

by Co

enzyme

suggesting

This

of

in the presence

on the therapy

observed

(4),

spleen.

2+

in the disease

on the acidic

were

Zn

capability

significantly

of the normal

(2-6, acidic

than

substrate

bearing

enzyme

is an inhibitor

effective

enzyme

ions

these

between

the hydrolytic

the natural

no changes

residual

and

The demonstration

been

bovine

neutral

the binding most

metal

with

activator

that

an important

has also

of these

I which

revealed

can be improved

2+ may have

of Zn2+

the low Co

activity,

of Zn 2+ and EDTA,

a-mannosidase

brain,

ions.

enzyme

acidic

2+

by

and the neutral

liver,

towards

enzyme

are in agreement

to be a better

the defective

metal

tissues

increased

ion improved

liver that

to bind

and

The neutral

with

mannosidosis

liver

indicate

capacity of this

activity

from

the diseased

human

tissues

found

is activated

on the effects

significantly.

was

from mannosidosis

These

defect

enzyme

. Furthermore,

enzyme

tissues

(2-6).

Our results

and Zn2+

tissues

in the presence

from normal

Co2+

enzymes,

acidic

or EDTA

Zn 2+ is associated

that

acidic

mammalian

slightly

13).

activity

both

residual

dase

(2-6,

in mannosidosis

the normal

2+

and mannosidosis

However,

activity

by Co

several

is decreased

but activated

13,14).

from

lower

in the normal

i S.

Vol. 67, No. 4, 1975

tissues storage. tissues tion

studied

BIOCHEMICAL

than

previously

The observation behaved

of its different

similarly origin

that under

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

observed this

(3).

activity

is probably

from both

the influence

from the acidic

This

the normal

of zn 2+ or Co’+

due to prolonged and mannosidosis is a further

indica-

enzyme.

ACKNOWLEDGEMENTS We are grateful nical assistance. Research Council

to Mrs. Munevera Mirazovic and Mrs. Sonja Glans This work was financially supported by the Swedish (grant No. 13X-22 2 2).

for techMedical

REFERENCES 1.

2. 3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 13. 14.

Carroll, M., Dance, N., Masson, P.K., Robinson, D., and Winchester, B.G. (1972) Biochem. Biophys. Res. Commun. 49, 579-583. Phillips, N.C., Robinson, D., and Winchester, B.G. (1974) Clin. Chim. Acta 55, 11-19. Chester, M.A., Lundblad, A., and Masson, P.K. (1975) Biochim. Biophys. Acta 391, 341-348. Phillips, N.C., Robinson, D., Winchester, B.G., and Jolly, R.D. (1974) Biochem. J. 137, 363-372. Snaith, S. M., and Levvy, G.A. (1969) Biochem. J. 114, 25-33. Sot. Trans. 2, 1014-1017. Patel, H. M., and Ryman, B.E. (1974) Biochem. Kjellman, B. , Gamstorp, I., Brun, A., &kerman, P.A. , and Palmgren, B. (1969) J. Pediat. 75, 366-373. S. (1974) Biochem. Biophys. Res. Masson, P.K., Lundblad, A., and Autio, Commun. 56, 296-303. Ackerman, P.A .,.. and Szab6, L. (1973) J. Pediat. 82, 686-688. Norden, N.E., Hultberg, B., Sjbblad, S.; and Ockerman, P.A. (1975) Acta Paediat. Stand. 64, 123-131. Norden, N.E., Lundblad, A., Svensson, S., &kerman, P.A., and Autio, S. (1973) J. Biol. Chem. 248, 6210-6215. Hultberg, B., Lundblad, A., Masson, P.K., and &kerman, P.A. (1975) Biochim. Biophys. Acta. In press. and Gourlay, G.C. (1971) Biochim. Biophys. Acta 235, 142-148. Marsh, C.A., Snaith, S.M. (1975) Biochem. J. 147, 83-90.

1479