Acid α-D-glucosidases from plant sources

Vol. 42, No. 2, 1971

BIOCHEMICAL

AC I D CX-D-GLUCOSI J. Department

of

J.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DASES

Marshall

FROM

and

Biochemistry, Miami,

PLANT

Pamela

M.

SOURCES

Taylor*

University of Miami Florida 33136, U.S.A.

School

of

Medicine,

and Lister Chelsea

Institute Bridge

of Road,

Preventive London

S.W.1,

Medicine, England.

Received November 30, 1970 Evidence is presented for SUMMARY: extracts of a-glucosidases with acidic enzyme has previously been described

Many

mammalian

(a--D-glucoside

of

from (5)

bovine

and

different

enzyme in

degrades

the

a serious

in

disease,

of

The

acid

liver of

reviewed glycogen

glycogen.

metabolism

(Type

r%glucosidase

This

rat

properties

and of

glycogen

(2),

been

maltose

contain activity.

spleen

have

degradation

to

(3,

this

4),

enzyme

(6).

It

and

which

Absence

of

II

enzyme

has

rabbit from

is

been

a number

a lysosomal

plays

an

this

important

enzyme

glycogenosis,

leads

to

Pompe’s

(7)). In

of

sweet

As

far

a previous

as

a plant

paper

(8)

we

corn

of

we

are

aware

now

purified

this

A-50

and

Sephadex

be

given

have

an a-glucosidase

mentioned

with

this

is

the

the

optimal

f i rst

presence

activity

in between

report

of

an

acid

column

chromatography

crude pH

extracts 3.1

and

a-glucosidase

3.8. from

source. We

have

DEAE-Sephadex procedure

will

hydroxylapatite

*

sources

shown 3.2.1.20)

and

(4).

both

oiuo

disorder

(I)

kidney ian

been E.C.

liver

human mammal

which

role

have

glucohydrolase,

purified muscle

tissues

the presence in plant pH optima. This t.ype of only in mammalian sources.

Presen’t

and

Address:

elution

The

enzyme

elsewhere. with

Pantiles,

by

Full

G-200.

Passage a gradient

Jordans

details of

the

of

phosphate

Way,

173

of

on CM-cellulose, this

resulting

Jordans,

purification preparation

buffer

Beaconsfield,

yielded

through three

Bucks,

England.

Vol. 42, No. 2, 1971

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

- 0.7 2 Z tfl g mM \ U 2 LL 600 k 5 z 400s *

2.4 -

5

0.8-

E B

200

E

lh1.2 -

5(

-

0.4 -

0 FRACTION

NUMBER

Fig. I Fractionation of purified sweet-corn @glucosidase on a column (12 x 2.5 cm) of hydroxylapatite (Biogel HTP). Elution was carried out with a linear gradient of phosphate buffer (- - -) of increasing molari ty (5 mM to 200 mM over I 1 i tre), fractions of volume 6.0 ml being collected automatically. Residual protein was removed by further elution with 500 mM phosphate buffer pH 7.5. The distribution of protein in the fractions was determined by measurement of the absorbance at 280 nm (-------) in a I cm cell. The activity ( > was measured by the release of glucose from maltotriose at pH 5.1 and 30°C using glucose oxidase (9). The fractions under the three peaks were combined, viz. 82-88, 92-108 and 112-120.

peaks

of

activity

17.0

and

II.5

with

the

activity

indicating activity as

substrate

they

were

with

specific

the

bovine-liver

amyl of

in

The

pH 5.1

70

225,

citrate

48

as

(I)

and

7.7

mg for for

measured were

on shown

periodate-oxidized

We have,previously amounts

of

pH/acti

vi ty

curve

enzymes for

in the

The

at

370C

the

three

exo major

174

units

per

total

When

the

in

pH

be

this enzyme fraction

(3)

free

from

a-amylase

and

crystalline

compares

and

at

the

34

pH

contamination

method

glucofor

detection

( IO).

ons

2)

for

optimum

fungal

a sensitive

(peak

buffer,

This

and

preparati

activities

ci,trate

37OC

is

ma1 totriose

specific

fractions.

at

of

using

3.0

13.3,

mg,

recovery

&glucosidase maltose

were compared

throughout

30°C.

to

that

0.059

fold.

amylose shown

endo

was

rat-liver

fractions

respectively,

determined at

three

protein

195

substrate

per

the

which

buffer

units of

mg of

were

fractions

with

288

activities

enzyme three

per

of

extract

maltose

and

activities

initial

activities

incubation

trace

The

with

64,

All

ase.

the of

52%. in

determined

The Units

purification

were

by

I).

International

was

values.

(Fig.

is

shown

in

Fi’g.

2;

Vol. 42, No. 2, 1971

BlOCHEMlCAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

pH

Fig. 2 (Fig.l)] from against water, The activity ml ). 0.2 M citrate (5 mg/ml, 0.05 at 300C for 60 oxi dase (9).

those

for pH value

are

essentially

in

the

The on

other

is

this

the

region

37OC.

The

unstable

at

this

pH

approximately pH 7.5

of

two

and

(at

2’C

with

In

all

shoulder

increasing

fractions

at

cases

at pH and

least

in in

under in

the

lengths

remaining

shown

resulted

a slight

various

3 hours

the

pH 4.25.

Above

all

fractions

three

was

Fig. the

of

3.

these

time

at

is

that

clear

of

of

the

as

all

fractions

the

half-life

of

activity

the

over

tested

temp-

maltose

Storage

loss

were

pH and

substrate),

conditions.

(3.0)

this using

of

no detectable

pH

measured

It

absence

optimum

subare

fractions a period

months. Polyacrylamide

was

three

After

are

with

similar.

pH 7.0.

activity

results

closely

sharply

the

percentage

strate.

3.0

above of

at

are

of

drops

inactive

the

at

fractions

activity

incubation

being

two

stabilities

erature

DIGEST

pH/Activity curve for activity in peak 2 [fractions 92-108, hydroxylapatite column. The enzyme fraction was dialysed then diluted 100 times with human serum albumin solution (0.5 digests contained maltotriose solution (10 mg/ml, 0.2 ml), buffer of measured pH (0.2 ml), human serum albumin solution ml) and diluted enzyme solution (0.05 ml). After incubation minutes the liberated glucose was determined using glucose

the

optimum

OF

carried

buffer

pH

out 7.5.

a single

protein

mobility

which

di-sc-gel as

described

The

gels

band was

electrophoresis by were

was

approximately

Ornstein

stained

observed. the

(II) with

in

7% gels

Coomassie

The

bands

same

for

175

o-glucosidase

were each.

using

Blue of

and

low It

is

fractions phosphate in

all

cases

el ectrophoretic clear

from

the

mg/

Vol. 42, No. 2, 1971

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0

120

60

DURATION

180 man

OF PRE:NCUBATION at 37Oc

Stability of sweet-corn @glucosidase fractions at 37’C and Fig. 3 Several 0.05 ml samples of each enzyme fraction (diluted optimum pH (3.0). 50-fold using a 0.5 mg/ml solution of human serum albumin) were preincubated at 37OC in the presence of 0.2 M citrate buffer pH 3.0 (0.2 ml) and human serum albumin solution (5 mg/ml, 0.05 ml). After incubation for various lengths of time the activity remaining was determined by addition of maltose solution (20 mg/ml, 0.2 ml) and incubation at 37OC. The amount of glucose liberated in 30 min was determined by removal of samples (100 ul) for glucose and the activity remaining after the various times of oxidase estimations, preincubation calculated as a percentage of the initial activity (o peak 1, fractions 82-88; LI peak 2, fractions 92-108; o peak 3, fractions 112-120).

the

stabilities, there

is

little

the

second

has

given

three

figuration This

was

shown

the

glucose

by

before

increased

the

rather

following

the

the

rate

of

liberated

al 1 three

enzyme

than

exo-a-glucanases

of

and must

fractions

are (14).

is

hydroxylapatite previously

We presume

hydrolysis the

liberated

shown

that

both

using

heating

(13).

oxidase the

Since specific

this

of

of

the

for

B-Dsuggests

ol igosaccharases), was

enzyme

mutarotation

This

(i.e.

con-

oxidase,

action

c+configuration.

of

of

molecule.

glucose the

is

CX-D-glucosidases Confirmation

retention

glucose

by

glucose in

with

substrate

by

1’76

This

enzyme.

We have

oxidation, as

be

that

forms.

of

mutarotation

oxidation

the on

(12).

about

atom rate

of

fractionation

isoenzyme

maltotriose

glucose

forms

electrophoresis

properties.

bring carbon

after

three

R-enzyme

of

anomeric

and

disc-gel

where

sweet-corn

from

the

similar

fractions

liberated

fractions

across

separations

the

the

closely

with

enzyme

at

come

with

are

and

between

have

phenomenon

fractionations

that

we

fractions

All

curves

difference

case

a similar

glucose,

pH/activity

obtained

by

Vol. 42, No. 2, 1971

measuring The

the

rate

was

BIOCHEMICAL

rate

of

release

found

to

decrease

glucosidases

corn

is

unusual

saccharide.

By

analogy

is

for

the

important

enzymes

might

therefore,

be to

maize and

substrate.

similar

to

those

of

glycogen maize

Figure

4 also

“Malt

the

that

it

for be this

from

various

maltodextrin

chain-length

substrates.

increased

for

determination

The

pH/activi

PF”

any the

we

as

is

expected

for

ty

corn

of

in

enzyme

the

also

has

order, seed

4,

and

are

theory of

waxy

the

that presence

maize

nor

polysaccharide.

activity a

of

after

Our

water-soluble maltose

these

using

Fig.

neither

this

enzyme

directly,

because

since

for

This

mature

activity

specifically

amount curve

used

shown

of

In

fractions.

invalid

significant

(WaIlerstein).

are

poly-

that

from

a-glucosidase

curves

type

suspected

were

c+glucosidase sweet

this

extracts

of

a reserve

phytoglycogen.

extracts

therefore

pH/activity

we

the

prepared

as

where

(7), of

crude

sweet-corn in

systems

breakdown

The

present

glycogen

glycogen

maize.

the

contains

Amylase

of the

contains

mammalian

hypothesis

plant

shows

the

in

this

might in

normal

of

as

degradation

dilution,

as

enzyme

with

normal

maltose

in

involved

test

and

dialysis

this

glucose

(14).

Sweet

waxy

of

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

low

in optimum

an

extract pH

(3.75).

pH

OF

DIGEST

pH/Activity curves for a-glucosidase activity in extracts of Fig. 4 and a commercial malt waxy maize (r), normal maize (-) The activity measurements were carried out as extract (-A-A--). 2 except that maltose was used as substrate. described in the legend to Fig. The buffers used were citrate (up to ptl 3.0) or citrate-phosphate (above pH 3.0).

177

Vol. 42, No. 2, 1971

It acid of

is

therefore

Zea

mcrys

enzyme

has

yet

been

function

established

but

the

enzyme

liberating

not

only

is

involved

with

in

glucose

pH

optimum,

in

the

retention

speculate enzyme,

as

that

degree

the has

been

a clearer

understanding

have

but

the the

also

are

the

(17),

the

buckwheat

however,

has

the

21)

of

not that

starch,

its

produced

favorable is

clearly

by

mechanism an

important

route

such

as

different fungal

as

of

0 and

before

in

progress

of

hydrolysis

interest

and

mechanism

is

place

tempting of

to

the

8-amylases. can

other

grouping

takes

action

this

experiments

acidic

hydrolysis It

from

glucoamylase, more

inversion.

for

has

(20,

breakdown

is

mechanism

detailed

made

a considerably

required that

enzyme

out.

insofar

(22)

of

cases,

been

most

ruled

than

Thoma

hoped

Only

pathway

that

by

is

mutants has

oligosaccharides

described

in

experiments It

be

differences done

accuracy.

not

rather

possible

have

a hydrolytic

suggests

site,

three

of

tomatoes

glucose-producing

maltodextrins,

configuration

further

of

not

clearly

we

which

active

of on

is

from

sources

in

(21). these

for

of

must

which

present

(17),

rice

suggestions

that

starch

only

C+Glucosidase

of

of

hydrolysis

possibility of

of

it

is

the

carrots

none

pathway

about

it

and

type

a non-phosphorolytic

degradation

enzymes

feel

this

bring

not

reported.

of

While

type

(20) In

been

that

(16),

purified. pH

are

preparation.

peas

significance

the

the

(19),

optimum

8-amylolysis.

The

of

radish

buckwheat

to

energetically, for

alfalfa

of

being

cx and

(15),

2)iUo

part

shown

in

clearly

forms

tissues

amylase

low in

We have

extensively

abnormally The

mammalian

malt

(18), been

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a commercial

reported

malt

that

activity.

and

been

barley

it

clear

a-glucosidase

also

an

BIOCHEMICAL

two

types

However, be

we

done

with

any

will

lead

to

by

these

enzymes.

ACKNOWLEDGEMENTS We wish during the work and

the gift was the

of

to

thank

course

of

Malt

W.

this

Department

by

J.

Whelan

work.

Amylase

supported U.S.

Dr.

We also

PF and grants of

for

Dr.

from Agriculture

E. the

his thank

E.

Smith

National (FG

1’78

Wallerstein for Science

UK-138).

encouragement Laboratories

helpful

for

discussion.

Foundation

This (GB

8342)

Vol. 42, No. 2, 1971

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. IO.

Auricchio, F., and Covelli, I., Bruni, C.B., J. Biot. Chem. 244, 4735 (1968). Fujimori, K., Hizukuri, S., and Nikuni, Z., B-&?hem. Biop&a. &a. Cam. 32, 811 (1968). Jeffrey, P.L., Brown, D.H., and Brown, B.I., Biochemistry 2, 1403 (1970). Auricchio, F., Bruni, C.B., and Sica, V., Bioehem. J. 108, 161 (1968). and Whelan, W.J., unpublished work. Palmer, T.N., in “Carbohydrate Metabol ism and Smith, E.E., Taylor, P.M., and Whelan, W.J., its Disorders” (F. Dickens, P.J. Randle and W.J. Whelan, eds.), Vol. 1, Academic Press, New York (1968). p.123. and van Hoof, F., in “Carbohydrate Metabol ism and its Disorders” Hers, H.G., (F. Dickens, P.J. Randle and W.J. Whelan, eds.), Vol. 2, p.151. Academic Press, New York (1968). Taylor, P.M., and Whelan, W.J., Biochm. J. 95, 26P (I 965). Anal. Biochem. 30, 467 (1969). Lloyd, J.B., and Whelan, W.J., Smith, E.E., Drunnnond, G.S., Marsha1 1, J.J., and Whelan, W.J., Fed. &oc., Fed.

Amer. Il. 12. 13. 14.

.Soc. Exp.

Ornstein, L., Lee, E.Y.C., in the press. Semenza, G.,

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J.J.,

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A. H.,

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E.T.,

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O.,

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Biochem. P.,

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Biophys. Mullet-,

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65, 16. 17. 18. 19.

20. 21. 22.

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Amagase,

M.,

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Okomura,

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Hutson, D. H., and Manners, D. J., Biochem. J. 94, 783 (I 965). Manners, D. J., and Rowe, K.L., Carbohydrate Res. 2, 441 (1969). Jorgensen, B.B., and Jorgensen, O.B., Acta Own. Scud. 17, 1765, 2471 (1963). Takahashi, M., and Shimomura, T., Agr. B
179