The enzymatic synthesis of a glucomannan

Vol. 23, No. 3, 1966

BIOCHEMICAL

The Enzymatic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Synthesis

of a Glucomannanl

Alan D. Elbein and W. 2. Hassid Department of BioLogy, Rice University Houston, Texas and Department of Biochemistry, of California, Berkeley,

Received

April

1, 1966

Previously, incorporated

it the

(Elbein,

et al --'

mixtures,

was shown that

glucosyl

an increased

was also

rated

that

communication

porated

into

method

(Roseman

(Barber

The data

shows

is

that since

glucose-

1961).

were

hexose-l-phosphate

D-mannose-l-phosphate

with

incorof

0-B-D-mannopyranosyl-(l-4)-D-mannopyranose -_

were Ottawa.

generous

A '@-mannanase

GMF-

was prepared

and O+-g-mannopyranosyl-(1-4)-

Council,

is

prepared

4-o-(p-g-glucopyranosyl)-D-mannopyranose

Research

presented

14C was also

4-0-(&-D-mannopyranosyl)-IJ-mannopyranose,

National

study,

an epimerization

(1962).

Bishop,

that

of GDP-D-mannose -

and GDP-g-glucose

the corresponding

of MacDonald

During

of the polymer.

- GDP--IJ-mannose

--et al:,

14C into

14 C was incorpo-

Evidence

1964).

component

occurs,

incubation

from GDP-D-glucose-

of GDP-D-mannose-

also

cellulose

to these

was observed.

-et al,

probably

products

by condensing

morpholidate

moiety

the mannose

to GDP-D-glucose

and Methods

chemically

polymer

polymer that

14 C into

was added

of glucose

the mannose

in the hydrolysis

Materials

the

incorporation

a glucomannan.

GDP-D-mannose found

When GDP-D-mannose -

an insoluble

in this

of mung bean seedlings

of GDP-D-glucose-

alkali-insoluble found

into

extracts

portion

1964).

a water-insoluble, it

University California

gifts

of Dr.

preparation,

1 Supported in part by Research Grant GB 3857, and in part Grant 623763, from the National Science Foundation.

311

by Research

by

C. T. which

Vol. 23, No. 3, 1966

had only

BIOCHEMICAL

slight

cellulase

U.S.

Army Quartermaster

were

obtained

from

pyridine:

O.lN

3. Ethyl

activity, Corp,

paper.

Paper

electrophoresis

pH 3.6,

or in

0.05 _M sodium

Total glucose

with

Enzymatic were (Barber

--et al,

in place

of Tris

x g contained

buffer,

about

seedlings

Ribbon buffer,

0.1 M phosphate

of Nelson

as previously

buffer,

fraction

1952),

(Phaseolus

pH 7.5,

which

sedimented

period,

(1944)

aureus) described was used at 30,000

by centrifugation,

content

As shown in Figure

was also

g-mannose-14

potassium

into 1, this

slowly

proportional

enzyme.

volume

phosphate At the

and the mixture

was

by centrifugation

for

2 minutes.

washed

several

The times

with

determined. was incubated

was incorporated

in a final

was collected

2 ml of 2% NaOH at 100'

obtained

and then

0.016;

was added

The precipitate

with

radioactive

20 minutes

(p,moles

cpm/pmole),

0.5 ml of water

2 minutes.

extracted

the following

5; and 0.1 ml of the particulate

- When GDP-+annose-14C

incorporation either

for

and its

polymer.

(Loewus,

by the method

prepared

The particulate

5; MgC12,

was again

radioactivity

sugar

extract

C (500,000

at 100'

Results

that

589 Blue

method

- Mung bean

GDP-FJ-mannose-14

and was then

water,

was done

pH 9.7.

by the anthrone

contained

end of the incubation

precipitate

chromatography

(3:1:3);

in 0.2 -M ammonium formate

mixtures

pH 7.5,

heated

water

the enzyme activity.

The incubation of 0.2 ml):

acid:

was performed

and the

except

buffer.

compounds

and Schuell

and reducing

the dark

1964)

other

1. -n-Butanol:

acetic

Paper

(12:5:4).

of Glucomannan

in

as follows:

acetate:

tetraborate,

oxidase

Synthesis

All

E. T. Reese,

or Schleicher

was determined

glucose

germinated

water

or 3MM paper

hexose

were

2. Ethyl

pyridine:

Whatman #l

by Dr.

Massachusetts.

systems

HCl (5:3:‘2);

using

supplied

sources.

solvent

acetate:

was kindly

Natick,

commercial

Chromatographic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

a water-insoluble,

off

during

to the protein

C, g-mannose-6-phosphate-14C,

312

enzyme,

alkali-insoluble

incorporation

leveled

the particulate

was linear

with

the next

hour.

concentration.

time The When

D-mannose-l-phosphate-14C,

for

Vol. 23, No. 3, 1966

BIOCHEMICAL

20

Figure

-D-glucose-14C, substituted into

poration are

first

1964). containing

60 TIME ldlWTES

Incorporation

14

of

into

the insoluble

time.

Reaction

in the

text.

GDP-D-mannose-

the polymer

occurred

of Mg++;

was found of glucose treated

from with

C from

mixtures

1).

were

C of

as described

The incorporation

14C into

volume

1, when GDP-D-glucose a marked

inhibition

313

of radioactivity

only

does not

in

the incor-

unless

(NH4)2S04

was added

was de-

20% as much

stimulate

cellulose

of saturated

were

of mannose

when Mg++ was omitted

GDP-D-glucose-

GDP-D-mannose-14C, -

as a function

or no incorporation

Mgtt

As shown in Table

14

GDP-mannose-

or ;-glucose-6-phosphate-14C

in tlhe product.

an equal

60

polymer

14C, little

(T.able

on the presence

radioactivity

40

D-glucose-l-p'hosphate-14C, for

pendent

1.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the particles

(Barber

to reaction

the incorporation

et al e-y mixtures of

Vol.

23, No. 3, 1966

BIOCHEMICAL

AND

Table

Requirements

for

Experiment

cpm in product

14 D-MannoseN"Z ir-Mannose-6-PO4-tiC B-Mannose-$ZP04E-GlucoseC E-G~ucos~-~--P~~-~~C F-Glucose-l-PO4-14C Boiled Enzyme

5832 0 5 22 21 20 1

C

2 Mg++

None Mg++ Mgtt

135 449 583

None 3x10-4pmole GDP-D-glucose 7~10-~urnole GDP-D-glucose 7x10V4umole GDP-B-glucose

508 329 96 82

1 kmole 5 bmole

-

Experiment 3 None None None None

Complete

reaction

mannose

was observed.

mannose

to reaction

stimulation The explanation

have

Synthesis

Additions

~',$~~;;~~~;14; GDP-rd-mannose-14C GDP-%-mannose-14C GDP-B'-mannose-14C GDP-&;;Iu-I~~-~~C

it

COMMUNICATIONS

1

None*

*

RESEARCH

1

Glucomannan

Omissions

Experiment

BIOPHYSICAL

is possible a greater

in

mixture

It

is

affinity

the

enzyme(s)

is

the addition 14 C resulted

GDP-D-glucose-

of radioactivity

results

for

shown that

containing

the incorporation

that

in the text.

was previously

mixtures

of these

described

not

clear

involved

GDP-D-glucose -

314

into

at the present

in the than

an insoluble

for

synthesis

time.

of GDP-Din a product. However,

of glucomannan

GDP-D-mannose. -

This

would

Vol.

23, No.

explain is

3, 1966

BIOCHE,MICAL

the inhibition

known

that

is

glucomannan.

This

GDP-D-mannose-1' glucose

isolated

is

from

C, indicating

based

that

is

observed

Distribution

polymer,

that

probably a 14 glucoseC and

both

in the presence of GDP-mannose

of

to GDP-

2

of

among Various

Radioactivity Fractions

Fraction*

Aqueous

Total

Phase

2% NaOH Phase 70% Ethanol 70% Ethanol Insoluble

In order was prepared

Insoluble Soluble

14C, 0.70

53,850

and 5 ml of particulate was treated in the

procedure

to characterize

(350,000

various

11,500 24,000

Product

as follows cpm);

cpm

260,000

Fractionation

vity

of

occurs. Table

mixture

it

into

in the presence

synthesized

an epimerization

Since

incorporated

of another

on the fact

the polymer

COMMUNICATIONS

by GDP-D-glucose. -

due to synthesis

hypothesis

RESEARCH

of GDP-D-glucose -

in incorporation

presumably

were

BIOPHYSICAL

incorporation

moiety

stimulation

GDP-g-mannose

mannose-14C

of mannose

the D-glucose

the

cellulose,

AND

is

as described

the product,

(pmoles

in a final

Mgft,

50; potassium

enzyme.

After

as previously fractions

volume

phosphate

shown in Table

incubation

of 10 ml);

incubation

315

the text.

a large-scale

described. is

in

for

buffer, one hour

The distribution 2.

Most

mixture

GDP-D-mannosepH 7.5,

20;

at 37O, the of radioacti-

oi‘ the activity

in

Vol. 23, No. 3, 1966

both

the

aqueous

BIOCHEMICAL

and alkali

fractions

as indicated

by their

immobility

matography.

Although

these

they have

not been

after

80-90x

treatment

a number pounds

with

were

of radioactive was then

product (0.2X,

became dialyzable.

identified

in

Isolated

dialyzable.

50')

were solvent

24 to 48 hours,

to mannose

and borate

detected. 2 (Table

for

How-

was concentrated

In addition

and glu-

electrophoresis),

Each of these 3).

In both

of Oligossaccharides from Polymer

Ratio

cellobiose, Solvent 2

Mannose:Glucose

Cellobiose

1.00

4-O-(&-D-mannosyl-mannosylmaYinose>

0.56

4-0-(S-g-mannopyranosyl)-D-mannopyranose

1.18

4-O-(D-~-glucopyranosyl)LJ-mannopyranose

1.08

Unknown

1

0.0

2.86

Unknown

2

0.21

3.75

Unknown

3

0.78

1.71

Unknown

4

1.10

1.04

Unknown

5

1.21

316

com-

solvents,

3

R Compound

was not

chromatography

Table Properties

chro-

and mannose,

pH 5.0,

1.

oligosaccharides

rechromatographed

or paper

glucose

The dialysate

solvent

by paper

both

compounds

as yet.

@-mannanase

in

weight

electrophoresis

contain

characterized

and chromatographed

case (which

upon paper

alkali-insoluble

of the activity

--in vacua

was in high-molecular

compounds

further

The water-insoluble, ever,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

No glucose

Vol. 23, No. 3, 1966

BIOCHE>MICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

two

of the radioactive

tic

4-0-(B-IJ-mannopyranosyl)-Q-mannopyranose

-D-mannopyranose, geneous in

compounds

respectively.

in solvent

3.

3N HCl at 100'

for

developments glucose of sugar either

in

oligosaccharide to mannose which

30 minutes

areas

and then

glucose

faster

were

oxidase). . = 1.10)

which

than

of 1.00

= 0.21)

@ cell. A faster

which

moving

tained

to 1.71.

that

recromatography

in

oligosaccharides

acid

1.

2 and 3.

procedure,

glucose 3, an

of glucose

= 0.78) . O+-D-mannopyranosyl-

.

to mannose

= 1.21)

of glucose

was also of 1.00

was also

detected

to 3.75.

detected

which

to be hydrolyzed units

is not

However,

was also

con-

further

by

of these of its

reaction

chromatography

as glu-

radioactive, has not yet

is

B since

been

determined.

the polymer

was susceptible

and one of the oligosaccharides

was tested

by emulsin.

known with

in several

by paper

oligosaccharides

by a @--mannanase,

identified

the product

identified

configuration

further

The glucose

to be radioactive;

which

in these

the anomeric

to hydrolysis

to

and the amount

oligosacchaxide

and mannose were

was further

(gluconolactone)

Presumably

glucose

solvents

oxidase

The linkage

and found

glucose

was found

glucose

conic

4

The

no glucose. In each case the

with

(Reel1

and mannose).

and had a ratio

of glucose

oligosaccharide

1 (3

(Rcell

of authentic

A slow-moving

had a ratio

in solvent

had a ratio

oligosaccharide

homo-

was hydrolyzed

As shown in Table

(1-4)-O-@-D-mannopyranosyl-(l--4)-D-mannopyranose to mannose

authen-

appeared

eluted

by the anthrone

and another

a mobility

with,

oligosaccharides

separate

(Rcell

to 1.04,

identical

chromatographed

(mannose

glucose

not

oligosaccharides

of the chromatograms

was isolated

exhibited

if

and 4-0-(@-LJ-glucopyranosyl)-

each readily

or with

of 1.00

to,

Each of the

each was determined

by anthrone

close

Each o-f the radioactive

of 12 hours

and mannose

ran

The exact

ratio

but it

appears

certainty

experimentation

is

necessary

of mannose

units

to be about before

these

to

3 or questions

can be answered. It

should

be mentioned

here

that

Perila

317

and Bishop

(1361)

isolated

and

Vol. 23, No. 3, 1966

BIOCHEMICAL

characterized polymer part

a p, 1-4 linked

of this

type

Summary -

moiety

was characterized

treatment these

with

oligosaccharides

of l:l,

contained

14 C is.

into

may be a

transferred

by a

a glucomannan.

of a number

The

of oligosaccharides

purified

glucose

it

a

plants.

seedlings

a partially

Although

mung beans,

of GDP-D-mannose-

by isolation

of the product

Jack Pine.

from

of these

enzyme from mung bean

product

from

been isolated

fraction

The mannose

particulate

glucomannan

has not

of the hemicellulose

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

p-mannanase.

and mannose

in

after Several

the approximate

of ratio

1:2 and 1:3 or 4, respectively. Since

rides,

it

converts

glucose-14C

was found

can be assumed

that

GDP-z-mannose-14C

in the hydrolysis

the particulate

Elbein,

A.D.,

Barber,

enzyme contains

to GDP-D-glucose-

LITERATURE

products

of the

an epimerase

14C .

CITED

G.A.,

and Hassid,

W.Z.,

J. Am. Chem. Sot.

A.D.,

and Hassid,

W.Z.,

J. Biol.

J.T.,

Moffatt,

86,

309 (1964). Barber,

G.A.,

Elbein,

Chem.,

4056 (1964). Roseman,

S.,

Distler,

J. Am. Chem. Sot., MacDonald,

D.,

J. Anal.

Org.

Loewus,

F.A.,

Nelson,

N.,

Perila,

0. and Bishop,

83,

J. Biol.

Chem., C.T.,

and Khorana,

H.G.,

659 (1961).

Chem., 2,

Chem.,

J.G.,

2,

1107 (1962).

219 (1952).

153,

375 (1944).

Can. J.

Chem.,

318

z,

815 (1961).

oligosaccha-

239,

that