Evidence for a buried location of Nigeran in the cell wall of Aspergillus niger

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 28, No. 4, 1967

EVIDENCE FOR A BURIED LOCATION OF NIGERAN IN THE CELL WALL OF ASPERGILLUS NIGER K. K. Tung and J. H. Nordin Department of Biochemistry University of Massachusetts 01002 Amherst, Massachusetts

Received

July 6, 1967

Nigeran,

an unbranched

of the Aspergillus a(1+4)

species

consists

associated

of alternating

with

a(l-+3)

fungi and

linkages

1957).

It

between D glucopyranose units (Barker -et al. = has not been clearly resolved whether or not this

polysaccharide

is a constituent

Mandels 1964, Johnston mycelia

by heating

tates

1965).

Since

it

isolated

insolubility.

to attack

involved

in such a position by a hydrolytic

(Reese and from

to 100' and quickly in hot water,

precipi-

to room temperature. it

is not known whether

in the architecture wall

material

We wish to present a constituent

wall

can be extracted

soluble

in water

along with

is in fact

is located

cools

is insoluble

isintimately

or merely

nigeran

Nigeran

an aqueous suspension

when the filtrate

polymer

of the cell

The polysaccharide,

filtering.

this

polysaccharide

direct

of the wall

by virtue evidence

of the cell

wall

as to be practically

enzyme (mycodextranase)

the

of this

indicating of A. niger

and

inaccessible specific

for

polysaccharide.

Materials

and Methods

Aspergillus surface

culture

niger

QM326 was grown from spore inoculum

at room temperature.

519

The medium consisted

in of the

Vol. 28, No. 4, 1967

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

following

in

(NH412N03

2.6,

0.4,

grams

FeS04*

washing

of

the

of the

product

mycelium

(Dubois using

et --

walls

2-5'

at

Whatman

grams

was accomplished

Dr.

al.

54:8:18

1950).

E. T.

study

istic rotation

[aID+

the

that

fact

using

of

hydrolytic

the

Examination

indicated

com-

glucose

and B. of

is

the

method

was done systems:

A.

isopropanol,

components silver

acetic

on chromato-

nitrate

standard

(Trevelyan

was a gift

from

Laboratories.

polysaccharide

0.5,

acid

solvent

alkaline

from

(c,

sulfuric

following

was based

products

phenol

v/v

as a function

222'

dried.

chromatography

U. S. Army Natick

water

walls

exhaustive

paper

Detection

as nigeran

in

and cell sonication

microscopy

The tetrasaccharide

Reese,

properties

the

6:4:3

v/v.

Identification present

in

water

water

freeze

(NH4)H2P04

to

Following

by the

Descending

1 paper

pyridine,

prior

homogenization,

were

2.7

Mg CO3 0.27 Just

cell.

they

acid

hyphae.

1956). No.

tartaric

and washed

and electron

the

acid,

et --

of

was measured

al.

l-butanol,

process pressure

Carbohydrate

0.047.

was harvested

a French

of

46,

K2C03 0.47,

and ZnS04

by light

breakage

sucrose

0.17,

by a sequential use

plete

(NH412S04

the

and the

of

liter:

7H20 0.047

sporulation prepared

per

liberated

on its of

(a)

in

unusual

temperature;

mycodextranase

solubility

(b)

character-

action;

(c)

NaOH) (Barker

et --

al.

1957);

sole

of

its

total

product

the

optical

and

(d)

acid

hydrolysis. Mycodextranase and Mandels and Nordin nigeran glucose)

was produced

(1964)

and purified

unpublished).

by this

enzyme

are

linked has

nigerose

(0-a-g

been

(tetramer) by an a(1+4) proven 520

to

30 fold

products

together not

about

The only

and a tetrasaccharide

nigerose units 1 This structure

according

the

method

of

Reese

prior

to use

(Tung

the

hydrolysis

of

of

glucopyranosyl consisting bond'

unequivocally.

(1+3)D of

(Reese

two and

=

Vol. 28, No. 4, 1967

Mandels

in

a highly or

to

clearly

determined

from

wall

Thus

since

it

only

is

the

has more

give

assay

these

for

the

quantity

total

the

of in

of

amount

of

Preliminary

ratio

identical

two

enzymatic

preparations.

that

one)

to

to

by measuring

demonstrated (about

walls.

found

possible manner

produced

disaccharide

unheated

is

specific

dimer

experiments

was also

It

exclusively.

tetramer

ide

Our enzyme

1964).

products attack

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

tetrasacchar-

heated

tetramer

carbohydrate

and

present

per

mole

was of

compound. Results The chromatographic in

comparison

I)

similarity

with

authentic

indicates

clearly

they

wall

as substrate.

cell

of

nigerose are

the

hydrolysis

products

and tetrasaccharide with

same

Table Chromatographic

the

either

(Table

nigeran

1

Mobilities of Compounds Released by Mycodestranase from Nigeran and A. niger Cell Walls

Compound

Solvent

Glucose Nigerose Tetrasaccharide

ARglcta)

Solvent

BRglc

1.00 0.74 0.32

1.00 .70 .33

Fast Slow

component component

from from

Nigeran Nigeran

0.74 0.33

.70 .30

Fast Slow

component component

from from

cell cell

0.73 0.32

.70 .33

(a)

Mobility

of

A four in

heated

this of

through bation

the

fold

difference

being

tetramer

enzyme

relative in

walls

the (Table

an integral

detected

preparation with

wall wall

compound

and unheated

polymer the

or

of

is the

should

to g glucose amount II)

of is

component from

walls, eliminate

521

of

exogenous one would

nigeran

strong the

evidence

for

If

part

wall.

nigeran

carried

expect

or decrease

hydrolyzed

the

that

preincu-

amount

Vol. 28, No. 4, 1967

liberated

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

from the unheated

walls

during

Table Enzymatic

Hydrolysis

Preincubation (min.)

incubation.

II

of Nigeran

time

the regular

in Cell

Wall

Preparations

M icro

moles of tetramer liberated per 10 mg of cell walls Unheated walls (a) Heated walls (a)

0 0 30

60 120

.07

.28

.07(b) .035 .035 .035

.27(b) .28 .27 .27

Cell wall suspensions (10 mg per m l.) in 0.02M citrate phosphate buffer pH 4.5 were preincubated at 30' with 0.1 unit (c) of mycodextranase for the times indicated. They were then washed three times in ice cold buffer and resuspended to the same concentration. Each suspension was divided into two portions, one of which was heated 10 m inutes in a boiling water bath and cooled. After equilibration at 40° 1.0 m l suspensions of walls were then incubated 20 m inutes with 0.1 unit of enzyme in a total volume of 1.1 m l. (The quantitative values above represent the product released during the second time period.) The reaction was stopped by heat and the water soluble material deionized and chromatographed in solvent B for 36 hours. The areas on the dry chromatogram corresponding to the tetramer were excised and eluted with water and the quantity of tetramer (above blank values) determined by the phenol sulfuric acid method using a glucose standard. (a) While these walls contain a very low level of reducing power (Nelson 1944) per unit weight prior to enzymatic attack, there is no detectable difference between heated and unheated walls, and neither contain any tetrasaccharide or nigerose as evidenced by paper chromatography. (b) 0.2 unit

of enzyme.

(c) One unit of enzyme will liberate 1.0 m icro mole of reducing equivalents expressed as glucose per m inute from a 0.4% suspension of nigran at 50“ and pH 4.5. This

was found to be the case.

did not reduce heated walls,

unheated centration released nigeran

the amount of nigeran indicating

Limiting

the enzyme. walls

In addition,

a definite

subsequently

more nigeran

does not change the quantity It

also

indicates

in the heated walls. 522

since

preincubation

liberated

inaccessibility

enzyme is not responsible

to yield

from them.

extended

from

of nigeran for

doubling

the failure enzyme con-

of tetrasaccharide complete

digestion

of

to of

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 28, No. 4, 1967

It

is probable

that

of tetrasaccharide unheated bation

walls

the continued

occurring

extended

is due to enzymatic

at a number of restricted To check this

the wall. which walls

for

incubated

directly

it

attack

of nigeran

without

In control completely

hours

experiments,

hydrolyzed

indicating

in unheated walls Further,

that

at an identical

nigeran

is alkali

soluble,

thereby

permitting

the wall

than heat will

the polymer likely

ionic

heat alone a possibility the wall resistance

frees that

and that

nigeran

at 40' during

this for

for

a heat labile

to enzymic hydrolysis.

523

four

Since from

Thus a method

another

of

is most molecule

attack.

arrangement

since

There is also

in some crystalline

molecular

dif-

the inaccessibility

enzymatic

may exist

pH.

inaccessibility

with

was

is

the polymer

hydrolysis.

to indicate

bonding

rapid

as compared to walls

agent extracts

This

a

walls

inhibitor

but neutral

enzymatic

the polymer

If

in 0.5N_ NaOH for

of nigeran

to mycodextranase.

not due to covalent

subjected

added to unheated

of walls

this

in

incubation.

strength,

suffice

both

would lead to the observed

in release

treated

20 m inutes.

heating.

no heat labile

preincubation

at O" resulted

and the

and become available

nigeran

in

and then

1944) after

additional

the final

in

was conducted

and those

were occurring

would have accumulated

ference.

exposed sites

power was present

60 m inutes,

by the enzyme during

present

the second incu-

at 40“ in the absence

(Nelson

of reducing

at 40° for

slow extraction

attack

during

of

enzymic preincubation

measured

level

to enzymatic

period,

attack

of up to 60 m inutes

power liberated

Only the low basal walls

preincubation

an experiment

through

amounts

Enzyme was again added to these preparations

of enzyme. reducing

periods

of small

or partially

point

were carried

incubated

other

with

release

array confers

in

Vol. 28, No. 4, 1967

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COAWJNICATIONS

Calculations tetrasaccharide

based on measurements released

up 7-8% of the weight

from heated walls

of these

material

liberated

from the wall

methanol

insoluble

fraction,

quantities

of glucose

of the wall. since

it

indicate

by heat

is a water

of this

other

equimolar

which makes up about fraction

in protecting

makes

soluble,

composed of approximately

Investigation

and

nigeran

The only

preparations.

and galactose,

may be involved

of both nigerose

LO-12%

is now underway

the nigeran

from enzymatic

attack. Acknowledgement This work was supported National

Science

by a grant

(GB 5162) from the

Foundation. REFERENCES

Barker, S. A., Bourne, E. J., O'Mant, D. M . and Stacey, M ., J. Chem. E., 2448 (1957). Dubois, M ., Gilles, K. A., Hamilton,.J. K., Rebers, P. A., and Smith, F., Anal. Chem. 28, 350 (1956). Johnston, I. R.,Biochem. J. E, 65 (1965). 375 (1944). Nelson, N., J. Biol. Chem.-E, M ., Can. J. M icrobial. 10, 103 (1964). Reese, E. T.-andndz Trevelyan, W. E., Proctor, D. cana Harrison, J. S., Nature 166, 444 (1950).

524