Starch in fungi IV. The lipoprotein amylose-precipitating factor

Vol.

93, No.

April

29,

4, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1980

Pages

STARCtI IN IWIG I\:. TllE ANYLOSEI’RllC I PITATING Derch

Received

A.

K3kMm11

Department

of

March

12,198O

and

Biological Norma

LII’OPROTF.TU FACTOR

1Jilliam

Sciences. 1 . Tllinois

1204-1209

!.I.

Rutherford

Illinois hl7hl

State

Ilnivcrsit)

The amylose precipitating factor from Il!eii2’,7sTi~‘~~ is :I lipoprotejn of 5?.800 m,\r . The lipid component, which is responsible for the amylose precipitntinc activity, is mainly polar lipid and constitutes l-1.5:( of the I ipoprotcin molec~~l ‘The binding of amylose to the amylose prccipitatin? factor possibly reslllts in the conversion of the amylosc molecule from R random coil to an interrtlptcd helix configuration. ISTRODUCTTON Certain

fungi

nmylose

molecules

Amylose

production

produce that

(EC

(unpublished

results).

rclntivcly

short

chain

1-phos~~hate tating

factor

factor

could

the

and

amylose

ted

was not

he

this

this

factor

to

the

or

of

characterized

these

[5,6). fungi,

In

3 preliminary in

we

which

co~iiil

even

of

describe possil>ly

starch/glycogen

hns

the

the

after

mcthotl

hc

with

ahi

I it!-

to of

incuhntion

in

211 nm~lose (-1 in

n simple

7.J.l.211

presence

hyphnc

difficulties

chain

I1 ,.?,?I.

(EC

report

tised

short

h>~phae

contains

enzymes

;I~izti~:+-.~?i,r

hccnl~sc

report

the

Therefore

these

present

of

of

glucos~ltransfcrase

molecules

he

composed

(4 I which

both

in

is

\
ADPglucose

molecules

shown

in

which

cytoplasm

13) . 1~3s unexpected.

In of

in

Either

nmylose

starch

dcr,osi

1.3.1.1)

long

prepnrations. szimplcs

are

occurs

phosphorylnse

form

n unique

onlb ~l~~cosc-

prcc tlo\iex,er,

obtaining

t-or

nht:lininq

other

snluhlc

1.

Supported

or frozen :!,;!2wqor,; 1.5% NaOtl overnight. at 20.000s g for i II part

b?

i’?7~:::‘8,: After 113 min

:I Rrovn-llnzcn

pure 13~13‘e lipopr0

(wild t>.pe OR2.7 I,\) mycelia were filtration through cheesecloth and the 1~11 of the resulting supcrnntant erclnt

from

Research

Corporntion.

this

teins.

Fresh floated in centrifugation

iI,i

c

Vol.

93, No.

4, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

liquid was adjusted to 7. During this pH change a precipitate formed which was composed of amylose precipitating factor and the amylose endogenous to /Jezirospc?ra (McCracken, Varkcy, & Rutherford, unpublished results). This preparation was centrifuged again and the supcrnatant liquid containing soluble amylose precipitating factor was adjusted to ptl 4.0. This step separated the amylose precipitating factor (insoluble) from glycogen (soluble). After centrifugation the pellet was redissolved in 0.1 M Tris-HCl buffer (pH 7.0) (10,000x g, 5 min) and used as the source of amylose precipitating factor for characterization. Amylose precipitating activity was measured as described previously (7). Protein was determined by the Lowry method (8). Aliquots containing 50 ug protein were run on disc polyacrylamide gel electrophoresis according to Maurer (9) using gels with 7% acrylamide, 0.29, bisacrylsmidc and Tris-borate-EDT.4 (pH 5.4). Elcctrophoresis was performed at 20°C and 3mA per tube. Gels were treated to visualize protein, amylosebinding ability, and lipoprotein. Protein was stained with coomassie hlue and lipoprotein with Oil red 0 (10). Amylose binding regions in the gels were demonstrated by incubation of the gel in 0.2”~ amylose for 1-2 h at room tempcrature. Aft.erwards the gels were rinsed in H20 and stained with 0.2: 17 - 2.0% KI. The electrophoresis was repeated with samples containing Triton X-100-(1 ul/lOO u To determine the molecular polyacrylamide gel electrophoresis and 0.2”” bisacrylamidc (11).

weight was

of the amylose run using gels

As the results of and bovine serum albumin activity was determined

observed interactions between standard on SDS electrophoresis, in the presence of 0.3: bovine

Amylose precipitating factor preparations in SDS elect,rophoresis.

factor ethanol-ether

The ethanol-ether nitrogen. In order to of amylose precipitating wore spotted on Silica

extracts determine factor gel TLC

The molecular determined

were

by

weights of sedimentation

apoprotein (12)

from

an

the

with

isolated

attempt

in to

2.

pure

isolate

Rutherford, activity

was

2.!4s ~sinr/s

LerztineZ

precipitation be

attempt

unpublished in

Molecular Department,

alkaline

fruit

amylose equilibrium

enough amylosc

made

sulfate

weights were University

precipitating runs

to

isolate (7)

or

.

low

allow

to

from

~U~Z~~YX,WY~I?

its

we observed of

the

determined of Illinois.

an Partial pll.

form

results) extracts

precipitating amylose precipitating serum albumin. by for

delipidation both regular

in

an

of and

under moiety extract

lipid

factor and ultracentrifuge’.

factor

its

%poprotcin

AND DISCUSSTON

bodies

ammonium

amylose

was prepared then used

and

factor SDSacrylamide

75

were reduced in volume by evaporation to which class(es) of lipids the belongs, aliquots of the concentrated plates (Supclco).

RESULTS Previously

precipitating containing

factor

was

by

, the

obtained

factor

CXWGS~Z

(McCracken,

Thomas

Adjustment Holzman,

could

During

characterization.

amylose

1205

precipitating

success fiowcver

mycelia. by

amylose

l’arkey

Precipitating of

not 3n

, CT factor

the

Biochemistry

pH

to

neutral-

Vol.

93, No.

BIOCHEMICAL

4, 1980

A

Fig.

ity

allowed

factor

the

as

(pellet)

removal

bl Tris

- HCl

band

which

co-migrated

SDS

electrophoresis

than

the

however, an

interaction

was

present

the

amylosc

upon

removal

esis

extracts

were

electrophoresed

band

was

of

these

activity.

system One

the

were

observed X-100

lipid

the

two

and

is

by

bovine

presence

treatment

two

pll

Triton

protein

bovine

1206

4 pellet as

Oil

X-100.

indicating

of

and were

amylose

is test

0. Oil

is

inactivated this

Also red

one

hypoth extracts

0 staining

amylosc-binding ohservcd,

(0.5”,)

observations

red An

did,

albumin

To

1).

observed factor

these

albumin.

(fig.

were

standard

in

protein

activity

which

with

solr~hle

a single

serum

for

factor

was

inhibition

protein bands

the

precipitating

a complete

stained

precipitating

separated

albumin

serum

the

amylose

hands

a lipoprotein

of to

The

protein

explanation

and

corresponded

no

When

factor

4.0

and amylose-

precipitating

serum

was

to

amylose

bovine

possible

moiety

the

of

proteins.

electrophortsed in

as

there

precipitating

of

amylose

presence

to

appear-cd

unsuccessful

The

assay

adjustment

with

(A)

complexed

elcctrophoresis

a band

mobility

protein

(supcrnatant). on

was

between

that

Triton

and

with

the

ptt

xlycojien 7.0)

the

in

amylose

COMMUNICATIONS

B

to visualize

endogenous

standards.

precipitating

After

(pH

affect

treated

Subsequent

endogenous

0.1

other

of

RESEARCH

A

gels (B).

a precipitate. from

BIOPHYSICAL

0

Polyacrylamide binding ability

1.

AND

hands. of

which

(the

Vol. 93,

No.

major

8lOCHEMlCAL

4, 1980

band)

ran

just

slow-migrating ment

of

band amylose

while

lipids

cipitants

lipid

soleIy

is

not

lipid of

moiety

polar

causing

the

of

precipitating

more

much

band

ethanol-ether

abolished

responsible

precipitating

factor

acids

are

Treatamylose protein is

activity. known

The to

be

The

not. the

a fast-migrating

its

slowly.

did

of

for

fatty

COMMUNICATIONS

fast-migrating

amylose

is

other

appearance

as

the

presence

of

phospholipids.

The 53,500

and

of

lipoprotein.

the

thin

molecular

with

the

molecular

that

the

polar

amino

acid

lipid

extend The

dextran

sulfate,

amylose

a sugar

from

the of

there

was

no

binding

precipitating

glycogen factor

sufficient

for ability

such

the

a involve-

amylose

pre-

to

together

plasma

high

amylose

forms and

residues

did

not

factor component

be

removed

the

indicate

the

is 14.5'

ethanol-ether,

serum

albumin,

bonds.

lipoprotein ionic

lipids.

constitutes

by

bovine

composed

lipoprotein

noncovalent

that

precipitating

precipitation or

By

(17)

with

nonpolar

the analogy

we propose

interactions

as

found

chains

charged of

the

binding

to requires

of

short

which

glucan glucan

configuration. chain

1207

are

seems

to

amylose

not

This (average

inhibitors

the

absence

which

residues. with

be

dextran,

indicates A (15)

an a-1,4

a helical very

glucose

conconavalin

terminal

amylose cellulose,

and

however, in

for

amylopectin,

Maltose (7‘1,

is

factor of

inulin.

apparently

precipitate

polar

density

moiety protein

for

and by

he

solvents

lipid

may

to

protein.

by

formation

the

water,

held

the

appears

precipitating

against

activity

site and

amylose

lipid

glycogen,

precipitating

amylopectin

moiety

the in

specificity as

lipid

for

chains

out

absolute

Thus

be

in

phosphate

46,000.

must

of

ran

is the

model

side

for

dialysis

head

only

the

Since

complex

factor

of

apoprotein

protein-lipid

lipid

plates

weight

the

the

layer

X-lOo,prolonged

Triton

amylose as

of

its

with

unexpected

lipids

Staining

by

the

the

RESEARCH

(13,14). The

of

but

and

factor

moiety

BIOPHYSICAL

front

Consequently

whose

of

dye

amylose

electrophoresis.

lipoprotein ment

bound

activity

on

the

precipitating

precipitating band

behind

AND

precipitates .4mylose

a chain is

length

demonstrated chain

length

of

Vol.

93, No.

4, 1980

12-20)

produced

maltose

(7).

sible

for

with of

known

precipitating chains

would

for

act

assume

the

the

as

the

stimulus

the with

other

density for

to

the

helical

molecule

in

an

interrupted

lipid

the

of

of of

helix

(14)

factor

the

for

the

The

result

would

each

helical

portion

one

in

lipid (13) much

a random of

be

an

or

by

is

portions

end

has

coil

chain

remains

further

action

free

random

lipid

molecule

the

proposed

(17).

amylosc

respon-

complcxing

moiety

been

plasma

convert

with

has

since

The

lipid

not is

acid) model

the as

but

factor (fatty

that

the

would

(16)

following

However,

configuration.

precipitating

of

a portion

chains

amylose

lipoprotein

much

COMMUNICATIONS

precipitating

lipoprotein

molecule lipid

mode

the

configuration.

amylose

amylose

developed

from

high

molecule

amylosc

and

RESEARCH

potato

We postulate

extend

helical

than

of

we have

the

BIOPHYSICAL

of

moiety

factor.

which

al.

Association

an

lipid

(14),

AND

hydrolysis

precipitation

is

et

smaller

acid

the

amylosc

free

chain

limited

Since

amylose

Scgrest

to

by

amylose

more

BIOCHEMICAL

coil.

the

amylos

amylose

complexed

with

molecule.

REFERENCES McCrachen, Dodd, Mecusc,

3.

D. .J.

B. 39-45.

McCracken, Rot. Bailey, Doi,

.J.

(1967)

McCracken,

D. 0.

klaurcr,

fl., Biol. Il.

10.

Chalvnrdjinn,

11.

Wcber, Il., Entcnman,

and

and

Whelan,

I,.

(1971)

Science

173,419.

I).

A.

(1973)

Mycologia

64,

D.

bl.

(1973)

Ann.

A.

Rosebrough, Chcm. 193,

(1971)

Osborn, R. L.,

and

(1961) .4cta Physiol.

N. .J., 265-290.

Far-r,

Lipid

(1975) Boeder,

Methods

in

Res.

N.

Dodd,

.J.

Biol.

54,

414-415.

L.,

and

pp. 12,

1208

Sci.

L.

4-1-35,

210,

(1973)

236,

Randall,

3,

299-317.

179-223, Press,

J.

969-973.

R.

de

Amer.

.J.

(1951)

Gruyter,

265-269

The proteins, pp. C., ed.) Academic Enzymology

1341-134.3.

Acad.

Chem.

603-605.

.A.

Y.

J.

136,

elcctrophoresis, .J.

M. and

.J.,

Plant

Disc

11'171)

.J.

Biophys.

(1974)

(1957)

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IZ.

Biochim.

A.

C.

[[all,

,J.

A., Nadahavuharen, 940-943.

R.

K. , and Hill,

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l\lcCrachen,

I,. , and

M.,

A.

J.

12.

and

I). 60, .J.

Lowry,

9.

L.,

A.,

New

(Ncurath, York.

Berlin.

Vol.

93, No.

4, 1980

BIOCHEMICAL

T. 1 2.5 3

15.

Schoch,

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Goldstein, I. .I. , Hollerman, s 7b - 8 i 3 .

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and

Sot.

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COMMUNICATIONS

Chem.

kc.

E.

(15157,

lilorrisett,

1209

RESEARCH

Sot.

Gotto,

its

66.

26,

1232-

186-190.

Riochem.

4,

1490-??!I;. A.

components,

bl.

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