Mannitol acetyl phosphate phosphotransferase of Aspergillus

BIOCHEMICAL

Vol. 29, No. 3, 1967

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MANNITOL ACETYL PHDSPHATE PHOSPHOTRANSFERASE OF ASPERGILLUS Wei Ewa Lee The Upjohn Co., Kalamazoo, Mich. and Dept. of Epidemiology & Preventive Medicine, U. of California, Davis, Cal., 95616

Received September 25, 1967

An Aspergillus

strain

that

medium has been previously in a medium with regulatory enzyme that mannitol

dehydrogenases,

mannitol

phosphoenopyruvate extracts

to1 acetyl

of this

phosphate Mannitol

A specific is

the

carbamyl

phosphate

Methods.

specific

can serve

for

Cultures

mannitol

g, CaC12*2H20

in

were shake

0.05

separately)

337

the

be detected an active

in manni-

+ Acetate l-PC4

enzyme but

(MlP).

either

AC-P or

donors. Aspergillus

an

by serial

at 28" shaker

for

3-4

0.01 per

species

(Upjohn

vegetative

trans-

days in a modified

at 100 cycles

90 g, MgS04 0.5 g, KC1 0.5

g, and FeC13*6H20

2 g, and KH2P04 1 g (sterilized

l-PO4

mannitol

maintained

medium on a reciprocal mannitol

for

from

flasks

to find

present.

this

as phosphate

not

however,

Mannitol

The enzyme was extracted

The medium contained 0.05

+

the

phosphotransferase,

could is,

grows

or particulate

pyrophosphate

phosphotransferase

substrate

The mold was grown

Soluble

There

also

In studying

it is of interest

kinase,

Phosphate

organism

of carbon.

of mannitol.

Asperpillus.

+ Acetyl

in a glucose

This

phosphotransferase

(AC-P)

No. 4177).

Czapek-Dox

source

enzyme assay was developed

Mannitol

mannitol

1967).

metabolism

mamitol

the breakdown

cell-free

fers.

of

free

(Lee,

as the sole

initiates

and mannitol

collection

reported

mannitol

mechanism

accumulates

g plus liter.

per minute.

g, ZnSO4*6H20

NIQNO3 3 g, K2HPO4

Vol. 29, No. 3, 1967

BIOCHEMICAL

Enzyme extracts O.@

potassium

Cell

(lo-15,000

were

obtained

phosphate

buffer

MlP dehydrogenase

purified

was dialyzed

was precipitated Kacetate

cific

2.7 4

and MlP phosphatase,

to 75% saturation

O.lg

with

the pH to 5.0 with

The supernatant added

by rupturing

the mycelium

in pH 7.0

EDTA in a cold

French

Pressure-

psi).

The enzyme was partially

by lowering

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

against

Interfering

in the crude

extract

acetic

acid

more inactive

at 100% saturation,

centrifuged,

and kept

of 0.48-0.61

frozen. units

Three (all

enzymes, were

inactivated

and the precipitate

50% (NH4)2SO4

to remove

buffer

activities

2.51

as follows.

(4")

removed.

and then

proteins.

Then

(NH4)2SO4 the enzyme

and resuspended

in pH 5.4

enzyme preparations

units

expressed

had spe-

are umole/min-mg

protein). MlP was assayed 0.2g

KHC03, and 5.4 +

and 2 pmole in O.D. genase

NAD were

to the

reliable.

It

stable

and detected Results enzymes mannitol particulate

for

in a 70% (NH4)2SO4

dehydrogenase mannitol

MlP formed

cell-free

by the method dehydrogenase

45-65% solution

and

to glucose

1967),

pH 7.0

6-phosphate,

phosphate. dialyzed

against

(NH4)2SO4.

It

in the cold.

in an enzymatic (Tanaka

reaction et al.,

was 1967)

1965). to demonstrate

were

by the method

the

unsuccessful:

of Edmundowicz

338

glycerol

to be accurate

(Lee,

attempts

extracts

it

change

MlP dehydro-

or u and f3 glycerol

(Stetten,

Repeated

The net

(1965)

respond

from a Dow 1-formate'column

& Discussion.

in Aspergillus

showed

against

chromatography

to be assayed

units

Wieland's

from Aspergillus

confirmation,

by paper

after

6-phosphate,

by dialysis

lg hydrazine,

MlP sample

0.05-0.1

MlP and does not

was obtained

and eluted

adding

MlP standard

sorbitol

when kept

For further

after

pmole

containing

and made up to 3 ml.

with

and precipitated

absorbed

separately

was modeled

is specific

6-phosphate,

0.05-0.2

which

tests

MlP dehydrogenase buffer

EDTA, then added

system,

Repeated

mannose

1.5 ml pH 9.1 buffer

at 340 mu was measured

assay.

very

by adding

Soluble

& Wriston of Arcus

following

(1963);

& Edson

(1956);

is

BIOCHEMICAL

Vol. 29, No. 3, 1967

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AC-P mM 92

t

0

Fig.

1.

mannitol

kinase

by the method

by the method

pyruvate

phosphotransferase

probably

these

Mannitol cell-free

enzymes

and substrates

2.5?$ kinetic rate trations salt ficity

of Stetten

40

(1965);

do not

exist

(Fig.

studies,

but

is practically were

the data limited

necessary KCl)

of this

was not

or a high enzyme is

derived

purified

enough

glucose

affinities

solubility. and could

(0.4$

much narrower

339

detected

from Fig. for

from

2 is

precise

about

enzyme

are weak and the mannitol

not be replaced environment.

the hexose

in

to enzyme

AC-P derived

High

osmotic than

Most

strain.

The Km for

show substrate

activity

(1967).

was proportional

mannitol

by mannitol

for

--et al.

was repeatedly

activity

phospho-

phosphoeno-

Aspergillus

1 & 2).

and the Km for

The enzyme preparation

of Tanaka

activity Its

pyrophosphate

and mannitol

in this

of Aspergillus.

O.lg

(1966);

by the method

concentrations

roughly

(1.35g

30

of Klungsoyr

AC-P phosphotransferase

extracts

1 is

20 MINUTES

Effect of AC-P concentrations on mannitol AC-P phosphotransferase activity. 2 ml of reaction system contains O.lg K acetate pH 5.4, 0.Q mannitol, LiAc-P (by assay) as indicated and 0.04 mg enzyme protein at the start of the reaction, 30'. The reaction was stopped by adding 1 ml 10% TCA, chilled and centrifuged (boiling produced assay active substance). See text for MlP assay.

transferase

Fig.

IO

concen-

by a high The speci-

AC-P phosphotrans-

Vol. 29, No. 3, 1967

t k

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

t 0.6

YANNITOL mM

600

l

l

0

A 400

0

2

P

0 200

A

A

0

IO

20

30

MINUTES

Fig.

2.

ferase

Effect of mannitol concentration transferase activity. Conditions LiAc-P and mannitol concentrations

(Kamel

as AC-P, Glucose could

& Anderson,

but Na4P207,

mannose-6-PQ4

and fructose not

extracts

(0.4g)

be involved contain

a very

no activity tivity

of 5.0.

active

Its

90 @ (NH4)2SO4. buffer

decreased

lower

concentrations

lation

phosphorylated

below

by the addition buffer

activity

60%.

could

were

and thus Aspergillus

the

in a pH range

decreased

pH values

than

was about

as active inactive. this

enzyme

cell-free

hexokinase.

Maleic

by end-product

not

phosphorylation.

activity

affected

phosphate

or phosphoenopyruvate

was active

at pH 7.0.

was not

were

in hexose

The phosphotransferase pH optimum

Carbamyl

1966).

on mannitol AC-P phosphoas in Fig. 1 with 92 + as indicated, pH 5.0, 37'.

could

rapidly 4.0 were

of 4 to 6 and had a above

not

tested.

replace

acetate

be significant

tion. 340

substrate

buffer

levels

in controlling

had

Enzyme ac-

of 5 @ CaC12, MgC12,

The enzyme was inhibited effective

pH 5.0 and it

EDTA, or but phosphate

by MlP at much (Fig.

3).

mannitol

Reguoxida-

BIOCHEMICAL

Vol. 29, No. 3, 1967

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AWE0 MIP mM

0.5 -

C 5 0.4U E” 3 z 0.3 41 + 9 E IL 0.2 5 I L =

O.I-

MINUTES

End-product Conditions

Acid This

phosphatase

phosphatase

active

than MlP

did

another

require

6-P04,

phosphatase.

A phosphotransferase

was also

cal.

The mannitol

isolated

strains

oxidation

rate

by growing catabolic

(0.05 cells

enzymes

6-W4,

341

1966)

both

by

purification is

is

a contaminant

or

phosphatase

is

not

producing

by the above methods.

strains

enough

mannitol

as the

activity

(3 n&ml/day could

was

p-nitro-

described

of this

specificity

from

units)

it

many substrates

from another

et al., A-

and substrate

The enzymes

that

of MlP synthesis.

AC-P phosphotransferase

of both

mannitol

One effect

NRHL 305 (Smiley

extracts

mannose

phosphatase

rate

in

hydrolyzed

Further

the acid

the measured

(UC-4177).

1967)

phosphotransferase

was to reduce

Aspergillus

enzyme preparation.

(Lee,

6-FO4,

of the enzyme itself.

candidus

in the

and it

fructose

whether

had the same pH optimum

other

MgC12,

an acid

to distinguish

Aspergillus It

not

was also

activity

activity

was detected

The pyrophosphate

(1965)

necessary

of mannitol AC-P phosphotransferase. 1 (92 e LiAc-P and 0.6_ mannitol).

from MlP phosphatase

(glucose

phenylphosphate). Stetten

activity

differed

at pH 5.0,

other

inhibition as in Fig.

are

enzyme from

probably

identi-

in the cell-free to account

or _+ 0.004 be found,

it

for

units). should

the mannitol Although not

be con-

BIOCHEMICAL

Vol. 29, No. 3, 1967

eluded

that

phorylation

mar&to1

on such negative

negative

mutant

was absent,

then

metabolized

solely

tionship

AC-P phosphotransferase

solely

mannitol

this

why free

would

enzyme.

mannitol

If

and the

constitute

catabolic

accounts

evidence.

of Aspergillus

by this

of mannitol

determine

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a direct

accumulated

mannitol

one could

phos-

isolate

a

phosphotransferase proof

Presently

that

activity

mannitol

we are studying

and anabolic is

for

was

the rela-

enzymes of Aspergillus by these

to

strains.

REFERENCES Anderson, R. L. & Kamel, M. Y. (1966), Wood, W. A., Vol. IX, p. 392, Academic Arcus,

A. C. & Edson,

Edmundowicz, Klungsoyr, Lee,

J. M. & Wriston, L.

(1966),

W. H. (1967),

Smiley, K. L., Am. Chem. Sot., Stetten, Tanaka,

J.

Biochim. Appl.

J. Biochem., C.,

Jr.

Biophys.

Microbial.,

64,

(1963), Acta,

122,

Lerner,

J. Biol. S. A. & Lin,

Wieland, 0. (1965), Methods H. U., Academic Press, N.Y.

J.

ed.

by

385. Biol.

Chem.,

238,

3539.

361.

in press.

Cadmus, M. C. & Liepens, P. (1966), New York City, Sept. 1966, p. Ql5.

M. R. (1965), S.,

N. L. (1956),

in Methods in Enzymology, Press, N.Y.

Chem. 240,

342

152nd

Meeting

2248.

E. C. C. (1967),

of Enzymatic

Abstracts

Analysis,

J. Bacterial. p. 211,

93,

642.

ed. by Bergmeyer,