Incorporation of mannose into mouse brain lipid

Vol. 36, No. 3, 1969

BIOCHEMICAL

AND BlOPHYSlCAl

RESEARCH COMMUNICATIONS

INCOBPOEATION OF MANNOSE INTO MOUSE BRAIN LIPID M. Zatz Departments Albert

ReceivedJune

and S.H.

of Pharmacology, Einstein

Barondes

Psychiatry

College

and Molecular

of Medicine,Bronx,New

Biology

York

10461

20, 1969

SUMMARY: Microsomal fractions from mouse brain incorporate mannose-C 14 from GDP-mannose-Cl4 into lipid material. The extent of incorporation is limited primarily by the endogenous acceptor. The incubation mixture includes phosphoenol pyruvate and Mn*. Protease has no effect on the product. The mannolipid is extremely sensitive to acid but is resistant to base. Mammalian been

brain

extensively

mannose.

mannose material

radioacttiity

spotted.

International

Chemical Swiss Co.)

and homogenized

describes

been

which

described

the enzymatic

mannose-C l4 from

lipids

have

contains in bacterial

incorporation

(GDP-mannose-C14)

of into

lipid

mouse brain.

mC/mM) was purchased

chromatography

corresponding

Breeding

however,

preparation

(151

one peak,

Male

described

these

AND METHODS:

GDP-mannose-Cl4 Paper

have,

diphosphate

by a microsomal

Although

none has been

communication

guenosine

MATERIALS

Corp.

in glycoproteins.

mannolipids

This from

rich

investigated,

Several

systemslr2.

is

albino

were

isobutyric

to GDP-mannose

& Nuclear mice

sucrose.

(48.8

the New England

1N NH40H, 5:3 and containing

m&M)

Nuclear

showed over

was purchased

only

98% of the from

Corp.

(25-30

decapitated

acid:

standard

D-mannose-l-C14

in 0.32M

Abbreviations used: PEP, phosphoenolpyruvic acid.

with

from

gm.,

3 months

and their

cerebral

Microsomes

C, chloroform, M, methanol, acid, TCA, trichloroecetic

511

old,

from The Charles

hemispheres

were

prepared3

River

wereremoved end resuspended

HAc, acetic acid, acid, PTA, phosphotungstic

Vol. 36, No. 3, 1969

in 10 ml 0.32M preparation

sucrose.

Reaction

(approximately

Mannolipid described washed

BIOCHEMICAL

at least

twice

with

were

washed

the

theoretical

1.

twice

mixtures

by incubation

The acid with

6% TCA-0.5%

0.2 volumes upper

The distribution

scintillation

courting.

Incubation

Reaction Mixture Corn lete system k -MD -MI+, +Mg* ,= PEP - PEP, + pyruvate - PEP, + ATP - PEP, + P04= - GDP-mannose-C14,

products

were

PTA and the pellet

2~1,

v/v.

0.5 ml of this

as described

of Mouse Brain

+ mannose-C

with

by Folch,

of radioactivity

mixture precipitated

Lees,

was followed

Microsomes

with

and

extracted

The organic

of 0.9% NaCl and then

phase

Stanley4.

TABLEl-

contained

of the reaction

insoluble

2 ml chloroform-methanol with

routinely

350 pg protein).

was prepared

in Table

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

extracts 0.2 volumes

of

and Sloaneby liquid

GDP-Mannose-C

14

.

cpm Incorporated 1120 232 672 78 308 46 721 90

14

The complete reaction mixture contained , in the final volume of 1 ml, ~JIM MnC12; ~SJIM Tris ii01 pH 7.4; 0.5p PEP; 2Om)lC GDP-mannose-Cl4 (approximately 20,000 cpm) 0.16 mM surcose; and 0.5 ml of microsomes. In other reactions MnC12 was replaced by 5~.@l MgC12; PEP by 5p pyruvate, 54ff ATP, or 5p phosphate buffer pH 7.4. In still other reactions GDP-mannose-C was replaced by 200 WC mannose-l-Cl4 (48.8 mC/mM). Reactions were incubated for 60 minutes at 370. Reactions were stopped by bringing the reaction mixture to 5 ml 6% TCA-0.5% PTA. After precipitation at Oo the mixture was run through a Millipore HA filter and washed 4 times with 6% TCA-0.5% PTA. The filter was placed in a counting vial, scintillation fluid was added, and the sample counted in a liquid scintillation counter.

ProCease protease

from

treatment Streptomyces

pH 7.3 to the reaction thehcubation albumin

for

was added

as described

consisted

of adding

griseus

mixture

to experimental

(Sigma-Iype

after

2 hours 8 at 37O.

1 ml (200 pg/ml)

30 minutes At the

and control

above. 512

IV)

of repurified

in 0.2 phosphate

incubation

and then

end of the incubation tubes,

buffer,

and analysis

counting

1 mg of proceeded

Vol. 36, No. 3, 1969

with

BIOCHEMICAL

Thin

layer

0.25

mm silica

65:25:4;

chromatography

(B)2

gel

C:M:

(D)2 C:M HAc:H20, H20,

40:25:3:4.

on Silica

paper

butanol:pyridine:

methanol minutes solution of

phases

ethyl

base

E and F were

cm glass

systems

(C) C:M:

C:M:HAc:H20, run on Silica

chromatography

hydrolysis

at 37'.

in 0.2 ml water

was neutralized

separated

5:3:2;

(H>7 isobutyric H20,

was performed

Three

then

was performed

acetate:pyridine:

to the sample

EtCHand

(E)5p6

0.l.N HCl,

(I)2

Mild

Solvent

7N NH4 OH, 12:60:10; 60:40:3:5;

on 20x20

plates

used were

coated

(A)C:M:N20,

15N NH40H, 75:25:4; 60:30:8:4; gel

and (F>5 C:M:HAc: H, the other

systems

G.

Descending

57:4:39;

was performed

(Brinkmann).

Systems

gel

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

by adding

solvent

acid:

15N NH40H: H20,

, and (J)

were

added,

Dowex 50-H+. added,

phenol:H

and incubating

the pH was above After

systems

0.8 ml of 0.125N

ml chloroform-methanol

4 ml of CHC13 were by brief

12:5:4

using

removal

the mixture

(G)

0, 5:2. 2 NaOH in for

20

11; and the

from

the resin,

shaken,

and the

2 ml

centrifugation.

RESULTS AND DISCUSSION: Requirements

for

the incorporation

of mannose

into

acid

precipitable

.

4111 1 1530

I 60

I 120 MINUTES

I I80

I 300

FIGURE 1 - Time Course of Mannose Incorporation into a Lipid Soluble Fraction. Reaction mixtures were those of the complete system described in Table 1; except that they contained 5p Tris buffer. Reactions were incubated at 37' for various times, stopped, and assayed as described in Table 1. 513

BiOCHMCAL

Vol. 36, No. 3, 1969

material for

by mouse brain

maximal

activity

unexplained.

microsomes and cannot

Since

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

inorganic

phosphate

for

is

by ADP, ADP + pyruvate

each for

inhibit its

mannose,

even

in

The time

course

The extent about

of PEP is

than

ATP,

required

pyruvate

in

Incorporation

GTP, UTP, and CTP

of PEP, and none

compensates

as the mannosyl

donor

by

concentrations.

than

is shown

5 minutes

of incorporation

protein

in Figure

1.

It

has reached

and 75% of maximum in 15 minutes.

was linear

1 mg of microsomal

is

donor.

or ATP.

be replaced

of incorporation

50% of maximum in less

Mn"

The role

as a phosphate

kinase,

cannot

at much higher

by Mg*.

the presence

GDP-mannose

1.

was more effective

may be acting

incorporation

absence.

shown in Table

be replaced

substituting inhibited

PEP, it

are

with

(Figure

microsome

2).

Figure

concentration

up to

3 illustrates

the

14 effect

of GDP-mannose-C

30 and 60 minutes the reaction

incubation.

mixture

incorporation.

If

after

system

is

data

limited

endogenous

acceptor.

sufficient

to accept

additional

without

These

on the extent

60 minutes

IncuSation

enzyme activity. this

concentration

GDP-mannose

primarily This

that

there for

approxtiately

is

30 @i

added

60 minutes

per

in destroy

of mannose

by

of

to be present

mannose

to

did not

or accessibility

calculated

after

is no increase

the incorporation

by the quantity

acceptor

14 is

GDP-mannose-C

incubation,

indicate

of incorporation

in quantity

mg of microsomal

protein. Almost material

all

is

lipid

the organic methanol 5 minutes, extract

of the

Neither

after

30 minutes, the radioactivity

or ethanol chloroform

with

incorporated

into

acid

Over 90% of the radioactivity treatment

with

a small

volume

1:l.

This

is

after

or ethanol:ether

to coprecipitate methanol

soluble.

solvent 2:l

radioactivity

or 60 minutes. from proteins

Tris

the acid

true buffer

pellet.

in TCA and require

to release

them from

nor petroleum

ether

their

readily

514

precipitable is

extracted

of chloroformincubation

pH 7.4 fails Polar

lipids

solvents

for to are known

such as

association

with

solubilised

the

proteins.

into

Vol. 36, No. 3, 1969

BJOCHEMICAL AND BIOPHYSJCAL RESEARCH COMMUNICATIONS

0.5

1.0

2.0 ml MICROSOMES

4.0

FIGURE2 - Effect of Microsome Concentration on the Extent of Incorporation. Reaction mixtures contained 5~rMMnC12, 5p PEP, 2074uCGDP-mannose-Cl4and varying quantities of microsome suspension. Microsomes from 2 brains were suspended in 10 ml 0.32M sucrose and an aliquot was diluted with an equal volume of 0.32 surcose. All reactions were brought to a final volume of 2.24 mlwith 0.32M sucrose. Incubation was for 60 minutes at 37O and incorporation was assayed as described in Table 1. The abscissa refers to volume equivalents of the more dilute microsomal swpension.

I500 r

-----w-e---

60 ml,, --o 30 rn2

/d

P

I 000 P”

5 0 v

d 500

1

II

I

5 IO

I

I

20 m UC

50 GDP - mannose - Cl4

I 100

FIGURE3 - Substrate Curve. Reaction mixtures were as described for Figure 1, except that the quantity of GDP-mannose-C14 was varied. Volumes were brought to 1 ml with water. Reactions were stopped after 30 minutes (0 -- 0 > or 60 minutes (0 -- 0 ) and assayed as described in Table 1.

515

Vol. 36, No. 3, 1969

incorporated

radioactivity

Non-lipid lipids

30 minutes

solvents.

incubation

with

extractable

did

from

to acid.

All

hydrolysis

2 minutes

There

I).

Unhydrolysed

were

material

the acidic

system This

lability

systems

further

in its

after

treatment

particulate

with

Jackson fractions

to be similar

it

also

acid

in its

0.l.N

beyond

with

mobility

the

from

have

liver,

in system

thin

observed

for

significant

soluble

mannose

glycoprotein

516

diglycerides

lipids.

of

It base.

The

phase

peak in system

a mannolipid

J

using

cells.

They consider precursor

and Sweeley7, containing synthesis

in

bond.

layer

the upper

and tumor

Lennara,

.J but

phase.

synthesized

by Scher,

phosphate.

the manno-

to the mannosyl-1-phosphoryl-polyisoprenol described

for

and ran with

by mild

into

the lower

oviduct,

as mannose

the mannosyl

of those

partitioned

in

and Eylar'

(O.OZNHCl,

from

in the

analysis

solvent

a phosphodiester

differs

by

aB mannose

fucose

and from

labile 14

in three

was released

consistent

quite

the mannolipid-C

NaOH gave the same single

material

is

migrated

to saponification

that

Furthermore,

hydrolysis

which

spot

1,5,6

resistance

may be a precursor

We have not

from

by mouse brain

of material

mannan biosynthesis that

is

used for

the unhydrolysed Caccam,

Mild

of material

lysodeikcticus2

proportion

microsomes

85% of the radioactivity

of Mycobacteria

chromatographic

it

traces

of

of radioactivity

Chromatography

at 100').

mannose.

synthesized

phosphoinositides

small

or proportion

after

chloroform-methanol.

ran as a single

acid

differs

material.

G, 50% of the radioactivity

The mannolipid

Micrococcus

precipitable

was released

about

mixture

the extent

by mouse brain

only

may accompany

reduce

the amount into

(60 minutes

released

not

acid

reduce pellet

revealed

at 100')

mannose.

not

material,

of the reaction

did

into

synthesized

lN HCl

(G,H,I)

as did

Treatment

of the radioactivity

in

pellet.

proteinacious

protease

the acid

The mannolipid

(System

the acid

of radioactivity treatment

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

including

organic

incopporation

systems

from

material,

into

protease

BIOCHEMICAL

of

and suggest

glycoproteins. in our system.

BlOCHEMlCAL

Vol. 36, No. 3, 1969

AND BlOPHYSlCAL

RESEARCH COMMUNICATIONS

However, the rapid rate of synthesis and the fact that mannose-containing lipids

have not been observed in mammaliansystems may indicate

mannolipid is an intermediate

that this

in the synthesis of someother product.

Whether the mannolipid synthesized by mousebrain microsomes is the same as that synthesized by the preparations from Micrococcus lysoaeikticus 7 or from liver'

is currently

not knwn.

Acknwledgment: - This work was supported by grants MH 12773, NIH 5T5 GM 1674, and Career Development Award K3-MH 18232 from the U.S.P.H.S.

REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8. 9.

Lee, Y.C.,and Ballou, C.E., Biochem. L 1395 (1965). Lennarz, W.J.,and Talamo, B., J. Biol. Chem., 241, 2707 (1966). Barondes,S.H., J. Neurochem., l5, 343 (1968). Folch, J., Lees, M. and Sloane-Stanley,G.H., J. Biol. Chem., 226, 497 (1957). Brennan, P., and Ballou, C.E., J. Biol.Chem., 242 3046 (1967). Brennan,P., and Ballou, C.E., Biochem. Biophysxes. Communs., 30, 69 (1968). Scher, M., Lennarz,W.J., and Sweeley, C.C., Proc. Natl. Acaa. Sci.U.S. 59, 1313 (1968). Barondes,S.H., J. Neurochem., l.5, 699 (1968). Eylar, E.H., personal communication (1969)

517