Galactosamine: A precursor of glycogen glucosamine

Vol. 23, No. 1, 1966

BIOCHEMICAL

GALACTOSAMINE: Frank

March

During

that,

of the metabolism

in addition

z.ble radioactivity

nor

component

galactosamine,

E:lucosamine

these the

DelGiacco

of galactosamine-l-

Department

with

products,

the glycogen

contained

the

glucosamine. part

be presented

(Maley

consider1966).

revealed

of the glycogen.

in this

, it

To

was neither

studies

by which

liver

et al.,

radioactivity

Further

and the mechanism will

14C in rat

of acid-soluble

as an integral

findings

glycogen

that but

to be present

supporting into

and Rudolph

to a number

was associated the

clur surprise,

:.ted

John F. McGarrahan,

4, 1966

a study

b#as found

E,lucose

A :PRF,CUWOR OF GLYCOGEN GLUCOSAMINE*

of Laboratories and Research, New York State of Health, and the Department of Biochemistry, Albany Medical College, Albany, New York

Division

Ileceived

Maley,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

The evidence

glucosamine

is

incorpor-

report.

RESULTS AND DISCUSSION Glycogen 14

mine-lfound

Labeling

C (DelGiacco

in the glycogen,

--

In contrast

to our

previous

and Maley,

1964),

where

little

perfusion

of rat

liver

with

studies if

with

glucosa-

any radioactivity

galactosamine-l-

was

14c lea to

. the

incorporation

that

in

Ective

the

liver

compound

of radioactivity acid-soluble to be glucosamine

into

the

glycogen,

The evidence

fraction.

glycosidically

linked

' amounting indicating to glucose

to 10% of the racliowas as

follows:

3, Supported by research grants from the National tiealth Service (CA-06406), and National Science

Cancer Institute, U.S. Foundation (GB-4589).

Public

1 Obtained by perfusing a liver for 3 hrs with 56 pmoles of D-galactosaminel-14C (New England Nuclear Corp.) (8.1 X lo5 cpm/pmole) in an enriched perfusate. The glycogen (9 ml) was precipitated from the acid-soluble fraction and dialyzed in the cold against five changes of water (2 liters each) for 2 Clays . The radioactivity retained by the glycogen was 3.1 X lo6 cpm.

85

Vol. 23, No. 1, 1966

1. G-100

BIOCHEMICAL

Chromatography

demonstrated

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the glycogen

the radioactivity

2.

Extensive

3.

Nitrous

dialysis

on a 90- x 1.2-cm to elute

did

not

remove

with

the

the

column

of Sephadex

glycogen.

radioactivity

from

the glyco-

gen.

al.,

1953; 4.

acid,

Yosizawa, Acid

since

of the

that

cleaves

1,4-hexosaminidic

was found

the radioactivity hexosamine

radioactive

for

12 hrs)

on a modified

verified

&

the presence

by Dowex 50-H+.

Garde11

hexosamine

compo,Jnd

(Foster

most of the radioactivity.

was retained

of the N-acetylated the

linkages

to solubilize

(4 N HCl at 100'

eluted

and electrophoresis evidence

1964)

hydrolysis

of hexosamine, tography

which

in

Chroma-

column

(Pearson,

1% borate

provided

incorporated

into

the glycogen

CI-amylase

solubilized

the

solubility

data

1963)

was

glucosamine. 5. ity

Treatment

associated

in Table

I.

of the

with

it,

Similar

glycogen

with

as seen in the

results

were

obtained

Table Solubilization

of Glycogen

time

versus with

radioactivpresented

@-amylase.

I Radioactivity

Minutes

with

a-amylase

cpm

0

807

2

1,590

5

3,620

10

4,600

60

20,800

300

26,600

The reaction mixtures contained 0.1 ml of glycogen (10.7 mg; 31,920 cpm); 0.1 ml of enzyme solution consisting of 0.02 M potassium phosphate, 0.02 M NaCl; and 0.36 i.lgm of a-amylase (Worthington Biochemicals Corp.). The incubation temperature was 25O and at the indicated times 0.4 ml of 95% ethanol was added. After centrifugation, a 0.2-ml aliquot was taken from the supernatant fraction for radioactivity determination.

86

BIOCHEMICAL

Vol. 23, No. 1, 1966

Chromatography

of the

possibly

three

radioactive

glycogen

(Fig.

1).

sugars

(glucose,

finding

The above unit the

results

in the di-, labeled

product

glucose

in this

on CL-amylase

resulting

with

the

of a single

produced

on enzymic

with

the

observation

chromatographic

did

for,

maltose. glucosamine

digestion that

system

similar

treatment,

than

presence

of the

reducing

A somewhat

more slowly

by the

two and

digestion

from @-amylase

migrated

be explained

mainly

oligosaccharides).

and are consistent

than

revealed

coincided

and tetrasaccharides

glycogen,

moves more slowly

the

radioactivity

could tri-,

apparently

and higher

with

1, the

components to be formed

None of these

was obtained

as seen in Fig.

solubilized products

maltose,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

glucosamine

(Fig.

1).

Figure 1. Descending chromatography (75% isopropanol-acetic acid (9:l)) of the alcohol-soluble fractions from a- and @-amylase-treated glycogen. The dark areas adjacent to the CX-amylase-treated glycogen signify the reducing sugar regions, as do the shaded areas in the chromatogram of the known sugars. The radioactive regions were located with a Nuclear Chicago Corp.actigraph III, paper chrornatogram scanner.

Studies assumption tively which

on the

B-amylase

was correct,

digested retained

with almost

Product

a sample S-amylase all

(@-A.P.)

--

To determine

of glucosamine-labeled and passed

of the radioactivity.

87

through

if

glycogen a column

The column

the

above

was exhaus-

of Dowex 50-H+, was then

eluted

Vol. 23, No. 1, 1966

with

BIOCHEMICAL

35 ml of water,

20 ml of 0.05

90% of the

radioactivity

the

(4 N HCl at 100°

p-A.P.

chromatography other

in

coinciding

the mixture

assay

revealed

these

assumed

glycosidically

for

both

last

(4:1),

hexoses

composed

>4)-D-glucopyranose

with

the Rg's

disaccharides

(0.27 However,

the @-A.P. to 0.45,

which

N-acetylation

compared

CX-D-glucopyranosyl-(1 for

(Reissig for

obtained

saminide.

Nitrous

acid

Thelatter

dehydrogenase

and the

as well

migrates

to the

of the

theoretical

an Rg of 0.47

treatment

former

by the

by the

indole

as by electrophoresis

amount

(1964a)

in

The yield expected

if

the

associated

glucopyranose,

with

a compound

yielded

for

these for

altered

1964a). 15-20%

the Rg

Assay of the

favorably

mainly

with

anhydroinannose

hexokinase-glucose-6-P

(Dische

and Borenfreund,

in which

glucose, @A.P.

of

the glucosyl-N-acetylgluco-

coupled

deoxy-O+D-glucopyranosyl-(l----X)-D-glucopyranose. probably

agrees

1% borate,

of free

et al.,

which

assay

Comparison

0-2-acetamido-2-deoxy,

only

for

recently

reported

yielded

of the @-A.P.

was estimated

cathode.

1955)

were

1956)

for

(Wolfram

et al.

the

an Rg of 0.15-0.25

and Daffner,

N-acetylglucosamine,

by Wolfrom

and glucose.

1950),

free

If

O-2-amino-2-

and b).

revealed

et al.,

2).

and glucosamine,

(4:1:5)

--->4)-D-glucopyranose

anticipated

the results

with

(Fig.

disaccharides

(1964a

(Roseman

favorably

N-acetylglucosamine

color

et al.

respectively)

et al.,

possible:

in n-butanol-ethanol-water

and 0.32,

of

and O-CX-D-glucopyranosylBoth

by Wolfrom

and the

dehydrogenase

ratio

are

of

Analysis

of glucose

two sequences

than

on paper

glucose

(Reissig

in a 1:l

(l--->4)-2-amino-2-deoxy-D-glucopyranose.

the @-A.P.

with

hexokinase-glucose-6-P

to be present

9 (l---X),

spots

and the radioactivity.

coupled

More

hydrolysis

two reducing

one coinciding

to be a disaccharide

and characterized

Acid

for,N-acetylglucosamine

deoxy-SD-glucopyranosyl-(l---

prepared

eluent.

yielded

glucosamine

by the

linked

and 20 ml of 0.5 N HCl.

by the

17 hrs)

N-acetylation

glucose

is

was eluted

with

for

B-A.P.

N HCl,

isopropanol-H20

after

1955),and

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

however,

was entirely

anhydromannose was only

70-80%

O-2-amino-2-

The residual

20-30%was

O-CX-D-glucopyranosyl-(l---->4)-2-amino-2-deoxy-Dthat

should

be resistant

88

to nitrous

acid

cleavage.

Vol.

If

23, No.

this

1, 1966

BIOCHEMICAL

assumption

is

valid,

by acid

sodium

p-A.P.

followed

active

N-acetyl-2-amino-2-deoxy-D-glucitol,

N-acetylglucosamine.

AND

hydrolysis

Fig.

BIOPHYSICAL

borohydride

RESEARCH

treatment

of the N-acetylated

and re-N-acetylation in

2 preesents

should

addition

the results

COMMUNICATIONS

yield

radio-

to the major

of such

a study.

product, As seen,

+ N -Acetyl

f3 A P

GO&AC

/,,

&,

,

4

86420246’3

IO

12

14

I0

16

cm

Figure 2. One-percent borate electrophoresis (4 hr at 17 v/cm, 10") of 1?-acetylated p-A.P. before (upper strip) and after (middle strip) reduction with sodium borohydride (2.5 mg to about 0.25 pmole of disaccharide in 1 ml), "he latter was also hydrolyzed in 4 N HCl for 3 hrs at loo", followed by reIT-acetylation (lower strip). The peak of radioactivity (dashed line) indicates the migration distance of a known sample of N-acetyl-2-amino-2-deoxyl)-glucitol-l-14C. Known samples of N-acetyl-D-glucosamine (G~cNAc) zld Nacetylgalactosamine (GalNAc) were also included. acid

hydrolysis

of the N-acetylated-reduced

glucosamine, migrates

exactly

experiments mentioned limited which

a small

as a known

would

thus

disaccharides. by the

of unhydrolyzed sample

indicate

that

Attempts

the

was of the order

of 2-3 pmoles.

products

resulting

disaccharide,

B-A-P.

available

Incorporation from

is

at more definite

of disaccharide

of Glucosamine

yielded

mainly

N-acetyl-

and a product

that

of N-acetyl-2-amino-2-deoxyglucitol.

quantity

Mechanism soluble

amount

@-A.P.

into

the metabolism

89

a mixture methods from

These of the two above-

of analysis

each perfused

GlycoRen

were liver,

-- One of the

of galactosamine-l-

acid-

14C by rat

BIOCHEMICAL

Vol. 23, No. 1, 1966

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Transfer

of Glucosamine

II

from UDP-Hexosamine

to Glycogen

Reactions Complete

cpm

system

4,600

II

II

plus

0.025

,I

II

plus

0.25

pmole pmole

2,236

UDPG UDPG

460

The reaction mixture contained the following components in umoles: UDP1.80 x lo6 cpm/umole (galactosamine-glucosamine, 78:22), 0.015; hexosamine, glycine (pH 8.7), 12.5; glucose-6-P, 0.83; glycogen, 0.67 mg; UDPG as indicated; protein from partially purified glycogen synthetase (Friedman and Larner, 1963), 0.1 mg.; final volume, 0.16 ml. After incubation at 37O for 5 min, the reaction was stopped with 1 ml of 0.6 N perchloric acid. Glycogen The precipitation procedure (4 mg) was added, followed by 3 ml of ethanol. was repeated three times, the final precipitate being dissolved in 2 ml of water and aliquots taken for scintillation counting. liver

was shown by us (Maley

samine

and UDP-glucosamine,

since

been

tions:

established

with

that

and Maley,

1959),

in press)

the

former

to be a mixture

nucleotide

the UDP-galactosamine

UTP + galactosamine

(Maley

et al.,

predominating.

arises

is

It

by two enzymic

and UDPG + galactosamine-1-P. UDP-glucosamine

of UDP-galactohas reac-

As shown previously

a product

of the UDPGal-O-epimer-

ase reaction. On the synthetase vided

system

for

tention,

assumption

that

(Leloir

and Goldemberg,

the incorporation glycogen

synthetase

and Lamer

Cl4 -labeled

UDP-hexosamine.

had been

perfused

was partially

(1963)

with

of the

labeled

chromatography

revealed

The inhibition

of incorporation

from rat

was purified

in into

a ratio glycogen

followed

the radioactivity

of 78:22. in the by acid

from

by UDPG suggests

90

that

con-

as described

activity

with

a rat

liver

of a mixture

of

As seen in Table

in vitro hydrolysis

to be associated

be pro-

this

muscle

14C and consisted

galactosamine-l-

glycogen,

would

To test

glycogen.

The UDP-hexosamine

was incorporated

dialysis

into

transglucosylase

II,

in the glycogen

an explanation

for

and UDP-glucosamine

sive

reactive

purified

and assayed

UDP-galactosamine radioactivity

is

1960),

of glucosamine

by Friedman

that

the UDP-glucosamine

with

system.

Exten-

and paper glucosamine.

the same enzyme is capa-

BIOCHEMICAL

Vol. 23, No. 1, 1966

blr: of utilizing

both

nucleotides

The incorporation to the metabolic substitute

for

be impaired

if

storage

defect

co\,ered

is

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

as substrates.

of glucosamine

significance

into

of the

glycogen Since

reaction.

glucose-l-P

in the phosphorylase

glucosamine

units

may thus

currently

arise,

in the

are

questions

pertinent

glucosamine-1-P

cannot

reaction,

interspersed

but whether realm

raises

in

glycogenolysis

the glycogen.

such cases will

of speculation

may A glycogen

actually

be dis-

and experimentation.

SUMMARY 14 C was found

Galactosamine-las glucosamine was shown containing

to be glycosidically glucose

The incorporation for

following

perfusion

UDPG in the glycogen

or intraportal

linked

and glucosamine appears

to be incorporated

to glucose after

@-amylase

to be mediated synthetase

into injection.

by isolation treatment

by UDP-glucosamine,

rat

liver

glycogen

The glucosamine of a disaccharide of the which

glycogen. substitutes

reaction.

BIBLIOGRAPHY Del;iacco, R., and Maley, F., J. Biol. Chem., 239, PC2400 (1964). Dische, Z., and Borenfreund, E., .I. Biol. Chem., 184, 517 (1950). Foster, A. B., Martlew, E. F., and Stacey, M., Chem. Ind. (London), 825 (1953). Friedman, D. L., and Lamer, J., Biochemistry, 2, 669 (1963). Leloir, L. F., and Goldemberg, :S. H., J. Biol. Chem., 235, 919 (1960). Maley, F., and Maley, G. F., Biochim. Biophys. Acta, 3l, 577 (1959). J. F., and DelGiacco, R., Fed. Proc., in press (1966). Maley, F., McGarrahan, Pearson, C. H., Biochem. J., g, 540 (1963). Reissig, J. L., Strominger, J. L., and Leloir, L. F., J. Biol. Chem., 217, 959 (1955). I., Anal. Chem., 28, 1743 (1956). Roseman, S., and Daffner, Wolfrom, M. L., El Rhadem, H., and Vercellotti, J. R., J. Org. Chem., 2, 3284 (1964a). J. R., and Horton, D., J. Org. Chem., 29, 547 Wolfrom, M. L., Vercellotti, (1964b). Yosizawa, Z., Nature, 201, 926 (1964).

91