Octulose phosphates from the human red blood cell

~01.3,No.S

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OCTULOSE

PHOSPHATES Grant

R.

Scripps

Received

We have

per

been

by the red

in air

known

phosphate

eluted

This

sample

(1958)

and Schroeder

for

BLOOD

CELL1

Foundation

a spectrym

0 to 1 N pH 3.0

peak

was

preceded

(Fig.

in the red diphosphate

smaller

acid,

cell

under

from

1 -formate the large

1. Supported in part by Grant H-1071, Institutes of Health. 2. Generously supplied by Dr. N. H.

Racker

compound

Richtmyer.

by the

5-phosphate, of our

gradient

fructose

National

described

suspected,

the red cells

containing

monophos-

as did a

of this

with

of un-

reaction

the conditions

phosphorus

474

to that

and ribose

fraction

formate,

a peak

acid

During

of Dowex

and hexose

was

yield

diphosphate

of Dowex

ammonium

by two

a small

incubated

1959).

on a column

-sulfuric

were

and 40 pmoles

1) similar

8-phosphate

obtained

was

(Bartlett,

fraction

in the cysteine

on a column

from

ml of inosine

the sedoheptulose

Octulose

which

blood

0 to 1 N formic

gave

on fructose

the hexose

chromatographed

from

before

(1957)‘having

human

per

monophosphate

‘.

intermediates

previously

just

octose

present

When

National

described

octoses

of transaldolase

intermediates

RED

Bucolo

heparinized

15 pmoles

elution

material

of authentic

action

normal

of the sugar gradient

by Dische

when

methods

with

phate s.

and Giovanni

the carbohydrate

at 38O with

using

the separation 1-formate

HUMAN

Clinic and Research La Jolla, California

isolating

cell

for 4 hours

ml of Pi,

Bartlett

THE

19, 1960

October

formed

FROM

Nov. 1960

was

re-

elution diphosphate

peaks. Heart

experiment.

The Institute,

Vol.3,No.5

BIOCHEMICAL

middle

one proved

reported was

to be predominantly The first

separately).

in the position

(1958)

evidence

diphosphate To obtain

preparation

of octuloee

of dihydroxyacetone incubated 7.4

Tris

moval

for

color

eluted

When

an aliquot

at 38O.

with

of Dowex

from

strongly

The

the same

manner.

in acid

(Fig.

prepared

unknown

aldolase

color

in the same

duct

for

By analogy

of the acid

were

chromatographed acid

phosphate.

1.2 mmoles

5-phosphate

were

salts

chromatographed

of a phosphate

treated

giving

with

was

compounds

were

DPNH

and an equival-

5-phosphate

diphosphate

diphosphate.

in 340 rn).t ab-

phosphate

were

was

and re-

the octose

aldolase,

decrease

N pH

on a

of fructose

the compound

the monophosphate

0 to 1 N formic appeared

both

that

the

in 300 ml of 0.02

was

ribose

aldolase.

unknowns

of the barium

of dihydroxyacetone

when

obtained,

octulose

cleaved

with

hydrolyzed

I,8 -di aldolase

in

at the same

2).

with

and the products

aldolase

was

for

with

cell

of ribose

the expected

reaction

diphosphate

attempted.

of 0. 12 mmoles

diphosphate

Moreover

To prepare phate

cell

mmoles

mmoles

ahead

the conclusion red

was

the mixture

dehydrogenase

in the orcinol

phosphate.

rate

peak

of the octose

supporting

the red

and

Dische

incubated

precipitation

0.23

the reduction

ent increase

50,

1 -formate.

as a narrow

with

muscle

After

Dowex

and glycerolphosphate sorption

and 2.0

reaction

of octulose were

comparison

color

(to be

diphosphate.

and diphosphates

mono-

3 hours

an octulose

5-phosphate

for

diphosphate

the octose

the formation

80 mg of crystalline

of barium

column

for

phosphate

with

gave

for

and ribose standards

sedoheptulose

peak

to be expected

has presented

fructose

Nov. 1960

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

hydrolysis

hydrolyzed

for

on a column

elution.

with

120 pmoles

elution

54 pmoles position

the properties of octulose

1 hour

of Dowex

diphos-

in 1 N HCl

at 100°

1 -formate

using

giving

the octose

unknown

mono-

of a phosphate as the red

cell

of fructose

diphosphate

1,8-diphosphate 475

of the octulose

would

the pro-

be octulose

Vol.3, No.5

BIOCHEMICAL

8-phosphate.

Acid

were

consistent

and a stable

phate.

Surprisingly,

relatively might

hydrolysis

with

labile

AND BIOPHYSICAL RESEARCH COMMUNICATIONS curves

the view phosphate

group

Figurr

contained

the monophosphate curve

from (Fig.

(Fig.

2)

a relatively

and the monophosphate,

and its hydrolysis

be the octulose

compounds

that the diphosphate

however,

acid-labile

on the purified

Nov. 1960

a stable the red

phos-

cell was

2) indicated

that it

1 -phosphate.

I

Figure

0

OCT 1,8-P{

0

OCT I,8-P(ALDOLA8E)

2

RED CELL)

m OCT 8-P (ALDDLASE) 0 OCT I-P (ALOOLASE) 0

OCT I-P(RED

A

D-ERYTHRO-GALACTDDCTOSE ka I uM)

CELL)

I//

330

400

450

300

330

800

OCT 8-P

ALDOLASE

l

850

A (md

Fig. 1. Spectra acid reaction: 3% cysteine-HCl,

sulfuric 0.1 ml

Fig. at 1OOO.

2.

From some

octulose

ribose

Rate

of hydrolysis

the study

of Jones

1 -phosphate

and dihydroxyacetone

phosphate

and 200 pmoles

10 mg of crystalline ed with rated

of octulose phosphates (1.0 PM) in the cysteine1.5 ml sample, 4.5 ml 95% H2SO4, 3’ A 100°, 30’ p 60°.

ethanol, on a column

muscle the barium of Dowex

of. ochilose

and Sephton

would

be. formed

were

1 -formate

from

incubated The barium

aldolase. removed

(1960)

with

Dowex

by gradient

476

in 1.0 N HCl

it was

expected

the action

100 pmoles

phosphate. of ribose

phosphates

that

of aldolase

on

of dihydroxyacetone overnight salts

at 38O with

were

precipitat-

50 and the products elution

with

sepa-

0 to 1 N

Vol.3,No.5 formic

BIOCHEMICAL acid.

5 tJ,moles

correct

position

latively

labile

short red

for

pared

octulose

thesized

with

ethanol,

H2O (40:11:19).

to sucrose) sprayed

with

1960;

Jones

under

were

eluted

Its phosphorus

the rate

the same

slowed

re-

considerably migration.

as did the octulose

in the

was

The

cysteine-sulfuric

per

in animal

gave

acid

1 -phosphate

of potato

and diphosphates

pre-

three

gave

and the diphosphate

identical

the characteristic

and Sephton,

acid phosphatase

on paper

using

Rf values

crimson

a trichloroacetic-orcinol

spot

reagent

syn-

n-butanol, (1.2

compared

changing

(Charlson

to grey

and Richt-

1960).

of octulose experimental

mono-

and diphosphate

conditions

was

0.06

in the red

and 0.2

pmoles

ml of red cells.

as we know tissue.

(D -glycero -D -manno

is the first

above,

on the enzyme

synthesis

from Dische

(1958);

of several

diphosphate

and free

aldolase

and the intermediates of the red

Jones octuloses

pentoses. probably cell

octuloses

lose.

477

of the presence (1960)

avocado

of octulose,

fructose

report

and Richtmyer

-octulose)

quoted

the preparation

this

Charlson

the references

the configuration

by the action

chromatographed

The

the above

As far

cribed

curve

color

due to phosphate

gave

obtained

concentration

respectively

lose

perhaps

mono-

aldolase,

and each

The cells

although

hydrolysis

sugars

on the red cell

myer,

the octose

Nov.1960

aldolase.

The free

when

giving

monophosphate.

monophosphate

and acid

with

an octulose

hydrolysis,

octulose

spectrum

of a phosphate

to acid hydrolysis

of complete cell

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

isolated

and sedum. Racker

by the action

involved would

an octulose

In addition

and Schroeder

and Sephton

From

of octu-

the known it would

( 1960)

have

to (1957), des -

of aldolase

on

properties

of

be expected

be D-glycero-D-altro-octu

that

Vol. 3, No.5

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Nov. 1960

REFERENCES Bartlett, Bartlett, Charleon, Dische, Jones, Racker,

G. R., J. Biol. Chem., 234, 449 (1959). G. R. , J. Biol. Chem. , 234, 459 (1959). A. J. and Richtmyer, NT., J. Am. Chem. Sot., 282 3428, (1960). Z., Ann. N. Y. Acad. Sci., 2, 129 (1958). J. K. N. and Sephton, H. H. , Can. J. Chem. , 38, 753 (1960). E. and Schroeder, E., Arch. Biochem. BiophG., 266 241 (1957).

478