2-Keto-3-deoxy-octonate, a constituent of cell wall lipopolysaccharide preparations obtained from Escherichiacoli

Vol. 10, No. 4, 1963

BIOCHEMICAL

2-KETO-3-DEOXY-OCTONATE,

A CONSTITUENT

PREPARATIONS Edward Rackham Arthritis

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

OF CELL WALL LIPOPOLYSACCHARIDE

OBTAINED FROM ESCHERICHIA s*

C. Heath**

and Mohammad Ali

Ghalambor

Research University

Unit and Department of Bacteriology, of Michigan, Ann Arbor

this

paper

is

acid

(Compound

The

January 25,1963

Received

The purpose

of

2-keto-3-deoxy-onic in preparations --ichia

colt

of cell 0111-B4

niques,

purified

an enzyme

of Pseudomonas

product

Materials commercial

was a gift

+ Pi.

except

All

from

the

studies,

Compound

compounds

Dr.

Saul

3-deoxy-D-arabino-heptulosonate Dr.

compound

was converted

phatase,

and

David

B. Sprinson,

to the

the dephosphorylated

following

potassium product

salt,

techL is

and Racker from

in(1959)

extracts

D-arabinoseto the dephos-

and Racker. were

Crystalline Roseman of T-phosphate

Columbia

(Heath

reaction:

and enzymes

below.

from

Levin

Escher-

J-5

calorimetric

to be identical

by Levin

as indicated

E. coli

synthetase)

I appears

obtained

from

by specific

(KDO).

catalyzed

(KDG) was a gift

barium

degradation

of a

constituent

obtained

organism,

obtained

Compound

and Methods---

deoxy-gluconate Synthetic

which

of KDO-8-p

sources

of this

(2-keto-3-deoxy-phospho-octonate

aeruginosa

the occurrence bound

(LPS)

2-keto-3-deoxy-octonate

5-P + PEP + KDO-8-P phorylated

a mutant

and chemical

from

for

as a glycosidically

Based on results

chromatography,

distingufshable

I)

evidence

lipopolysaccharide

and from

1962).

and Elbein,

wall

to pres.ent

University, treated

with

(KDH) was isolated

obtained calcium

this

from 2-keto-j-

University. (Sprinson,

New York. potato

acid

1960) This phos-

by ion-exchange

* The Rackham Arthritis Research Unit is supported by a grant from the Horace H. Rackham School of Graduate Studies of The University of Michigan. This investigation was supported by grants from the National Institutes of Health (A-2963) and from the Michigan Chapter, Arthritfs and Rheumatism Foundation. * Research Career Development Awardee, U.S. Public Health Service. 340

Vol. 10, No. 4, 1963 and charcoal

BIOCHEMICAL

chromatography

KDC was prepared

sulfate,

the fraction

tained

was inactive

or D-arabinose

were

fraction

with

material

formed

extract

precipitated

was isolated

relatively

crude

talline

as substrates.

and Racker,

was fractionated

D-glucosamfne-6-P

University) (1955),

followed

and precipitation arabinose-5-P

obtained

4-P was a gift

1952);

extraction (California with

formed

up*

for

acid

Biochem.

(Csborn,

according (TBA)

methods:

method

paper

employing

acetic

water

(8:l:l);

saturated

the detection

1962).

phenol;

the

LI, n-butanol,

pyridine,

on paper

341

chromatograms

D-Brythrose-

(Westphal, ether

with

Pronaee

by precipitaof LPS was per-

(1959)

as modified

was performed systems: acid, 0.1

of

The thiobarbituric

and Saslaw

acetic

resin

1933);

followed

(1956).

solvent

this

of Pontis

cells

degradation

following acetate,

of

crys-

Berkeley.

et al.,

Los Angeles),

chromatography

this

from

extraction

(Boivin,

Paper

I& ethyl

of compounds

phenol

--et al.

in

yield

804.

of acetone-dried

of Waravdekar

(1959).

The overall

of California,

Alkaline

of Neter,

used was that

No. 1 filter

5, water

Research,

to the method

and Hurwitz

acid,

digestion

et al.,

by Weissbach

extraction

reactive

on ion-exchange

was approximately

by the following

The

as the phos-

to the method

ethanol.

University

1959);

I)

C. W. Jourdian,

phosphate

C. E. Ballou,

et al.,

Corp.

with

procedure

trichloracetic

(Ribi,

salt

in this

from Dr.

LPS was prepared et al.,

of the pentose

of the barium

Compound

according

con-

acid

was prepared

ninhydrin

by isolation

for

by Dr.

ammonium

protein

D-Arabinose-5-P supplied

with

this

of phosphatases

(kindly

by degradation

of

due to the presence

enzyme preparation.

treat-

activity.

thiobarbituric

below

after

1.

D-erythrose-4-P,

incubation

80% of the

with

and 70% saturation

50

D-ribose-5-P,

After

1959)

with

synthetase

(as described

presumably

of Compound

as follows:

between

PEP and D-arabinose-5-P,

compound,

for

the isolation

obtained

when PEP and either

used

phate-free

acid

Olll-B4

the crude

which

for

most of the 2-keto-3-deoxy-phospho-octonate

preparation

tion

below

and PEP (Levin

from -E. coli

protamine

sulfate;

as described

from D-arabinose-5-P

an enzyme preparation ment with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A, 2-butanone, water

(3:1:3);

N HCl (5:3:2). were

on Wbatman

as follows:

Methods 2-keto-

Vol. 10, No. 4, 1963 3-deoxy-onic method

BIOCHEMICAL

acids

by the method

of Cordon, Isolation

solved

hydrolysate

--et al.

I from

LPS---

N H2SO4 and heated

was neutralized

centrifuged,

resin

in a boiling

mesh).

acid

material

in a symmetrical

fractions

compound mesh,

were

Pittsburgh

ammonia

purified

ft was nitrogen

All mutant) from

of

and phosphorus

free

the LPS samples

ially

Compound

I contained

(100

precipitated

Compound carbohydrate cated

examined,

enzyme

electrophoresis

that

volts/cm,

to polymer)

Compound

LPS preparations in rapid

could

constituents

(Pronase) in not

with

with

eluate

Dowex-50-e

preparation

indicated

that

the phosphatase-treated fsolated

and complete removal

contained

Compound

did

of these not

be separated 2 hours).

with

by this

pro-

(parent

or

from

alter

located

those

exchange

resin

I before

the liberation

part-

antiserum, as other

studies

thus,

(Painter,

paper

were

same degree Preliminary

comp-

treated

voltage

of specific

in the polymer;

alkali

their

When LPS preparations

such as colitose.

ranging

with

LPS by high

to the

342

in amounts

(including

was precipitated

of Compound

I,

significantly

quantities

cation

source

preparations

limiting

terminally

soluble

of their

the preparations

for

of LPS,

I is

methanolic

The charcoal

treated this

The

CAL, 12 x 40

with

MI4OH, 3).

of

and the

24 hours.

(Type

regardless

Treatment

by treatment

I (bound

for

KDO preparations

of preparation,

or Pronase)

acid

and phosphorus).

a proteolytic

alkali

(0.2

by elution

(Similarly,

synthesized

1 to 4s of the dry weight.

osition.

ether

in water,

Analysis

free.

of nitrogen

or the method

or with

with

KOW.

concentrations The TBA-reactive

on charcoal

followed

solution, of Dowex-

N formic

with

50; cont.

dissolved

with

KDH and the enzymatically were

extracted

0.5

The

to a column

the column.

peak with

each dis-

10 min.

Ba(OH)2

of increasing

through

Co.),

for

was applied

by adsorption

to dryness,

and neutralized

cedure

passed

(MeOH, 47; water,

was concentrated resin,

were

Coke and Carbon

solution

by the

were

bath

of saturated

fluid

continuously

was further

water

Solutions

to 0.5 N) of formic

pooled

2-deoxy-aldoses

The LPS preparations

supernatant

(200&C

was eluted

(1960);

by the addition

and the clear

1-formate

of Warren

RESEARCH COMMUNICATIONS

(1956).

of Compound

in 0.1

AND BIOPHYSICAL

indi-

hydrolysis

1960)

resulted of any of

of

Vol. 10, No. 4, 1963 the other

BIOCHEMICAL

sugar

components

Compound

1 and polymer

lowing:

acid

group

to reduction

lability;

alkaline with

(Weissbach

tion

was obtained

with

semicarbazide

(MacCee

typical

and Doudoroff,

Analysis

those

Waravdekar

a-keto

with

an absorption

KDO, KDH, and KDC indicated KDO (Table

by oxidative cedure

NaBH4, followed aldoses

ranged

obtained

with

1959);

NaBH4.

maximum at 250 w. systems

that

distinguish

identically

1

Chromatography

of Compound

in

I

Solvent

Systems C

D

I

.56

.70

059

.51

KDO

.56

.70

- 58

.52

KDH

.79

-85

.81

.71

KDG

1.00

1.00

1.00

1.00

by oxidation from

was sought

to establish

to the corresponding of Compound with

ceric

absorbs

the

identity

sulfate.

by the TBA method alkali

I

The pro-

compounds

The yields

at 532 mn and is 343

of Compound

2-deoxy-aldose.

I and the reference

30 to 40% as determined

2-deoxy-aldosea

between to enzymat-

B

reduction

I reacted

derivative

A

evidence

no TBA reac-

Compound

semicarbazone

I migrated

&)G

decarboxylation

involved

2-keto-3-deoxy-onic

1).

Compound

Additional

with

with acid

Compound

Table Paper

fol-

I by the TBA meth-

and Saalaw,

of solvent

that

of the

of the carbonyl

of Compound

obtained

a typical

1954)

on the basis

hydrolysis.

I was reduced

in a variety

synthesized

I---

1959;

when Compound

of the bond between

and resistance

to acid

of

and Hurwitz,

Chromatography

ically

stability;

of Compound

to give

The nature to be glycoaidic

NaBH4 prior

a chromogen

acids

was detected.

was concluded

Characterization od produced

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

of 2-deoxy(the

chromogen

stable).

Two of

Vol. 10, No. 4, 1963

BIOCHEMICAL

the products were further KDG) calorimetrically

AND BIOPHYSICAL

characterized

RESEARCH COMMUNICATIONS

as follows:

(1) 2-deoxyribcae

(Dische, 1930); (2) 2-deoxy-D-glucose

glucose oxidase (Sol6 and Fuente, 1957).

(from

(from KDH) with

Paper chromatography of the oxidation

products obtained from CompoundI and the reference compoundsgave the results shown in Table 2, and indicate

that CompoundI is,

indeed, KDO.

Table 2 Paper Chromatography of Degradation Products*

Degradation Product of 2-Deoxy Ribose

Solvent System

2-Deoxy Glucose camp. I

KDO

KDH

KDG

A

1.00

.65

.37

.36

.64

.97

B

1.00

.75

.54

.54

.75

.98

* Mobilities

expressed as R+deoxyfibose

All of the evidence listed

above, and that presented in the accompanying

paper, are consistent with the conclusion that CompoundI is identical KDOoriginally

obtained by Levin and Racker (1959).

that stereoisomers of KDOwere not available

to the

We should note, however,

for examination,

and it

is there-

fore conceivable that CompoundI is such an isomer. Discussion---

The evidence presented in this paper clearly

the occurrence of 2-keto-3-deoxy-octonate uent of cell wall lipopolysaccharide 0111-B4 and of g. calf material

J-5.

as a glycosidically

preparations

This compoundmay be similar

We have also observed (by calorimetric

of a nitrogen-free,

KDG-like substance(s) in a variety

Salmonella typhi-murium,

2. typhosa, and 2. adelafde. 344

bound constit-

obtained from -K. coli to the TBA-reactive

observed by Sundararajan, -et al. (1962) in a variety

g. coli K-12.

establishes

of strains of

analyses) the presence of strains of E. &, It

is of interest

to

Vol. 10, No. 4, 1963

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

note that Osborn" has observed the presence of a KDO-like compoundin oligosaccharides obtained by mild acid hydrolysis The previously ed lipid,

described constituents

of LPS from e. coli

phosphorus, hexosamine, heptose, glucose, galactose,

(Westphal and Luderitz,

1960).

that KDOis indeed, an integral proof is necessary to definitively the criteria

of LPS from 5. typhi-murium.

While all

0111-84 includand colitose

of the data presented above suggest

component of LPS of these organisms, further establish

this point.

presented above rule out the possibility

For instance, none of

that a KDO-containing

substance, other than LPS, may be present in these preparations.

BIBLIOGRAPHY Boivin, A., Mesrobeanu, I., and Mesrobeanu, L., Compt. rend. Sot. Biol., 114, 307 (1933). Dische, Z., Mikrochemie, 8, 4 (1930). Gordon, H. T., Thornburg, W., and Werum, L. N., Anal. Chem., g, 849 (1956). Heath, E. C. and Elbein, A. D., Proc. Nat'l. Acad. Sci., 48, 1209 (1962). Levin, D. H. and Racker, E., J. Biol. Chem., 2& 2532 (1959). MacGee, J. and Doudoroff, M., J. Biol. Chem., 210, 617 (1954). Neter, E., Westphal, O., Luderitz, O., Gorsynski, E. A., and Richenberger, E., J. Inrmunol., @. 377 (1956). Osborn, M. J., Rosen, S. M., Rothfield, L., and Horecker, B. L., Proc. Nat'l. Acad. Sci., 48, 1831 (1962). Painter, T. J., Chem. Ind., 1214 (1960). Pontis, H. G., J. Biol. Chem., 216, 195 (1955). Ribi, E., Milner, K. C., and Perrine, T. D., J. Immunol., 82, 75 (1959). Sols, A. and Fuente, G., Biochim. et Biophys. Acta, 3, 20-6(1957). Sprinson, D. B., in Advances in Carbohydrate Chemistry, edited by M. L. Wolfrom, Academic Press, 5, 235 (1960). Sundararajan, T. A., Rapin, A. M. C., and Kalckar, H. M., Proc. Nat'l. Acad. sci., g, 2187 (1962). Waravdekar, V. S. and Saslaw, L. D., J. Biol. Chem., 234, 1945 (1959). Warren, L., Nature, 186, 237 (1960). Weissbach, A. and Hurwitz, J., J. Biol. Chem., 234, 705 (1959). Westphal, 0. and Luderitz, O., Angew. Chemie, 72, 881 (1960). Westphal, O., Luderitz, 0.. and Bister, F., 2. Naturforsch., E, 148 (1952).

* Private York.

communication from Dr. M. J. Osborn, NewYork University,

345

New