Isolation of cytidylyl-(5′ → 3′)-adenosine-5′ phosphate (pApC), adenylyl-(5′ → 3′)-cytidine-5′ phosphate (pCpA), uridylyl-(5′ → 3′)-adenosine-5′ phosphate (pApU) and adenylyl-(5′ → 3′)-uridine-5′ phosphate (pUpA) from embryos of the copepod Euchaeta japonica Marukawa

BIOCHEMICAL

Vol. 26, No. 5, 1967

Isolation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of Cytidylyl-(5'+3')-adenosine-5'

Adenylyl-(5'+3')-cytidine-5'

Phosphate

(5'+3*)-adenosine-5'

Phosphate

uridine-5'

(pUpA)

Phosphate japonica

Euchaeta

Department

of British

ReceivedJanuary80,

Marukawa reports

of the

(Hepner

ribonucleotides

(numbering

is

and Michael

SmithX

Faculty

Copepod

of Medicine Vancouver

8,

Canada

shown that

marine

soluble

pCpA,

cm) using

The present

from

communication of

were extracted

a linearly pH 8.0,

and Smith,

1966).

+ Holder

of National August 24,

Research 1966. Associate

japonica

the

di-

5 gm of embryos

of diethylaminoethyl

(Oikawa

x Medical Research of Canada.

Euchaeta

of

pApU and pUpA.

50,000)

(45 cm x 1.2

nucleotide

same organism,

nucleotides

approximately

bicarbonate,

the

soluble

copepod

1967).

from

pApC,

adenylyl-(5'+3')-adenosine-

acid

calanoid

and Smith,

on a column

Died,

of the

the major

fractionated

ammonium

Embryos

Columbia,

isolation,

The acid

and Adenylyl-(5'+3')-

Columbia,

been

(pApA)

the

Uridylyl-

1967

has recently

embryos

from

Hepner+

British

the

(pApU)

of Biochemistry,

University

5' phosphate

(pCpA),

(pApC),

Marukawa

A. Sheri

It

Phosphate

cellulose

increasing to elute

The elution Council of the

584

and then

the

concentration nucleotides

profile

is

of Canada Medical

shown in

Bursary.

Research

Council

of

Vol. 26, No. 5, 1967

Figure

1.

ammonium Peak

The major

7, eluted

After

M ammonium

acid:

su1fate:O.l

as judged

M phosphate,

1.

Euchaeta

Separation japonica

cellulose at

20 minute

(approx.

peak

9, pApU (approx.

pApA (approx. contained peaks

5 vmoles);

25 pmoles;

guanosine-5'

6 and 10 have not

by paper

262 mu. the

chromatography

(5:3)

and ammonium

(30 gm:50ml:l

soluble

cm).

nucleotides

ml).

Peaks

(20 ml)

were collected

1 to 5 contained phosphates.

peak

from

of diethylaminoethyl

Fractions

5' and deoxyribonucleoside-5' p~pc

at

a

NUMBER

(5 gm) on a column

intervals.

1967).

cellulose,

pH 6.8:n-propanol

of acid

(45 cm x 1.2

M

contained

in water

hydroxide

FRACTION

Figure

bicarbonate,

maximum

M ammonium

by 0.12

and Smith,

on diethylaminoethyl

was homogeneous

isobutyric

RESEARCH COMMUNICATIONS

was eluted

9 (Hepner

an absorption

rechromatography

nucleotide

pApA,

in peak

by 0.11

with

AND BIOPHYSKAL

component,

bicarbonate

nucleotide

in

BIOCHEMICAL

Peak

8, pCpA (approx.

1 pmole),

pUpA (approx.

Hepner triphosphate.

and Smith,

7 contained 5 pmoles);

1 vmole) 1967).

The contents

been characterized.

585

ribonucleoside-

Peak of

and 11

Vol. 26, No. 5, 1967

The ratio to total

BIOCHEMICAL

of phosphate phosphate

AND BlOfflYSlCAL

liberated

(Ames,

1966)

was 0.9:2.0:1.0.

The nucleotide

phosphodiesterase

I from

1963)

to yield

equimolar

and cytidine-5' periodate yielded 3',5'

1966)

nucleotide identical consistent 5' phosphate

cytidine

amounts. with with

E.

the

(pApC);

of the phosphatase

1.

OH

586

and

diphosphate

reaction

of the

was chromatographicall! These

being

HO

1964)

T2 (Uchida

and adenosine-3',5'

nucleotide formula

phosphate sodium

ribonuclease

alkaline

(Razzell,

and one of adenosine-

cytidylyl-(5'+3')-adenosine.

with

with

by

(Neu and Heppel,

The product

coZi

venom

of adenosine-5'

of lysine

1957)

degraded

amounts

Degradation

phosphatase

(Ashwell,

was rapidly

Similarly,

yielded

equivalent

to pentose

of cytosine

diphosphate.

alkaline

adamanteus

presence

one equivalent

Egami, in

the

coZi

Crotalus

phosphate.

in

by E.

RESEARCH COMMUNICATIONS

data

cytidylyl-(5'+3')-adenosine-

are

BIOCHEMICAL

Vol. 26, No. 51967

Peak further

8, eluted purified

cellulose

degraded

by 0.115

M ammonium

by rechromatography

and by paper

M ammonium

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

hydroxide

(5:3).

This

phosphate

and cytidine-5'

with

periodate

and lysine

of

adenine

and a nucleotide

cytidine-3'

,5'

alkaline identical are

with

consistent

cytidine-5'

yield

in

phosphate

the

a product

587

Degradation amounts

expected

of

yielded

the

T2

amounts.

(CPA).

being

formula

HO

of

E.

co-ii

chromatographically

nucleotide (pCpA);

amounts

properties

equal

:

was also

equimolar

adenylyl-(5'+3')-cytidine with

acid

phosphate.

Ribonuclease

and adenosine

phosphate

isobutyric

nucleotide

yielded

with

diphosphate.

same nucleotide

in

I to equimolar

adenosine-5

was

on diethylaminoethyl

chromatography

by phosphodiesterase

sodium

bicarbonate,

dH

2.

These

adenylyl-(5'+3*)-

data

BlOCHEMlCAL

Vol. 26, No. 5, 1967

The early as in

the

with

fractions

of peak

purification

maximum

by phosphodiesterase

3',5'

I yielded

equal

phosphate

uridine-3' the

amounts

phosphate.

nucleotide

containing

in

pUpA,

pApC,

amounts

1 umole;

200 mpmoles.

embryos formed However, cannot

It

are others from

the

they

the

with data

in

of

are consistent

(pUpA)

the

five

Possible

roles

of organic synthesis

this

connection

it

pUpC can promote

is the

transfer-RNA

phosphates. this

than

study

in Euchaeta

type

are not

of molecule

and Smith, that

binding

and

50 mpmoles/gm.

function

this

the

can be

or as regulators

of interest

(Rottman

in

which

dinucleotides

for

200 m~moles;

present

in

biological

are pApA,

pApU,

that

greater

(Hepner

specific

of

and uridylyl-

embryos

1 umole;

phosphate

or protein

amounts

dinucleotides

16 dinucleotides

and their

of

with

equimolar

japonica

the

expected

(pApU). of

amounts

yielded

adenosine-

properties

been detected

acid

Serine

adenosine,

is possible

have not

embryos

storage

of uridine,

4 common ribonucleoside-5'

The biosynthesis

understood.

T,

pCpA,

of the

be present

japonica

Ribonuclease

approximately

1 gm of Euchaeta

5 umoles;

phosphate.

phosphate

The approximate

Degradation of adenosine-

phosphate

(5'+3')-adenosine-5'

material

amounts

These

adenylyl-(5'+3')-uridine-5'

present

equimolar

and a nucleotide

,5'

nucleotide

at 260 rnp in water.

and uridine-5'

approximately

9 were rechromatographed

of pCpA to yield

an absorption

5' phosphate

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

lay

in

of nucleic

1967).

In

dinucleotide

to ribosomes

and Nirenberg,

yet

of

1966).

Acknowledgement This Council

investigation

was supported

of Canada. 588

by the

Medical

Research

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 26, No. 5, 1967

References Ames,

B. N.

(1966).

Methods

and V. Ginsburg, Ashwell,

G.

Vol.

in Enzymology,

and N. 0. Kaplan,

Vol.

in Enzymology, 3, p.

73.

M.

(1967).

In press.

Oikawa,

T. G. and Smith,

M.

(1966).

Biochemistry,

(1963).

S. P. Colowick New York: Rottman, Uchida,

Methods

and N. 0. Kaplan,

Academic

T. and Egami,

New York:

ed.

M.

Vol.

F.

(1966).

(1966).

by G. L. Cantoni

Harper

Academic

Press.

2, 1517. ed.

6, p.

by 236.

Press.

F. and Nirenberg,

Research,

in Enzymology,

Press.

by S. P. Colowick

New York:

A. S. and Smith,

W. E.

Academic

ed.

Hepner,

Razzell,

by E. F. Neufeld

New York:

8, p. 115.

Methods

(1957).

ed.

and Row.

589

J. Mol.

Procedures

Biol.

2,

in Nucleic

and D. R. Davies,

555. Acid

p.

46.