Ribonucleotide reductase activity in green algae

BIOCHEMICAL

Vol. 70, No. 3, 1976

RIBONUCLEOTIDE

Received

Chemie,

March

RESEARCH COMMUNICATIONS

REDUCTASE ACTIVITY

Wolfgang Fachbereich

AND BIOPHYSICAL

Feller

IN GREEN ALGAE

and Hartmut

Arbeitsgruppe 3550 Marburg,

Follmann

Biochemie Germany

der

Universitat

23,1976

SUMMARY. A ribonucleoside diphosphate reductase is demonstrated in the algae, Scenedesmus obli uus and Chlorella tEr;;;;d;,";; In synchronized cultures an----+ac ivity maximum at of the cell cycle coincides with maximum DNA production.Induction of reductase activity is prevented by cycloheximide. The enzyme requires dithiols for reduction of CDP in vitro; it is not significantly stimulated by iron or magnesium= nor dependent upon deoxyadenosylcobalamin. ATP stimulates the reaction but dATP or dTTP act as inhibitors. The ribonucleotide reductase enzyme characterof green algae differs from the Bq2 -requiring ized in Euglena gracilis.

INTRODUCTION The enzymatic

conversion

deoxyribonucleotides, has

(1,2)

reductase in

root

bean

of

callus

the

plant

enzymes

tide

reductases

in

lis

chartarum

Copyright All rights

in

but

low

is

(Vicia -the

amounts.

comparison

because

longa possess

(coenzyme eucaryotes.

(6,7,8),

0 1976 by Academic Press, inc. of reproduction in any form reserved.

germinating

of these

of

the Bq2) Thus,

as the

dif-

of

ribonucleo-

important

function

distribution

requirement the

soyis

knowledge

uncertain

reductases,

752

growing

and animal their

wheat,

enzymes

detailed

of

as well

B,2-dependent

Ribonucleotide

and in

bacterial

and because

in

study

to 2'-

reductases

faba),

A more

with

necessary

reduction

(9)

be demonstrated

(3,4,5)

proliferation

and Astasia

cells.

bean

of deoxyadenosylcobalamin nucleotide

in plant

broad

their

5r-phosphates

by ribonucleotide

studied

could

tissue

due to

cell

been

activity

ficult

in

catalysed

rarely

tips

of ribonucleoside

algae fungus while

for

ribo-

Euglena

praci-

Pithomyces deoxyade-

BIOCHEMICAL

Vol. 70, No. 3,1976

nosylcobalamin yeast

is

or the

Microalgae

higher offer

ductases

without

because cells,

and because

synchronous

many aspects

of nucleic

various

algal

scribe green

proliferation

species, has

some characteristics algae,

MATERIALS

a high

acid

Scenedesmus

of

leads

growth

is

of

obviously of

easily

the

formation

reof 8 or

of DNA precursors, achieved

have

and Chlorella

investigated pathway

neglected.

ribonucleotide

(lo).Although

been

reductive

been

obliquus

in

ribonucleotide to

supply

metabolism

study

reduction

above.

as a source

requiring

deoxyribonucleotides

on ribonucleotide

mentioned

themselves

16 daughter

in

effect

plants

cell

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to

We here

reduction

in

dethe

pyrenoidosa.

AND METHODS

Synchronised cultures of S.obliquus (strain D3) and C.Eyrenoidosa (strain 211-8b) were-derived from stock cultures maintained in the collection of Dr.H.Senger, Botany Department, Marburg. Cells were grown in a light thermostat in inorganic, aerated (3 % CO > media at 28'C essentially as described (lo); synchrony was 1-8 duced in a periodic light-dark regime of 14:lo hours. Algae were harvested at the desired time by centrifugation at 2,500 rpm. The cells (750 - 1000 fil packed cell volume) were suspended in 1.5 ml of potassium phosphate buffer (0.05 M, pH 7.5) containing 5 mM mercaptoethanol; the mixture was frozen in liquid nitrogen and ruptured in a high-frequency disinte rator (Micro-Dismembrator, Braun-Instrumente, Melsungen, Germany ti using a Teflon capsule and steel balls.The homogenate was diluted with buffer (2 ml) and centrifuged for 30 min at 30,000 x g. Unless otherwise stated, the green supernatant was directly used for enzyme assays. Nucleotides and other chemicals were of highest purity available, deoxyadenosylcobalamin was a gift of Dr.H.P.C.Hogenkamp, Iowa City, U.S.A. Solutions of the radioactive substrate, CDP (Amersham; specific activity, 23 Ci/mmole) had to be freed from ethanol prior to use by evaporation --in vacua. Ribonucleotfde3reductase assays contained, in a total volume of 0.3 ml: L5- H]cytidine diphosphate, lo Ci; lo mM dithiothreitol; 3.5 mM ATP; 5 mM Mg++; 0.06 mM Pe++/Fe+++; and 0.2 ml of an enzyme extract; final pH, 7.2. After incubation for 1 hr at 3o°C the reaction was terminated by addition of 1 ml perchloric acid, nucleotides were h drolysed to the monophosphates and separated on Dowex 50 (Hf 4; columns (11). The eluted fractions corresponding to CMP and dCMP were pooled, and radioactivity was determined in a liquid scintillation counter. Although under the above conditions the substrate (CDP) concentration was not saturating and specific enzyme ac-

753

BIOCHEMICAL

Vol. 70, No. 3, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2600

200

600 700 600

_ P

0.3-

?%

500

_

f

400 300

_

s I0

::

f 2

0

I ‘

I 6

t 10

1 6

I 12

Figure 1. a. Development of cell ( W ) and cell number ( A ) in a , growing in Scenedesmus obliauus hrs). b. Ribonucleotide reductase increase of DNA content ( 0 ) (12)

tivities formation to better

cycle in 6th

I 20

I 22

200

_

100 .

24

time [hours]

volume (pcv, logarithmic) synchronous culture of light-dark regime (14:lo activity ( A ) and relative in the same culture.

of product reproducible

AND DISCUSSION

The green

algae

degree

course

t 16

could not be obtained, relative values (dCMP, based upon unreacted CMP) were than + lo %.

RESULTS

good

t 16

14

_

of

of cell

shown in

extracts and 16th

of

used cell

in

this

study

synchrony

can be cultured

(lo),

as is

volume

and cell

Figure

1, a. Ribonucleotide

Scenedesmus

hour

and is

number

obliauus seen

to rise

754

with

a very

evident

from

the

the

24-hr

cell

during

reductase

was measured sharply

from

activity

between low

the levels

time

Vol. 70, No. 3, 1976

Table

Ribonucleotide

1.

Source

BIOCHEMICAL

and assay

Chlorella Standard culture

AND BIOPHYSICAL

reductase

RESEARCH COMMUNICATIONS

activity

conditions

in

algae

Relative

activity

pvrenoidosa culture, standard assay* + cycloheximide (lo ,uM)

(96)

loo 18

Scenedesmus obliquus Standard assay* - r'H]CDP, + [3H ] Cm or L3~]C~P b - dithiothreitol - dithiothreitol, + NADPH (5 mM) - Fe++/Fe+'+ - Mg++ + + +

extracts

loo -= lo 44 75 105 97 67

ATP ATP, -Mg++ hydroxyurea (12 mM) dATP (3.5 mM), - ATP dTTP (3.5 mM), - ATP

38 61 8 15

* Absolute values for standard assay (see Materials and Methods): 4.7 pmoles dCMP/mg protein with Chlorella extract, 2.0 pmoles dCMP/mg protein with Scenedesmus extract; approximately 5 % product formed from labeled ribonucleotide substrate. to a maximum This

at

activity

related

vity

is

12th

peak

DNA synthesis ly

the

coincides

during

the

species, also

present

chloroplast

DNA occurs

hour, more pendent

nuclear

Cycloheximide, reduced than

the

For further

with In

(12).

12th

during

12th

hour.

the

characterisation

extracts

the

whole to

indicating result

the

that

the 755

are

cycle

culture

at

reductase

of --de novo of

reductase

production

cell

the

cell

enzyme

reductase,

rate

of the

The enzymes since

(Fig.l,b).

maximum

high

hr-ribonucleotide I),

the

pyrenoidosa,

when added

is

by a decline

DNA synthesis

80 % (Table increase

S phase

at the

for

followed precisely

Chlorella

responsible

algae.

hour,

of

closeacti-

clearly of

in

these

the

9th

activity

by

cycle-desynthesis.

Scenedesmus

cells

Vol. 70, No. 3,1976

harvested

BIOCHEMICAL

at the

soluble

12th

protein

because

supernatant

of

diphosphate

reductase

servation

cell

better

reduction

than

(Table

1).

However,

the

substrate

The time

most

centration mulate

of

contrast,

rapid

at

as an affinity

lution

was free

dependent

upon

Dithiols (Table ce of (at

such 1); enzyme

5-10

extracts existence

is

CDP but

not

lipoate

as yet

reduction coli.

supports

and the

independently:

off

(product)

to light

initial

formaCDP con-

did

not

had no effect. by partial

stiBoth

purification

on P 3-(6 -aminohex-l-yl)dATP-Sephaafter

(13),

which

weight

upon

the

coenzyme

serve

as reductants

much less

dithiothreitol

similar

to the

dithiol

reductant addition

756

--in vitro dependen-

requirement tissues in

donor

and

the

of NADPH to

reduction, hydrogen

B12.

has a maximum

in mammalian

ribonucleotide

and was only

efficient.The

added

observed

enzyme so-

colpaunds

added

however,

of a thioredoxin-like

during

leveling

(259)

The physiological

unknown;

CTP)

chromatographical-

when the

confirmed

is

upon

mM concentration)

also

time

as dithiothreitol

Escherichia

algae

an earlier

low-molecular

activity

(CMP,

activities,

deoxyribonucleotide

adsorbent

of ribonucleotide

(followed

ob-

much

as an effector

of monophosphate

reductase

reduced

the

yields

was confirmed

and exposure

from

from

and phosphatase

level

g

a ribonucleoside

(CDP)

added

a

a 1oo,ooox

or triphosphates

kinase

were also

rose

in

follows

Deoxyadenosylcobalamin

Scenedesmus

found

is

CDP, CMP, and CTP concentrations

in

reaction,

enzyme

likely

this

ATP is

increase

specificities

of the

most

CDP as substrate

is high. the

these

in

both

dependence

is

is

mono-

because

with

is

diphosphate

phosphorylation

30 min;

tion

It

do the

shows an initial

after

activity

cytidine

contains

an incubation ly)

total

The algal

(EC 1.17.4.1);

labeled

extract

were used.

extracts.

that

cell

hour

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

green crude

suggesting system.

the

Whether

BIOCHEMICAL

Vol. 70, No. 3, 1976

mercaptoethanol after

further

The metal extracts

alone

requirement differs

reductase

content

an iron assay.

containing

(Table

1).

if

Ribonucleotide

ribonucleoside purified

as low as 0.03 @f in the the algal

enzyme is an iron-

effecters

of all

bound. ribonucleotide

enzymes make no excepof 3.5 mM, stimulates inhibifor

the

the action

in some organisms.

organism,

in green algae obviously Euglena gracilis

algae

differs

is well

from

known as a

and a deoxyadenosylcobalamin-dependent

triphosphate

Scenedesmus obliouus

tightly

and mammalian enzymes while

reduction

from this

mg/g)

compound is also inhibitory

of the Euglenophyta.

B,2-requiring

the (14).

but dTTP and dATP are strong

The latter

yeast, wheat, --E.coli, of dTTP is stimulatory

that

(0.14-0.19

algae

of

may explain

of dried

ATP, at an optimum concentration

tors

any effect.Addi-

in Chlorella

and the plant

the Scenedesmus reductase

many bacteri-

as is typical

the metal must be very

known so far,

in that

by hydroxyurea

act as allosteric

reductases

if

is inhibitory

concentration

Therefore,

protein

Nucleotides

for

enzymes. This observation iron

in the algae

organisms

have little,

however,

of DNA synthesis

The endogenous

reduction

of most other

reductases

the iron-containing

would yield

to be established

ions which are stimulatory

of hydroxyurea,

inhibition

remains

of ribonucleotide

from that

al and eucaryotic

tion.

is a reductant

purification.

magnesium and iron tion

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

reductase (6,8).

(EC 1.17.4.2)

In contrast

also contains

to higher

considerable

has been plants,

quantities

of

reductase does not depend vitamin B,2 (15) but its diphosphate upon B,2 coenzyme. Judging from our present data the enzyme is similar

to the ones found

in wheat or broad bean.This

757

dissimi-

BIOCHEMICAL

Vol. 70, No. 3, 1976

larity

between

with

the

outside

proposed the

main

The results of

ribonucleotide

and fall tion

of

with

longer

line

above

reductase cerevisiae enzyme

generation

time.

of ribonucleotide

eucaryotic

bear

(16).

It

latter

resemblance

in

accord

on a branch

to the

synchronous However

during are

of the

is

evolution.

in

activity

and Euglena

place

of plant

DNA synthesis

role

or Chlorella

phylogenetic

described

Saccharomvces

key

Scenedesmus

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in

the

even more

cultures

distinct

justified

reduction

for

of yeast,

Scenedesmus

S phaee

appears

induction

to cell

the

rise

and its

correla-

despite

the

much

the

same

expect

division

in

all

cells.

ACKNOWLEDGEMENTS. We are greatly indebted to Dr.H.Senger and his staff at the Botany Department, University of Marburg, for providing advice and culture facilities. This work is supported by Deutsche Forschungsgemeinschaft, SFB 103. REFERENCES I. :: 4. 5. 6.

Hogenkamp,H.P.C. and Sand0,G.N. (1974) in: Structure and Bonding vo1.2o,pp.23-58; Springer-Verlag,Heidelberg Follmann,H. (1974) Angew.Ch emie Int.Ed. 13, 569-579 Miiller,H., Wahl,R., Kuntz,I. and Follmann,H. (1973) Hoppe-Seyler's Z.Physiol.Chem. 354, 1299-1303 (1976) unpublished results Hovemann,B. and Follmann,H. Cowles,J.R. and Guevara,J.G. (1973) Plant Physiol. 51 (Suppl.) 24 Gleason,F.K. and Hogenkamp,H.P.C. (1970) J.Biol.Chem. 245,

4894-4899

Gleason,F.K. and Hogenkamp,H.P.C. (1972) Biochim.Biophys. Acta 277, 466-470 Hamilton,F.D. (1974) J.Biol.Chem. 249, 4428-4434 ;: Stutzenberger,F. (1974) J.Gen.Microbiol. 31 501-503 lo. Pfau,J., Werthmiiller,K. and Senger,H. (1971) Arch. Mikrobiol. 75, 338-345 11. Reichard,P. (1958) Acta Chem.Scand. 12, 2048 12. Premer,S. and Senger,H. (1975) unpublished; Senger,H.and Bishop,N.I. in: The Cell Cycle, pp.179-202; Academic Press, New York 13. Pries,M. and Follmann,H. (1976) unpublished results 14. Wanka,F., Moors,J., and Krijzer,F.N.C. (1972) Biochim. Biophys.Acta 269, 153-161 15. Fink H., Herold,E. and Lundin,H. (1958) Z.Naturf. 13 b,

7.

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Lowdon,M. 158,

and Vitols,E.

(1973)

177-184

75%

Arch.Biochem.Biophys.