Photochemical activities of a particle fraction P1 obtained from the green alga Chlorella fusca

BIOCHEMICAL

Vol. 49, No. 6, 1972

PHOTOCHEMICAL

ACTIVITIES THE

OF A PARTICLE GREEN

L.H.

CSIRO,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ALGA

Grime+

Division

CHLORELLA ---

and

of

FRACTION

N.K.

Plant

P,

OBTAINED

FROM

- FUSCA

Boardman

Industry,

Canberra,

Australia.

Received

October

31,1972 SUMMARY

A procedure is described for the careful breakage Chlorella fusca by short treatment in a Nossal shaker, of a chloroplast preparation (P,) which resembles the absorption and fluorescence chakacteristics at liquid derived from autotrophically grown cells exhibits i!z;ivities . Fractionation has with

been

hampered

high

break

of

the

cell

preparations

of

or

by

with

Chlamydomonas extensively chloroplasts

is

tough

and

thick

break

the

Chlorel

IOSS

+

of

Permanent

tightly

photochemical

address:

with

photosynthetic

studies,

wall. ccl

as

the

The

rather

but

have

been

been

in

Copyright 0 19 72 by Academic Press, Inc. All rights of reproduction in any form reserved.

lnstitut West

1617

to

(l),

obtained

only

cells to

have obtain

for been highly

successful. a single and

methods

chloroplast

activi.ty.

Botanisches 852 Erlangen,

algae

chloroplast

attempts

membrane drastic

Press

la

contains

cell

resulted

Chlorel but

Chlorella

with

1 wall

not

procedures

French

(3),

Intact

have

such

associated

la

activity

green preparations

describe the

beads

(3).

Scenedesmus

alga

cell

glass

unicellular

chloroplast

reports using

photochemical

of

obtaining

organisms

preparations

A unicellular which

of A few

shaking

and for

systems

difficulty

these

high

(1)

photochemical

activity.

wall

(2)

active

the

photochemical

sonication

used

by

the

of the green alga and the isolation whole cells in its nitrogen temperature. good photochemical

der UniversitHt, Germany.

is

chloroplast, surrounded

used damage

by

previously and

a heavy

a to

Vol. 49, No. 6, 1972

In for

the

the

BIOCHEMICAL

present

careful

chloroplast

work,

rupture

we of

fragments

have

the

with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

developed

Chlorella

the

Algal

Culture

flasks

in

NaCl,

470;

were

fitted

them

with 1800

high

I .5% by

two

two

made

chlorophyl

rpm,

MSE

and

centrifuge

20 40

(Commonwealth

a final

dry

and

added

to

for

three

periods,

The

contents

of

and

filtered

through from

beads

of

agitate

the

were

(NH~)~

each

range

of

harvested once

with

each

of

bovine

of

- 3.3.

by

centrifugation 0.05M

were

2 layers by

sizes

(12

of

in

a Nossal

added Miracloth

centrifugation

1618

to

of

viscous Shaker

20

ml

of

and

300

4 ml

mixture

the

pH and

in

was

of

0.35mm)

was

shaken

a cold

room.

homogenising Whole

x g

(TES),

suspension

(Calbiochem). at

containing:

(BSA)

cell

(5)

min,

buffer

5% Ficoll

albumin

O.lmm

2

(5

a buffer

The

le

phosphate

isoascorbate,

ml

The

steri

acid

ia).

one

chlorophyll

2.9

in

intensity

fresh,

ratio

serum

Austral

capsule.

5 set

capsule

5 mM

an of

into

ml

flasks

provide

consisting

with

4.5

The and

illuminated

the

of

6.3.

cultures

Transfers

washed

0.5%

two

steel

filtrate

pH was

initial

the

Melbourne,

a stainless

the

The

volume

and

Labs.

the

Mo7024.4H20,

I mM CaC12,

(Pharmacia),

glass

0.2;

methyl-2-aminoethanesulfonic

mM MgC12,

Erlenmeyer

ZnS04.7H20,

were

I I I),

in

CaC12.

conditions

wt.)

MARK to

Serum

with

removed

fresh

27OC

15 from

-

250;

lights

in

211

MgS04.7H20,

tubes.

and

(hydroxylmethyl)

Dextran

mixed

constant

at

per

360;

fluorescent

these

strain

grown

I igrams

cultures

Under

activities.

810;

to

fluorescent

- 2.59

mM sucrose,

2.5%

of

white

resuspended

50 mM N-tris

The

banks

daily.

(1.5

tubes

of

KNO3,

Na2HP04.12H20,

9.3.

release

1 iter):

mil

0.5;

the

Krauss,

were

470;

ai r.

I b-was

Cells

250

in

cool

were

7.2

CO2

et

(in

bubbling

and

method

METHODS

Gtfttingen

H3B03,

reproducible

photochemical

Shihira

Na2EDTA,

gas

AND

containing

0.5; 6.3;

with

ft.C

medium

pH

at

NaH2P04.2H20,

and

4000

Collection (4)

FeS04.7H20,

warm

fusca

MnC12.4H20,

0.02;

of

Chlorella

a medium

6~~0,20;

to

of

cell,

reasonably

MATERIAL

Cultures

a fast

for

IO

buffer cells

min.

were The

7.5,

BIOCHEMICAL

Vol. 49, No. 6, 1972

supernatant

from

nearly

clear

this

in

phosphate

buffer,

suspension

was

resuspended

the

was

following

0.5% at

same

extracted were

serum

estimate

chlorophyll

-

8.3

chl

b

(pg/ml)

=

33.8

x

0.D650

-

12.5

77°K (7).

62% glycerol in

sensitivity

on

source (8).

Jagendorf

and

are

in

given

reductase

were

ies

legend

to

prepared from

from

Nossal they

showed

cell

suspension

with

glass

with poor

Ferredoxin and

to

the

A degree

of

of

5600 of

from

the ccl

15 set,

Btfger

(6).

chlorophyl

I b

62%

glycerol spectra

and

measured

Shaw

(10).

from

77°K

photomul by

were

for

the

Details

and

a Cary

at

correction

were

also

with

t ipl methods

of

ferredoxin

ier of

assay

media

- NADP+

Chlorella.

Plastocyanin

two

AND DISCUSSION

Chlorella beads,

cells but

photochemical

beads

squeezed

of

glass

at

damage

of

0 - 4°C.

spinach.

breakage

shaker

at

a and

response

2.

spinach

pellet

method

automatic

and

RESULTS

Complete

in

with

Vernon

Table

the

emission

activities and

The

performed

the

M

0.0~~~

x

monochromator

(8)

0.05

x 0.D650

recorded

a spectrometer

intensity,

Margul

were

Fluorescence

Photochemical

the

isolated

were

MKCl,

and

by

x O.D665

in

10 min

The

min.

(P,)

(BSA).

methanol

16.5

spectrophotometer

0.01

into to

20

pellet

operations

=

14R

green

for

albumin

x 1 for

(us/ml)

variations

time

bovine

x p

All

used

at

the

M sucrose,

2

spectra

1,000

0.3

chl

Model

major

and

10,000

buffer.

equations

at

discarded,

and

centrifuged

in

centrifuged

containing

pH 7.2

Absorption

was

was

a medium

Chlorophyll

made

was

supernatant

resuspended

The

spin

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

cycles/min

divided

of into

shorter

sizes It

to

obtained

the

local

60 -

80%

3 periods

1619

that

pressure was

achieved

of

5 sec.

small

cell

walls

the

chloroplasts the

buffer without were

glass

a total

the and

of

a viscous

the

with

in

oscillation in

around

2 min were

rapid periods

ruptured seems

within

fragments

However, for

different

I due

I breakage

resulting

activity.

chioroplasts. ccl

was

beads. shaking

a

Vol. 49, No. 6, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE CHLOROPHYLL

CONTENT

OF

Fraction

Whole

(300

x cJ,)

5890

63

3.20

in

the

constricted

3.20

of

was

b

for

of

These

were

broken

3.20

10.2

3.22

as

at

bands.

different the

photochemical

French

the of

2,500

the

Michel

25

cells;

of

P,

no

of

Chlorella

chl

composition

a/chl

then

b

of

very the

(12.5 the

the

were

same

therefore,

and

was

particles

77°K

during

- Wolwertz

on

force

analyses

evidence,

phase-

Furthermore, at

showed

The

fragments

their

cells.

min

by

chloroplast

Pigment

is

chlorophyll

smaller

washed

1.

osmotically

centrifugation

x ‘3 for

Table

centrifugal

whole

2).

in

examined

but

into

whole

gradient

There

were

whole

spectra

1 and

photosystems

Previously,

The

that

density

shown

A higher

of

(Figs.

P,

the

shaker.

The

consist

emission

sucrose

all of

with

same

cells

washed

through

to

fragments.

fluorescence

of

fragments

51

(P,)

buffer.

3.28

are

x 3 pellet

chlorophyll

the

whole

fractionation

cells

found

the

the

by

+/chl

Nossal

of

obtained

sucrose)

particles

1,000

and

and

those

chl

original

resediment

51% of

bands

chloroplast

sucrose-phosphate

absorption to

the

chloroplasts.

to

ratio

of

microscopy

contained

4.7

4800

b

3.21 3.22

yields

required

a/Chl

100

960

with

Chl

20.5

Supernatant

washing

FUSCA

1940

(washed)

contrast

% total

of

440

particles

CHLORELLA ---

x 5)

Supernatant

The

FROM

9400

(1000

P,

ISOLA.TEO

Chlorophyll content (pg)

cells

Porn3 P,

FRACTIONS

1

similar different

- 50% ratio for

of a

fragmentation

Michel by

passage

P,

fracticn

(11)

in

the

obtained of

Chlorella

Press. activities

of

1620

the

washed

are

summarized

Vol.

49,

BIOCHEMICAL

No. 6, 1972

AND

436 1

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

-1 0 9

4

8 7 .3

6

& z

11

5

1.0

4

2

3

m

.’

g

.8

2

ii Q

7

I

.6

,3

.l

.4

3

400

1

1

1

/

,

450

500

550

600

650

.2

700

A(nm) .1

Fig.

1.

. 660

680

700

720

740

760

A (nm)

Fig. Fig.

Absorption spectra at 77°K and chloroplast particles,

1.

Fluorescence cells and wavelength, 0.2 Ab36.

Fig.2.

in

Table

donor

2. were

High

or

NADP+

reductase

with

the

Hill

activity

rates

obtained

spinach

had

was

fusca

emission spectra at chloroplast particles, Concentration 436nm.

77°K

of

NADP+

provided

Chloreila)

behaviour

(b)

of C. P,.

and no of

effect

on

chloroplast

observed

with

with

amounts

plastocyanin

were the

rate

of

of

ferricyanide

or

trace)

(a) C. fusca Excitation or particles,

water

Addition

NADP+ from

(upper

as

electron

ferredoxin

added.

particles

1621

of P,. ceils

of

photoreduction,

saturating

cells

2.

reduction, Scenedesmus

2,3’

(either of

from

ferredoxin

which (3).

,6-trichlorophenolindophenol

contrasts good

-

Vol.

49,

No. 6, 1972

BIOCHEMICAL

AND

BIOPHYSICAL

TABLE PHOTOCHEMICAL

React

ACTIVITIES

OF CHLORELLA

PARTICLE

Activity moles/mg reduced)

(p

- DCMU + NADP

H20

+ TCIP

H20

+

COMMUNICATIONS

2

ion

H20

RESEARCH

FRACTION

Chl/hr

+ DCMUa

1.7

122b

69’

FeCy

P,

0.9

1 52d

0

176 Asc/DCIP DPC +

Sf

07

1179

31

+ NADP DCIP

a.

Concentration

b.

Reaction mixture contained (in 0.6 ml): 8,119 chlorophyll and Tris HCI (pH 8) 8; NaCl 14; MgC12 2; NADP 0.12; and saturating of ferredoxin and plastocyanin. Illumination: 2 min saturating 1 ight. Reduction of NADP was calculated from the absorbancy at 340nm.

C.

Reaction Tris HCl measured

mixture contained (pH 7.8) 40; NaCl at 620nm after 45

d.

Reaction Tris-HCl decrease

mixture contained (in 2.2 ml): (pH 8.0) 29; NaCl, 51; MgCl was measured at 418nm after2;

e.

Reaction mixture with a Clark-type

as for (d). electrode

f.

Reaction

mixture

as

9.

Reaction phosphate Absorbancy

mixture contained (in 3 ml): IO pg chlorophyll buffer (pH 6.7) 90; sucrose, 750 and diphenyl decrease was measured at 550nm after illumination

(TCIP) not

as as

high

: 1 x

oxidants, as

diphenyl

inhibition

for

the

rates

by

3 ml): 20 TCIP 0.06: illumination.

of

(DPC)

3(3,4-dichlorophenyl)-I

ug

20 7.3; min

chlorophyll Absorbancy

but

with

rates

NADP’

(in

reduction

about

25% ,l

1622

measured U.K.).

ascorbate

Somewhat were of

the

dimethylurea

u moles): amounts white increase

and 1.

(in pmoles), Absorbancy

polarographical

and

0.6

umole

chl/hr) higher

observed activity

DCIP.

were

rates in

was (DCMU).

ly

and (in pmoles) carbazide 1.5. for 45 sec.

p equivalents/mg

reduction.

(DCIP) but

2 pmole

(in

and (in pmoles) decrease was

ug chlorophyll ferricyanide, i I luminat ion.

Oxygen evolution was (Rank Bros. Cambridge,

the

indophenol carbazide

(in 70; set

(b),

although

2,6-dichlorophenol of

10e5M

the

of presence

resistant

to

Ascorbate

- DCIP

Vol.

49, No.

served

BIOCHEMICAL

6, 1972

as

an electron

reaction.

Thus

reactions

of

the

those

water

of as

the

P,

by

on

Chlorella

higher

I and

donor

of

autotrophic

detergent

plant

activities

of

the

and

in

levels

P,

of

to

elucidate

membranes

the

which

are

generally

lack

for

the

partial

not

as

in

progress

mode

of

action

on to

of

characteristic

high with

studies

are

the

insensitive

particularly

advance

Studies

-

showed

activity

a significant

COMMUNICATIONS

a DCMU

Chlorella

I I.

Chlorella.

treatment thylakoid

from

Photosystem

represent

RESEARCH

photoreduction

particles

chloroplasts,

electron

BIOPHYSICAL

NADP+

photochemical

spinach

photosystems

for

chloroplast

Photosystem

Although as

donor

AND

the

fragment

detergents grana

of

the

chloroplast.

ACKNOWLEDGEMENTS

The authors are indebted to Mr. S.W. Thorne for spectra and to Dr. Jan M. Anderson for helpful advice. gratefully acknowledges a postdoctoral training award Forschungsgemeinschaft.

the

fluorescence One of us (L.H.G.) from the Deutsche

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Boschetti, (Metzner,

3.

Pratt,

4.

Kessler,

E.

5.

Nossal,

P.M.,

6.

Btlger,

7.

Boardman,

N.K.

8.

Boardman, 56, 586,

N.K., Thorpe, (1966)

9.

Jagendorf, (1960)

A. H., L.H.

P.,

Levine,

and ed.)

Grob, Vol. Bishop,

N.I,

and

Czygan,

F-C.,

Austral.

A.T.

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l& and

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Vernon, (1969)

and

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Michel - Wolwertz, Year book 67, 514

Acad.

Sci.

Margulies,

E.R.,

M.R. and (1968)

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(1970)

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R.P.,

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