Catalysis of photophosphorylation by allagochrome

BIOCHEMICAL

Vol. 4, No. 2,196l

CATALYSIS

OF

PHOTOPHOSPHORYLATION

Helen

M.

Department

Received

versible

oxidation-reduction

plasts.

Of

which

has

c

gested

that

been

yellow by

The

resistant

on

complex

from

ascorbate,

may

evidence plant

is

has of

its

and

to

reduction

led

of

been

Depts. of Medicine,

of

red

as

expect

Biochemistry 1600 N. W.

109

(1960)

sunflower

leaves This

other

named

in

acid

and

the

and lOthAve.,

color

from

is

reduced

re-

cyto-

properties oxidase

this

in It

terminal

Its

pigment

Foundation Service

in

sugintact

oxidase

in press). that

(mean-

solution.

a terminal

and

allagochrome

These

is

species

changes

(Habermann, to

chloro-

Habermann

reversible

allagochrome

us

a re-

ubiquinones

several

by grants from the National Science H. and from the U. S. Public Health K. and (RG 7659) to H. M. H. address: School

and

autoxidizeable.

respiration

by

dithionite

function

that

or

oxidation-reduction.

borohydride, form

reduction

from

leaves

pigment

reduction

reduced

is

and

Recently

because

allagochrome

cyanide

leaves.

a reversible

The

pigment”)

There

* Supported to H. M. to A. R.

College

either

flavins

isolated

seeds.

tissue.

oxidation

Krall’

nucleotides,

in green

of undergoing

vitro

chrome

2 Present Miami,

pyriaine

found

since

to --in

R.

photophosphorylation

pigment-protein

“changeable

duced

of

are

sunflower

blue-green

ALLAGOCHROME’

Goucher Avenue

of undergoing catalyze

the

capable

complex

ing

capable

a blue-green is

from

these,

1959)

isolated

A.

and

of Biological Sciences, and RIAS, 7212 Bellona Baltimore, Maryland

compounds

(Bishop,

Habermann

BY

13, 1961

January

Several

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

ease

could

(RG

Psychiatry, Miami

(G8972) 5583

Univ. 36.

c-l)

of

Vol. 4, No. 2‘1961

catalyze flavin

BIOCHEMICAL

an oxidative

photophosphorylation

mononucleotide

firm

these

or menadione.

and Methods:

chilled

sucrose,

leaves

0. 05 M trishyroxy at 200 x g for

1000 x g for

7 min.

resedimenting

Total

was

either

stated.

The

reactions

the bottoms

of experimental

through

the first

by 50% acetone

was

added

3 hours.

here

by con-

was

were

Preparations

were

and consisted

1. 0 ml.

and

The

at 15OC.

uptake

funnels)

Incident

1500 fc. was

at

deter-

of the isobutanol

and Lehninger

(1955)

in which

counted.

removed

described step.

resulting

acetone and dialyzed purified

Impurities

preAcetone

blue-green

removed against

precipi-

by evacuation, cold

by continuous

di- and tri-phosphate: inorganic phosphate: 110

(Habermann,

by centrifugation.

The

further

* Abbreviations used: Adenosine phenazine methosulfate: PMS;

at

at concentrations

Phosphate

precipitation

in water

the brei,

(5 wales)

was approximately

by centrifugation, dissolved

MgCl2 was

as previously

acetone

(0. 35 M

filtering

mixture

5 to 10 minutes

of Nielson

to 90% by volume.

collected

reaction

(use of separatory

by

chloroplasts

or allagochrome

bulbs

prepared

the supernatant

(8 PMoles)

for

was prepared

cipitated

the precipitate

run

in the water

Allagochrome

tate was

catalyzed

in ice cold buffer

at pH 7.8),

The

vessels.

method

remaining

sand

of the reaction

incandescent

by a modification extraction

7 min. KH2PO4

were

were

the sedimented

0. 03 JIM PMS

from

then

reported

, centrifuging

volume

illumination

1960)

1 min.

radioactive

catalyst

ATP32

results

chloroplasts with

, resuspending

(5 poles).

benzene

to that

aminomethane

at 1000 x g for

of chloroplasts,

mined

Spinach

in a mortar

centrifuging

ADP”

The

similar

expectations.

Materials grinding

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

water flow

ADP Pi.

for paper

and ATP;

2 to

BIOCHEMICAL

Vol.4,No.2,1961

Pigment

electrophoresis.

The

concentrations standard

density

at 67 5 mp.

obtained

by drying

and weighing

molecular

weight

of allagochrome

(Northrup

and Anson,

Results were

in these

allagochrome hour

a rate

was

per

ml.

(cf.

Fig.

observed. were

Thus,

on a molar than

PMS,

density

vs.

aliquots

of known

optical

density.

from

Pi esterified

a rate

I).

were

basis

appears

Catalyst*

freshly

known

on the rate esterified

the effect

per

mg.

chlorophyll

Pi per

of increasing

hour 111

pigments this

rate.

or more

time.

allagochrome

mg chlorophyll

with 0. 2 ml catalyst

was

per

hr)

with 0. 3 ml catalyst

86

146

89

101

32

53 372

7 seed allagochrome per ml. ;

crude mg.

concentrations

A maximum per

PMS

I

@Moles

of photophosphorylation.

per

PMoles

to achieve

up to this

seed

chlorophyll

to be as effective

with 0. 1 ml catalyst 51

1 shows

or leaves

prepared

required

Crude seed allagochrome Crude leaf allagochrome 63 Electrophoretically puri-fi_e_d_Ie-af- &+agoc _-----------_-_-------------------------------------hr ome Control rate with 0. 03 pM PMS Control rate (no added catalyst) *Concentrations of preparations were as follows: 0.61 mg. per ml. ; crude leaf allagochrome, 0.90 purified leaf allagochrome, 0. 35 mg. per ml. Fig.

The

PM crude

mg.

of esterification of Pi by chloroplasts using as the catalyst of photophosphorylation Rates

was

technique

that 0.004

With

0. 0012 ph4 pigment

catalyst

weight

seeds

0.0024

per

reported

of 90.

allagochrome

either

With

of 86 FMoles

(1957)

optical

50,000.

Preparations

TABLE Rate

by a diffusion

is approximately

the best

from

of optical

(Table

for

determined

curve

determined

Jagendorf

RESEARCH COMMUNICATIONS

were

experiments

required

1) about

effective

1929)

and Discussion:

active

AND BlOPHYSlCAL

rate

attained

of allagochrome of 158 PMoles with

il. 024 wale

Pi

Vol. 4, No. 2,1961

BIOCHEMICAL

AND BIOPHYSICAL

ml.

mixture.

allagochrome

per

250 in this

experiment.

trations

was

chrome

which

reaction The

probably

fall

lowered

0. 03 pMole

in rates

a consequence

at high

of strong

the intensity

RESEARCH COMMUNICATIONS

of light

PMS

gave a rate

allagochrome

light

concen-

absorption

impinging

of

upon

by allagothe chloro-

plasts.

Figure

,l

Effect of concentration of seed allagochrome on rate of photophosphorylation.

g : f 20. ?O

0

.002 .004 .006 .008 Allagochromo cont. (PM per ml.1

Preliminary

experiments

and production Brown

of oxygen

--et al. (1952)

and equivalent the experiment slightly fate,

greatly

the rate

reduced (or

that

there

phosphorylation

in contrast

phosphorylation

which

18 in which

by methods

with

described

by

of oxygen

uptake

nitrogen,

all

with

to catalyze other

in a system 112

Running

a condition

known

which

phenazine

of photophosphorylation

is known

function

rate

uptake

photophosphorylation.

of phosphorylation

with

simultaneous

was a high

during

flushed

the rate

pyocyanine)

0

determined

production

in a system

increases

PMS

were

showed

oxygen

with

methosul-

with a cyclic catalysts

capable

allagochrome.

photoof photo-

of oxygen

exchange

BIOCHEMICAL

Vol. 4, No. 2,1961

(Nakamoto

--et al.

results

with

the

allagochrome

, 1959;

AND BIOPHYSICAL

Krall

mass

--et al.

spectrometer

catalyzes

the

latter

RESEARCH COMMUNICATIONS

, in

press).

and

N2

flushing

an

oxidative

type,

The

preliminary indicate or

that

non-cyclic

photophosphorylation. References Bishop,

N.

Brown,

A. H., Physiol.

Proc.

I.,

Nat.

Acad.

Nier, A. 0. 27, 320 (1952). -

C.

Sci. and

Van

Habermann, Helen M., In: Comparative Photoreactive Pigments, M. B. New York, p. 73 (1960). Habermann, Congress Jagendorf,

A. p.

Krall,

236

M., Tn: Photobiology,

T., (1958).

In:

A. R. , Good, (in press).

Nakamoto, 235, Nielson,

Helen on

T., 1843

S. (1955).

Northrup,

0.

J.

H.

N.

E.

and

and

D

Lehninger,

Anson,

M.

(1959).

Norman,

Allen,

R.

W.

and

A.

L.,

L.,

(1929).

113

in Biology

B.

, Plant

Mayne,

J.

3.

Plant

Biol.

Gen.

Press,

International 1960 (in press).

Symposium

Mayne,

W.,

Biochemistry of ed. Academic

of the Third Copenhagen,

Brookhaven

Krogmann, (1960). and

Proc.

1696

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No.

Physiol.

B.,

Chem.

Physiol.

J.

11,

36 -

Biol.

215,

12, -

Chem.

571

543