A naturally occurring cofactor for photosynthetic phosphorylation

Vol. 7, No. 1,1962

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ANATURALLYOCCURRINGCOFACTORFOR PHOTOSYNTHETIC D. W. Department

Received

January

from

that with

chloroplasts

water

the whole

The water

during

of chloroplasts

from

lyophilized

described

While

nor

chloroplasts

which

isolated

are

phorylation measured method

isotonic

assays

were

by the method (5).

FMN,

activity

solution

show found

were

and SubbaRow and caffeic 46

of

media. as the source

prepared

and washed of

of photophosphorylation

the exception

Inorganic

were

that

phosphate

(4) and chlorophyll acid

of

(2).

to the procedure

(3) with

out aerobically.

neither to

of Stocking

for measurement

and Avron

catechin

served

according

has been

activity

to the procedure

The conditions

of Fiske

the preparation

been

preparative

media

of cofactor

carried

for

preparations

aqueous

presumably fraction

the photophosphorylation

in nonaqueous

of Jagendorf

are

solvents

they have

be

of ATP

particulate

a method

immedi-

must

rate

nonaqueous

activity,

sucrose

leaves

cofactors

of this

using

according

(3).

soluble

chloroplast

support

in the assay

those

of Arnon

will

prepared

and Avron

essentially

leaves

isolated

used

once in aqueous, Jagendorf

Illinois

it was

spinach

recently,

in the conventional

and were

Chloroplasts

More

such nonaqueous

Chloroplasts cofactor,

Chicago,

if a vigorous

isolation

photophosphorylation

a cofactor

from

(1).

homogenate.

(2).

isolated constituents

out of the chloroplast

contain

2, Michigan

phosphorylation,

soluble

is to be observed

activity

Detroit

of Chicago,

of photosynthetic

leaf

Hill

Stiller

University,

University

the discovery

supplemented

washed

State

L.

4, 1962

recognized

synthesis

and M.

Wayne

of Biochemistry,

With ately

Krogmann

of Chemistry,

Department

PHOSPHORYLATION

purchased

all phoswas

by the from

VoL7,No.

1,1962

BIOCHEMICAL

the Nutritional

Biochemical

the California supplied

The data once

in aqueous,

the absence observed.

Research

in Table isotonic

per

the chloroplasts

activity

is heat

stable

The ion exchange

cofactor resin

water

water

bath was

of FMN,

was

for twenty

is seen

by suspending giving

one milligram

twenty

minutes

That

the cofactor

of activity

after

minutes.

to surpresa

dry,

In other

to

incubation experi-

photophosphorylation

tested.

was not absorbed be extracted TABLE

from

on Amberlite an aqueous

MB3 solution

I

PHOTOPHOSPHORYLATION

WITH

CHLOROPLAST

NONAQUEOUS

EXTRACT

lo. moles Pi taken up per mg. chlorophyll per hour

Cofactor

None

0

FMN

92

Extract

55

Boiled

is

chloroplasts

prepared

fragments.

in

phosphorylation

at a concentration

found

and washed

of photophosphorylation

by the retention

of the extract

and could

acid was

prepared

at 20, 000 x g. for

is indicated

activity

from

Chlorogenic

of nonaqueous

extract

and chloroplast

atmosphere

at all concentrations

extract

centrifuging

in a boiling

a nitrogen

incapable

On addition

This

then

remove

of the extract

are

in cold

ml,

Research.

that the chloroplasts

cofactor.

photophosphorylation.

chlorophyll

ments,

I show

an aqueous

chloroplasts

and quercitrin

Laboratories.

sucrose

of an added

nonaqueous

and quercetin

for Biochemical

Similarly,

to elicit

Corporation,

Corporation

by the Mann

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

54

Extract

The amount of extract is equivalent to 0.1 mg. chlorophyll in nonaqueous chloroplasts. 47

mixed into

bed

BIOCMEMICAL

Vol. 7, No. 1,1962 butanol

or ethyl

graphy

on Whatman

ascending this

acetate,;

Eighty

chromatogram

a purple fifteen

was

fluorescence percent

preparation

Further

3 h4M paper

solvent.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS purification using

percent

with

an area

are presented

in Figure

1.

350 rnp peak Similar

spectral

responses

> c z 3

to a longer

are

shifts

wave

length

(390)

are induced

suggestive

pH,

of a flavone

a visible was

peaked

color,

with

purified

spectrum

with shift

on the addition

and H3B03

type compound

from

obtained

a pronounced

is observed

as

yellow

of a partially

while

by A1C13

(4:1:5)

recovered

showing

Spectra A double

at 270 rnp and 350 rnp is seen at neutral

activity

An Rf of 0.83

as the solvent.

by chromato-

acid-water

of the cofactor

associated

acid

accomplished

butanol-acetic

and an Rf of 0.43.

acetic

was

cofactor maxima

of the of alkali.

and all of these

(6).

0.30.4 -

P 0.3 a 0

0.2-

ii 0

O.l-

220

250

280 WAVE

3lO

340

3m

400

LENGTH

FIG. I To further chloroplasts commercially

support

isolated available

the notion

#at

in nonaqueous

media

flavones

and flavone 48

the cofactor

activity

is due to a flavone, like

compounds

found

in

several were

tested

for

Vol. 7, No. 1,1%2 activity with

BIOCHEMICAL

ae cofactor6

these

As with

for

compounds

the cofactor

atmosphere cofactors

photophosphorylation.

as well

Table

as the optimum

activity

eurpreeeed

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

found

concentrations

in nonqueoue

photophosphorylation

II gives

date for

chloroplaete,

obtained

activity.

a nitrogen

at all concentrations

of these

tested. TABLE

II

PH~T~PHOSPHORYLATION ?ARIOUS

Cofactor

Optimum

WITH

COFACTORS

Concentration

p moles

Pi taken

per

chlorophyll

mg. per

Chlorogenic Caffeic

acid

-4

6.7x10

acid

-5

4.0x10

-5

hour

M

93

M

52

M

50

Quercetin

5.2x10

Quercitrin

2.7~10’~M

26

Catechin

4.0x

70

10 -5 M

up

REFERENCES 1.

Whatley, Acta, 2,

F. R., Allen, 605 (1955).

2.

Stocking,

C. IL.,

3.

Jagendorf,

A.

4.

Fiske,

C. H.

5.

Arnon,

D. I.,

6.

Geissman, T. M. V. Tracey,

Plant

T.

M.B.,

Physiol.,

and Avron,

and SubbaRow, Plant

and Arnon,

Physiol.,

34,

D. I.,

Biochem.

56 (1959).

M.,

3. Bio.

Chem.,

231r

Y.,

J. Biol.

Chem..

66,

24,

Biophys.

277 (1958). 375 (1925).

1 (1949).

A., Modern Methods of Plant Analysis, K. Peach and Editors; Springer-Verlag, Berlin (1955), Vol. III, p 450.

49