Light-induced potassium efflux from spinach chloroplasts

Vol. 17, No. 6, 1964

BIOCHEMICAL

LIGHT-INDUCED

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

POTASSIUM

SPINACH

CHLOROPLASTS

Richard Charles

EFFLLJX

A. Dilley

F. Kettering

*

’

Research

Yellow Springs,

FROM

Laboratory

Ohio

Received September 28, 1964 Several

laboratories

light-dependent

increases

--et al. , 1963; Jagendorf Itoh, --et al.,

have shown that chloroplasts in light-scattering

and Hind,

1963; Dilley

(1963) demonstrated

chloroplast

shrinkage.

(1964~) measured light- scattering (a) contraction from within

shrinkage

changes.

the chloroplast

observed

shrinkage

phenomena

changes related Dilley

could result

and Vernon as the

from:

We have observed which appears

in its kinetics

a light-

related

and response

to the to

inhibitors. METHODS Chloroplasts

sucrose

were prepared

- 0. 1M Tris-acetate

*Contribution Laboratory, tPost-doctoral Service.

No. 167 of the Charles Yellow Springs, Ohio. trainee

(Dilley

pH 7.5.

and Vernon,

Potassium F. Kettering

under sponsorship

716

to

or (b) loss of ions and water

structure.

K+ efflux from chloroplasts

1964a).

having the same kinetics

protein

dependent

selected

counter,

Such shrinkage

of a contractile

1963; Itoh,

and Vernon,

slow scattering

Using a Coulter

a rapid

(Packer,

exhibit

1964a) in

ion was measured Research

of the Public

Health

BIOCHEMICAL

Vol. 17, No. 6, 1964

potentiometrically and Northrup trode.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with a Beckman Model

The meter

“cation”

7401 pH meter

resistance

(100 to 300-h)

adequate

sensitivity.

Potassium

tatively

measured

trophotometer.

content

illuminated

and non-illuminated through

LKR.

A Corning (No. CSl-69)

The light

intensity

-2

by

Bowling Green State

medium

a Millipore

Spec-

performed

For these measurements

systems

was provided

absorbing

cm

Absorption

was determined

after removing filter

the for

the chloro-

having an average

of 0.65 p. Illumination

Model

Department,

using a

and quanti-

were graciously

of the suspending

plasts by filtration diameter

303 Atomic

Ohio.

elec-

output to give

was also qualitatively

Chemistry

Bowling Green,

reference

across the meter

These measurements

University,

using a Leeds

on a Brown recorder,

with a Perkin-Elmer

Dr. H. K. Anders,

potassium

with a Ag:AgCl

output was recorded

variable

electrode

set

-1

by a Unitron

2304 red filter heat filter

incident

Research

and a Corning

Illuminator infrared

were placed in the light beam.

to the suspensions

was 1.5 x lo5 erg

. RESULTS

Figure electrode

1 shows a typical

upon illuminating

of an electron’transfer is apparent

first

on the chloroplast

signal

a chloroplast agent (TMQHz*+

obtained

from

suspension

” Abbreviations used: TMQH2, Cl-CCP, meta-chlorocarbonyl 3-(3,4-dichlorophenyl)-l, l-dimethyl

degree

reduced cyanide

717

in the presence

in this case).

order with a t1/2 of approximately preparation,

the “cation”

of aging,

The reaction

5 set, depending etc.

A true first

trimethyl1,4-benzoquinone; phenylhydrazone; DCMU, urea.

Vol. 17, No. 6, 1964

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

loo5 m MOLES

K+

1

ON

+-I

w

IO SEC.

Fig. 1. Potassium efflux from chloroplasts. The reaction mixture contained 0.1 M Tris-acetate pH 7.0, 0.132 pm/ml TMQH2, and chloroplasts equivalent to 314 pg chlorophyll in a total volume of 15 ml. The light was turned on at the arrow indicated by “on”.

order

reaction

reactioq

is indicated

by two facts:

are the same for chloroplast

11 to 22 pg in the reaction + Ktoo- t ( [K+l linear

values for the

concentrations

ranging

from

and (2) a log plot of time versus

at the steady state minus

Cl@] at time t) gives a

plot. The signal

with K+ through Table

system,

(1) the tti2

obtained

from

the “cation”

the use of atomic

I shows that the suspending

plasts contained

significantly

The K+ efflux calculated chlorophyll. Beckman

Similar

chloro-

more K+ than a non-illuminated

control.

(see Table

shrinkage

inhibited

(TMQ)

I

with the

I

II).

is inhibited

reaction

II it is seen that DCMU acceptor

from

these data is 0.26 mole K+ per mole

The K+ efflux reaction

an electron

spectrophotometry. light-treated

from

medium

was identified

values for K+ efflux were obtained

K+ electrode

chloroplast

absorption

electrode

much the same as the

(Dille y and Vernon,

1964a) a In Table

the K+ efflux almost

is added. ‘718

However,

completely

when

if the reduced form

Vol. 17, No. 6, 1964

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table K+ Efflux from measuring

illuminated

spinach chloroplasts

K+ concentrations

Conditions

I

in chloroplast

Absorbance

153 -+ 4

Light

165 + 4

suspension

by

media.

K+ efflux in light

l

at 770 rnp Dark

determined

moles/mole

Chl

0. 26

Reaction mixtures contained chloroplasts equivalent to 7.3 pg chlorophyll, 0.1 M Tris buffer pH 7.0 and 0.132 mM TMQH2 in 3 ml total volume. Ten samples each were incubated in darkness and in the light (one minute at 1.5 x 105 erg cm- 2 set- ’ red light). Atomic absorption spectrophotometry was used for the K+ assay, making use of 770 mp emission line of potassium. Concentrations of K+were obtained from a standard curve prepared by adding known amounts of KC1 to samples under similar conditions. *x 10

-3

of the acceptor

is added (TMQH2),

cyclic

flow and phosphorylation,

electron

which is capable

only 33% by DCMU at 3.2 x 10m5 M. photophosphorylation,

reduced

but it did not measurably coupler,

inhibited

Ouabain, membrane

Quinacrine,

an inhibitor

an uncoupler

of

the extent of K’- efflux by about 40%,

both the initial

systems

the K+ efflux was inhibited

affect the tr/z value.

at a very low concentration

of catalyzing

Cl-CCP,

rate and the extent of the K+ efflux

(6 x lo-’

M).

of active cation transport

(Schatzmann,

also an un-

in mammalian

1953), did not affect the K+ efflux at

5 x 10-5M. If one assumes (Rabinowitch,

that chlorophyll

1945) the data in Table

mum K+concentration

is about 0.2M

II may be used to calculate

in these chloroplasts 719

in chloroplasts

(assuming

a mini-

all K+is effused)

Vol. 17, No. 6, 1964

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Effect of inhibitors spinach

and uncouplers

II upon K+ efflux from illuminated

chloroplasts. Conditions

poles

K+ effused

)Imole 1 Control (TMQ) plus 1.6 x lo-’ plus 3.2 x lo-’

Per Cent

chl.

Inhibition

M DCMU(TMQ)

0.21 0.025

90

M DCMU(TMQH2)

0.14

33

2 Control

0.20

plus 6 x lo-’

Cl-CCP

0

3 Control

100

0.26

plus 4 x lo-’

M Quinacrine

0.14

44

plus 8 x lo-’

M Quinacrine

0. 14

44

plus 5 x lo-’

M Ouabain

0.26

0

Exp. one: 0. 1 M Tris-acetate pH 7.0, 314 pg chlorophyll and 0.11 mM TMQ (or 0.13 mM TMQHl as indicated). Exp. two: 0.1 M Tris-acetate pH 7.0, 0.132 mM TMQHz and 180 pg chlorophyll. Exp. three: Same as exp. two except 340 pg chlorophyll were used. The final volume in each case was 15 ml. The illumination period was one minute, and K+ concentration was determined with a Beckman “cation” electrode.

of 0.04 M K+. tration

This is consistent

in chloroplasts

of higher

with published plants

values for KS concen-

(Saltman,

--et al. , 1962).

DISCUSSION The data presented effuse K+ ions through

above show that chloroplast

a mechanism

electron

flow.

The reaction

similar

to that of the shrinkage

efflux and chloroplast tron transfer

reactions,

is first

shrinkage

which depends on light-induced order and has a t1i2 value

reaction.

It appears

are coupled

since DCMU

720

fragments

inhibits

that both the K+

to photosynthetic both reactions

elecand a

Vol. 17, No. 6, 1964

differential

BIOCHEMICAL

effect is observed

The reduced form induced electron (Dilley

for systems

of the quinone transfer,

and Vernon, A further

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

containing

overcomes

KS efflux,

DCMU

the initial

completely

inhibits

shrinkage.

shrinkage

rate,

Quinacrine

(Dilley

The above evidence the chloroplast

shrinkage

are now in progress

Quinacrine

reaction

efflux to other energy-related

effect on

the extent of K+ does not alter

the until

1964b).

that the K+ efflux is related which in turn appears

mechanism

to further

and quinacrine.

rate to continue

and Vernon, indicates

to the energy conservation

and the K+

has no appreciable

but allows the initial

is complete

reaction

Cl-CCP

rate of K+ efflux but it does decrease

shrinkage

shrinkage

the rate and extent of both KS efflux

efflux by about 40% at 8 x 10w5M. initial

of light-

1964a).

analogy between the shrinkage

and chloroplast

inhibition

and chloroplast

efflux is found in the effect of the uncouplers The former

TMQ or TMQH2.

clarify

of chloroplasts. the relationship

chloroplast

to

to be related Experiments of the K+

phenomena.

ACKNOWLEDGEMENTS The author counsel rendered

gratefully

of Dr. L. P. Vernon by Mrs.

Inger

acknowledges

the encouragement

and the competent

technical

and

assistance

Stoughton.

REFERENCES Dilley,

R. A. and Vernon,

Dilley, R. A. and Vernon, 15, 473 (1964b). -

L. P., Biochem., L. P.,

Biochem.

Dilley, R. A. and Vernon, L. P. , Amer. Symposium, Cleveland, Ohio (1964~).

721

3, 817 (1964a). Biophys.

Res. Comm.,

Sot. for Cell Biol. ,

Vol. 17, No. 6, 1964

BIOCHEMICAL

Itoh, M. , Izawa, 319 (1963).

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

S. and Shibata,

K.,

Biochim.

Biophys.

Acta, -66,

Mechanisms Jagendorf, A. T. and Hind, G., in Photosynthetic Green Plants, Symposium. (AirI& Va. ) Washington, D. C. 1145 Nat. Acad. Sci. , Nat. Res. Coun. p. 599 (1963). Packer,

L.,

Bicchim.

Biophys.

Acta, -75, 12 (1963).

and Related Rabinowitch, E. , in Photosynthesis science Publ. , Inc., New York. Vol. 1 (1945). Saltman, (1962).

P. , Forte,

Schatzmann,

J. G. and Forte,

H. J., Helv.

of Publ.

Physiol.

G.,

Processes.

Exp. Cell Res.,

Acta, -11, 346 (1953).

722

Inter-29, 504