Kinetics of light-induced absorbance changes in Rhodospirillum rubrum extracts

Vol. 23, No. 4, 1966

BIOCHEMICAL

KINETICS

AND BIOPHYSICAL

OF LIGHT-INDUCED

RESEARCH COMMUNICATIONS

ABSORBANCE CHANGES IN

RHODOSPIRILLUM RUBRUM EXTRACTS" David

M. Geller

Department of Pharmacology, School of Medicine, St. Received

March

This induced

29, 1966

paper in

is a report

extracts

illumination 1960).

Washington University Louis, Missouri 6311.0

of R. rubrum

(Smith R. rubrum

of the

during

extracts,

1959;

purified

changes

which

activation

of photophosphorylation.

of absorbance

and following

and Baltscheffsky,

absorbance

common components

kinetics

infrared

Geller

and Lipmann,

by a novel

procedure,

strikingly

in the

course

The effects

suggest

that

are modified

may be involved

changes

in both

the

show

absorbance

of

changes

and photophosphorylation. Methods ---harvested,

- R. rubrum and crude

as previously

strain

extracts

described

Crude

S-l

extracts

made with

(Geller,

were

cells

partition

system

of Albertsson

R. rubrum

particles

the

chlorophyllous

bottom

phase.

Chemical

Co.)

and 1%

(w/w)

extract

to give

final

in

4000

(Fisher

were

added

the

to

crude

and 0.2% (w/w) 0.2

M glycylglycine

at the fuged *

same time.

The tubes xg.

by USPHS Grant

l$

Duplicate

pH 8 instead

20 min at 37,000 Supported

Aqueous

respectively.

the

light,

pressure

at O-5”,

In

trated

French

in

purified

cellulose

system

the

grown

cell

1962).

dextran-methyl this

were

of crude

using

(w/w)

Dextran

(1960). are

"blank"

2000

tubes,

extracts,

The resulting

bottom

concen-

cellulose (Pharmacia)

concentrations

thoroughly

415

biphasic

methyl

were mixed

GM-07023-07

the

of 0.68% containing were

prepared

and centrifluid

phases

Vol. 23, No. 4, 1966

were

BIOCHEMICAL

removed

by capillary

tube

extract

the

was diluted

of the

crude

with

extract

The precipitate

Absorbance

a "blank"

The bottom

phase

The washed

precipitate

dark

at O".

changes

were

measured

90" to

for

the

the

at 25"

analyzing

analyzing

obto that

1 hr at 100,000

repeated, in the

and

finally equal

same volume

path

tube,

to a volume

and centrifuged

each crude

of buffer

and

was stored

with

a cuvette

beam;

beam with

xg.

the

which

cuvette

infrared

was

light

path 3.5 mm). The infrared light, 0.7-1.0 ~1, 1.0 x 106 -2 -1 (measured with a YSI Kettering Model 65 Radiocm set

ergs meter,

Yellow

Springs

Instrument

lamp filtered

by flowing

This

maximal

produced

relative

light

oscope

will

Each cuvette

1951)

Bazire,

et al.,

1957))

at

Results R. rubrum

(band

contained

et al.,

sorbance

were

in

determined

with

the

detail

an oscill-

infrared

light.

(0.5

suspension

light as noted

induced in

previous

416

final

paper.

mg protein

bacteriochlorophyll

phosphate,

of the

in a subsequent

particles

in

a single

(Cohen-

1 ml of 50 mM glycylglycine,

880 rnp of this

- Infrared

were

spectra,

2 rn@) employing

R. rubrum

and 20 mumoles

No. 2600.

The kinetics

controlling in

Filter

determined

width,

and 5 mM potassium

extracts,

cuvettes,

be described

(Lowry,

10 ml'4 MgC12,

changes

by a shutter

equipment

by a tungsten

Difference

Spectrophotometer,

photometer

triggered

in absorbance.

reference

absorbance

beam recording

was provided

(2 cm) and Corning

changes

14 Recording

induced

Co.),

water

to unilluminated

a Gary Model

This

from

from

in the

had a 10 mm optical

(light

phase

phase

was resuspended

in glycylglycine

at

top

glycylglycine

used

centrifugation

irradiated

with

RESEARCH COMMUNICATIONS

bottom

was repeated.

tained

the

pipette;

was mixed

centrif'ugation

AND BIOPHYSICAL

5 mM ADP, pH 7.8.

The ab-

was 3.0.

absorbance work

changes (Geller

in

these

and Lipmann,

Vol. 23, No. 4, 1966

BIOCHEMICAL

1960) a, However,

unlike

sorbance

were

turned

changes off,

The djeficit

3 min.

for

provided in

previous only

changes

were

the

photo-induced

reversed

when the

period

reached

a maximum after

Upon addition

of

succlnate

irradiation,

almost

RESEARCH COMMUNICATIONS

illumination

absorbance

NO

results,

partially

the

1) or following

(Fig.

AND BIOPHYSICAL

the

completely

exceeded

ab-

light ten

was seconds.

illumination

(1 mM) either

prior

to

light-induced

absorbance

reversible.

SUCCINATE

ADDITION

A .A t.020

.ooo -.OlO

400

500

600

400 Am

500

600

P

Figure 1. Effect of succinate (1 mM) upon the reversibility of lightminus dark difference spectra. The base line ( A = 0.000) was obtained, followed by the difference of absorption during infrared illumination ("light"). Following 3-l/2 min illumination, the light was turned off and the difference in absorption again The "dark" curve was stable determined ('dark") within 2 min. for at least 9 min. The effects the

"light-on"

were analyzed

by measurements

and "light-off"

reactions

420 and 435 rnp (Bartsch,

1963),

19551) and 605 rnv (Geller

and Lipmann,

actiions which

at these ranged

The "light-on" longed

prior

wavelengths

from

500 to

constants

selected

428 rnlJ- (Smith 1960).

had first 700 set-'

were

at

of the

not

irradiation.

417

order

at the changed

light

kinetics

of

wavelengths:

and Baltscheffsky, The "light-on" reaction intensity

by succinate

re-

constants used. or pro-

Vol. 23, No. 4, 1966

On the

other

BIOCHEMICAL

AND BIOPHYSICAL

hand,

magnitude

the

was decreased

by continuous

succinate

DPNH),

(or

"light-off" slower

than

the

off"

"light-on",

by tracings reaction

changes

and increased

in Fig,

1.

by

The rate

of the

two orders

of magnitude

was markedly

influenced

by prolonged

(or

DPNH).

of an oscilloscope

at

"light-off"

was at least

and by succinate

irradiation, trated

which

reaction,

of the

irradiation

as reflected

RESEARCH COMMUNICATIONS

435 rniL (Fig.

The results

recording In the

2).

are

illus-

of the

"light-

of

succinate,

absence

I

NO ADDITION

SUCCINATE

I 4

I 2

I 0

I 8

I 6

I 0

SECONDS

1 .02

AFTER

I .06

I .04

I .08

“LIGHT-OFF”

Figure 2, Effect of prolonged infrared irradiation on the in signal (in millilight-off" reaction at 435 rnp: variation volts with time following "light-off" after a 10 set (curves 1 (curves 2 and 4) of illumination in the and 3 I or 3 min period presence (curves 3 and 4) or absence (curves 1 and 2) of 1 mM succinate. prolonged parallel rate

irradiation

induced

experiment was only

slightly

An example

of the

100 msec and the shown in Fig.

in the

3.

a profound

presence

depressed

(O-10

After

irradiation

succinate

by prolonged

evaluation

overall

of

depression

of the see)

in

418

the

rate,

A

that

the

showed irradiation.

kinetics

"light-off"

in

of the reaction

absence

initial is

of succinate,

Vol. 23, No. 4, 1966

the

BIOCHEMICAL

reaction

tion

followed

first

was irradiated

subsequent

AND BIOPHYSICAL

again

'light-off"

RESEARCH COMMUNICATIONS

order

kinetics.

When this

in the

presence

of succinate,

reaction

was polyphasic.

SECONDS

AFTER

preparathe

The Init-ial

100

“LIGHT-OFF’

Figure 3. The effect of succinate upon the kinetics at 435 ml-1, following after 3 min illumination. In the absence of succinate j the total change in absorbance (AA total) was -0.0038. Succinate (1 mM) was added and the cuvette was illuminated again for 3 min (curve 2); AA total was -O.OIOO,l The values of k (for thp first 20 msec) were 0.5 and 7.5 set respectively (0.24 set for 0 to 10 set in the absence of SW'cinate). LS,A total was determined as the magnitude of the "lighton" reaction after a 30 set dark "recovery"period following the radiation pretreatment. msec (2C$ of the initially in

following

rate

tion

rate

selected

order

Following

be noted

succinate,

(O-10

consisted

of a rapid

course, the

with

first

reaction

a distinct

100 msec,

break the

reac-

declined.

wavelengths

succinate, sec.-l

msec.

rapidly should

reaction) a first

at 20-40

It

with

total

6-8

O-4-0.5

that

(vide -set-l see-'

the

"light-off"

infra) (for (for

did the

the

set).

419

first first

not

kLinetics differ 20 msec) 20 msec)

at various

significantly: and without or 0.24

to 0.27

Vol. 23, No. 4, 1966

BIOCHEMICAL

The significance off"

reaction

(for

the

on the

(and DPNH) required

at 435 mp.

The rate

phase) in

(measured

given

in Methods

in

air

parallel

fashion,

requiring

a minimum

For

photophosphory-

maximum for

30 min,

This

level

to

that

not

affect

of

medium

and Lipmann,

of 20 and 80 IJM for

(by Lineweaver-Burk

note

in

a maximum velocity

TPNJJ (at

or stimulate

of TPNH did

the

(Geller

with

protein/hr

changes

using

had Km values

and DPNK, respectively,

photophosphorylatioh

effect.

techniques

same preparation

port

constants

of the

at 30"

was of interest

concentration

magnitude

and previous

I-lmoles ATP formed/mg

"light-

and the

or DPNH for

lation

It

succinate the

10 Ils\l succinate

succinate

of

by determining

increased

the

effect

RESEARCH COMMUNICATIONS

examined

initial

absorbance

1960),

of the

has been

of succinate

AND BIOPHYSICAL

the

plot).

10 @)

did

"light-off"

the

stimulatory

spectra

(Fig.

of 11

not

sup-

reaction. effects

of

DPNH.

Discussion longed the tion

irradiation

loss

of unsupplemented

of a portion

of the suggest

sents

the

that

the

effect

requirement

DPNH) suggests absorbance reaction

contribution

that

changes

similar

seen in the of inactive

particles

repre-

polyphasic of succinate

may be involved

might

or be otherwise

(or

in both

The slow

of activator

DPNH)

Furthermore,

concentrations

absence

(or

illumination

and the

and photophosphorylation.

"light-

then

be the

unrelated

photophosphorylation. Additional

evidence,

to be detailed

420

in

to

The restora-

activators.

common components

pro-

leads

by succinate

of continuous

of photophosphorylation for

extracts

reaction.

reaction

of endogenous

reaction

1) show that

R. rubrum

"light-off"

"light-off'!

consumption

"light-off"

off"

of the

complete

would

the

- The difference

a subsequent

paper,

to

Vol. 23, No. 4, 1966

strong:Ly tion

suggests

seen with

transport (1)

a istudy

tion

rnycin,

the

succinate

AND BIOPHYSICAL

rapid (or

concerned

phase

the

within

(2) and the

"light-off"

observation

a variety

with

the

flashing

than

(e.g.,

only

process:

light

the

has fully phosphoryla-

100 msec following antimycin

phenylhydrazones), change

reac-

of electron

(1962) that

of less

of inhibitors

"light-off"

phosphorylation

in

period

carbonylcyanide reaction,

of the

of Nishimura

a dark

RESEARCH COMMUNICATIONS

DPNH) is a measure

of photophosphorylation

occurs

a flash;

that

directly

confirmed

the

BIOCHEMICAL

rapid

which phase

A,

oligoaffect

(Geller,

1966). REFERENCES of Cell Particles and Macromolecules," Albertsson, P., "Partition Jc'hn Wiley and Sons, New York, 1960, Bartsch, R. G., in H. Gest, A. San Pietro, and L. P. Vernon (E:ds.), "Bacterial Photosynthesis", The Antioch Press, Yellow Springs, Ohio, 1963, p. 475. Cohen-.Bazire, G., Sistrom, W. R., and Stanier, R. Y., J. Cellular Comp. Physiol. 3, 25 (1957). Geller, D. M., J. Biol. Chem. 237, 2947 1962 . Proc. 25, 737 I 1966 1 . Geller, D. M., Federation Geller, D. M., and Lipmann, F., J. Biol. Chem. 235, 2478 (1960). Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J, J* Biol. Chem. 193, 265 (1951). Biophys. Acta 57, 88 (1962). Nishiriura, M., Biocm. Chem. 234, 1575 (1959). Smith:, L., and Baltscheffsky, M., J. BEl.

421