Initiation of succinate oxidation in aged pigeon heart mitochondria

Vol. 3, No. 1

BIOCHEMICAL

July 1960

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

INITIATION OF SUCCIXATROXIDATIONIN AGEDPIGEONREARTBlITOCHONDRIAl Rritton

Chance and Eunji Hagihara*

Johnson Research Foundation University of Pennsylvania Philadelphia, Pennsylvania Received June 14, 1960 In studies of the activation mitochondria,

it

of respiration

observed sometime ago that pretreatment of the mitochon-

was

dria with ADP for increasing intervals respiration also

by succinate in rat liver

caused an increasing inhibition

(B. Chance and-G. R. Williams, unpublished observations).

of It was

observed that the reduction of pycidine nucleotide by succinate was de-

layed.

I.&e recently,

WilMams (1960) has shorn this lag in mitochondria

incubated with high K', dinitrophenol, In a recent investigation

and in the absence of phosphate.

of the stability

against aging, under aerobic conditions,

of m$tochondrial preparations

without added substrate,

pigeon heart mitochondria have been examined in detail ration.

control,

maximal respiration investigated nication). activity

somedays after

Although these aged mitochondria showedgood respiratory

and respiratory

it was observed that a minute'or

with succinate was achieved.

by other workers (M. Wgenberg

prepa-

activity

so elapsed before

This poblem is also being

and L. Easter,

Since succinate is a substrate which elicits

personal corn-

a high respiratory

within a second after -its addition to non-phosphorylating

Hartree heart muscle particles,

a number of

Keilin

and

experiments have been carried out to deter-

mine the site at which the respiratory

inhibition

occurs.

This research was supported in part by a grant from the National Science Foundation. 2

PWmanent address: Japan.

Department of Biology,

1

University

of Osaka, Osaka,

Vol. 3, No. 1

BIOCHEMICAL

Materials

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Respiration

end Methods.

num microelectrode

was measured by the vibrating

(Chance and Willisms,

1955).

Pyridine

was measured by the double-beam spectrophotometer 374 mp as the measuring

fluoranetrically tion

and Rference

July 19do

nucleotide

plstireduction

(Chance, 1954) with 340 and

wavelengths,

respectively,

and also

(chance, Conrad, and LZ~~IJB~S, 1958) with 365 mp exita-

and 450 mpmeasuremsnt.

Flavoprotein

was measured spectrophotanetrically

With 460 and 510 rnp as the measuring and reference

wavelengths,

respectively.

Pigeanheartmitochondriewere~~edbyanerproceduredevelopedin this

laboratory

subtilus

(B. Esgihma,

~oteinase.

tury control

efficiency

1.0 mM "ia3,"

substrate

carriers.

details

of the experiments legend.

Themitochondria

Results.

were preserved to

in 0.23 M msnnitol,

of 5 mM succinate

platinum

microelectrode

0.g

M sucrose,

mitochondria

diluted

de-

and contained

exceptthatversenewas

on the figures

themselves

and in the

c concentration

(succinate, of

Other

state 3). the activation

in the aerobic

reaction

of

medium.

in state l(~~~~,butnocrd8edADPgrresent). produces no measurable

trace

(0.2 @d02/sec.)

of the carriers,

of the slope of the

f0r half a minute.

should result

At the end

f!rcea changes in the

the oxidation-reduction

consideredofmostsignificance. of flavoprotein,

increwnt

to 0.6 pi O&ec.

Since such changes in respiration

level

similar

used here, the c@xhrane

the rate has increased

levels

Intermediates

Fig. 1 shows the time course

are initially

Addition

steady-state

to about 2 and the

lhese activities

number being 5.5 sec. -'

in pigeon hew

pvteinwas

diminish

With one or

was 0.02 M phosphate 0r "trls."

are included

In the prepsration

EqxrJntental

steady-StstX

ratios

andhigh-energy

concentration

was 0.95 pM, its turnover

of a minute,

control

llrereactionmediumwas

and the buffer

respiraticm

of 6 and a P:O value of 1.8.

of the mitochondria

witted

figure

have respira-

and 0.05 ml4 versene at 0' C. Ihe addedmitochondriawere

pletedofenclogenous oxidized

the use of Racillus

these mitochondria

to about 1.5.

extent by aeration

involve

wed,

with succinate

the respiratory

phosph0rylation this

When freshly

ratios

two days' aging,

in preparation)

Level of flavo-

The lowest trace shows the sn upward deflection 2

carresponding

to a

Vol. 3, No. 1

BIOCHEMICAL

July 1960

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

5mM

4

Ftg. 1. are&iy

. 4

T Flovopmteh

0.003SpMhrc

Reduction

A study of the aela yea activation

p----SO%-+

of succinste respiration

in

aged pigeon heart mitochondri.8 (P. H. '21) suspended in the aerobic

pcdiumdescribed

inthetext.

measuredby the vibrating

The top trace represents reSpil'8tQty platinum microelectrode;

nucleotide reduction recorded fluorometricaUy; Hnetics

of rlavoprotein

11 cm-l mM-? D&Z?~C~

the middle trace pyridine

and the bottom trace the

reduction recorded 5pectrophotometricall.y.

rates or reduction of flavoprotein !Be rate of pyridine

The

are directly

calculated using A& =

nucleotide

reduction measured fluoro-

i5 Calculated fran independent Spec~OphOt~tXiC

0.95 * cytocti

activity

c. Optical path, 1 cm. 26' c.

@pt.

diminution 0r absorption at 460 rnp and to rlavoprotein able reduction occurs inuuediately following second5 elapse before an upward deflection

c8librations. 101-54).

reduction.

substrate addition,

Ho detectand several

or the curve is observable.

At

half 8 minute, there is 8 sharp break in the trace which moves upward at an acceler8ted rate until after

the steady state is reached, approximately

treatment of the mitochondria with succinate.

which flavoprotein

140 sec.

The actual rate5 with

reaches the Steady state are very slow campared to the

rate with which oxygen is being reduced.

In the initial

30 sec., the rate

is 3.5 x 10m3 pt4 fp/sec ., and the accelerated rate is 15 x 10m3 p&l fp/sec. The inhibited

phase of respiratory

activity

is characterized

by 8 scarcely

me8SLW8blereduction of flavoprotein. It has been noted that succinate increases the reduction 3

of pyridine

Vol. 3, No. 1

BIOCHEMICAL

nucleotide

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in mitochondria to a greater

(Chance and Hollunger,

1960).

extent than any other substrate

It is therefore

what point in the time course of respiratory is reduced.

of interest activation

Therefore a simultaneous fluorometric

nucleotide reduction

is included.

nucleotide reduction

deflection

Although the fluorometric

cate quantitatively

the amount of pyriaine

spectrophotometric

calibrations

to determine at

pyridine

nucleotide

recording of pyri.dine

In this case, an increased fluorescence

corresponding to pyridine of the trace.

July 1960

indicate

is indicated by a downward recording does not inai-

nucleotide

present, independent

that the amount,finally

reduced in

this recording corresponds to 8 times the amount of cytochrome c present or 7.8 @DPNIF. lhe reduction

of pyridine

nucleotide

following

the addition of succinate

does not proceed at a measurable rate for an interval after

an abrupt reduction at a rate of.0.25

noted that by the time pyridine rate,

the respiratory

pyridine

activity

nucleotide reaches its

In order to indicate

of flavoprotein

activated

respiration.

oxidation riers

is increasing significantly

It should be

and that the

steady state reduction prior to flavoprotein. activity

has been

ADP is added and the typical

cycles of

and pyridFne nucleotide accompanythe interval

The conditions that lead to an inactivation

are associated with an oxidized

state of the respiratory

cm-

Thus we may conclude that scmeessential ccmponent has

become oxidized.

The site of inhibition

of succinate oxidation

is at

the succinic dehydrogenase end of the chain since it is observed that flavoprotein

is not reduced on adding succinate.

Respiratory cleotide

activity

becamereduced.

increased as flavoprotein

and pyridine

It would appear that flavoprotein

started before that of pyridine

nucleotiae. 4

Au explanation

of

of succinate

(state 2) in which the mitochondria are depleted of high-energy

intermediates.

There-

nucleotide reduction has reached its maximal

achieved in this incubation period,

Discussion.

liM/sec. is recorded.

that a high degree of respiratory

oxidation

of half a minute.

nu-

reduction of the

vol. 3,No. 1

results

BIOCHEMICAL

July 1960

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is afforded by subsequent experiments showing that pyridine

nucleotide

reduction and respiration

can be reactivated

in such pre-

parations by the addition of ATP. A single explaaetion for the activation pyddine

nucleotide reduction

essential for optimal activity requiring reduction. oxidation

It is more likely,

and

is that high energy intermediates in both reactions,

ena pyriaine

activation

of respiration

succinate itself

nucleotide requiring

energy for its

however, that the activation

requires only the reduction of someoxidizable

of succinate

component

of the system, and that this reduction

is mediated by high energy

intermediates.

is consistent with the

The latter

explanation

observations that succinate can be rapidly

oxidized

mitochondria or in mitochon@ia pretreated

with amytal (Williams,

l@O),

whereas the former explanation is-not.

of pyriaine

nucleotide

are

in the uncoupled

However, the reduction

in the presence of succinate clearly

requires

ATP or endogenoushigh energy intermediates.

Chance, B. (1954).

Science, 1x), 767.

Chance, B., Conrad, H., -a Iegallais, V. (19%). Program ana Abstracts, Biophyslcal Society Meeting, Cambridge, Mass., p. 44. Chance, B. and HOI&anger, G. (1960). Chance, B. and Williams, G. R. (Lg55). WiUiams, G. R. (1960).

Nature, 2, J. Biol.

666. Chem. 22,

383.

ProC. Canadian Fed. Bi01. Sot. 1, 53.