Biochem. J. (1960) 76, 47
Barbiturates and Oxidative Phosphorylation
By W. N. ALDRIDGE AND V. H. PARKER
Unitfor Research in Toxicology, M.R.C. Laboratories, Carshalton, Surrey
(Received 23 October 1959)
It has been claimed that barbiturates uncouple
oxidative phosphorylations (Brody & Bain, 1954;
Brody, 1955). These authors have demonstrated
that during the oxidation of pyruvate by liver and
brain mitochondria, phosphate uptake was lowered
proportionately more than oxygen uptake. Support
for the uncoupling theory was derived from certain
similarities between the barbiturates and
dinitrophenol (Brody & Bain, 1954). 'The slopes of
the inhibition curves of these compounds are
remarkably similar although dinitrophenol is the
more potent agent. Both depress fatty acid oxid-
ation and stimulate oxidative rate in a phosphate-
deficient system. The addition ofexcess magnesium
ion does not reverse the uncoupling action of either
the barbiturate or dinitrophenol' (Brody, 1955).
Further, barbiturate hypnosis is potentiated by
2:4-dinitrophenol (Killam, Brody & Bain, 1958).
None of these arguments is conclusive evidence for
uncoupling, and they ignore the major difference
that, in vitro, barbiturates inhibit (Bain, 1952)
whereas 2:4-dinitrophenol stimulates respiration
(Loomis & Lipmann, 1948; Parker, 1958). This
difference is readily demonstrated in vivo, for after
administration of barbiturates oxygen consump-
tion (Costa & Bonnycastle, 1955) and body temper-
ature (Birnie & Grayson, 1952; Lessin & Parkes,
1957) fall, whereas 2:4-dinitrophenol causes a rise
of both oxygen consumption (Cameron, 1958) and
body temperature (Stoner, 1956).
ences in behaviour in isolated systems have also
been demonstrated (Johnson & Quastel,
Jalling, Low, Ernster & Lindberg, 1957; Messer,
1958). Although the supporting evidence for the
Bain (1954) have nevertheless demonstrated a
lowering of the phosphorylation quotient (P/O
Respiration, with pyruvate as substrate, of the
liver mitochondria used in this paper can be in-
hibited 50% without any decrease of theP/Oratio
(Aldridge, 1957). With such preparations we have
re-examined the effect ofoxy- and thio-barbiturates
upon oxidative phosphorylation.
dissociating the process of oxidation from that of
oxidative phosphorylation by the intervention of
1958), we have studied the effects of
is not conclusive, Brody &
A preliminary report of these results has already
appeared (Aldridge & Parker, 1958).
Manometric experiments. The reasons for the composi-
tion and pH of the medium have been discussed by
Aldridge (1957, 1958). For measurements of respiration
each flask contained 3 ml. of a solution containing adeno-
sine 5-phosphate (1-15 mM), adenosine triphosphate (ATP;
1.06 mm), KC1 (10 mm), MgCl2 (14mm), ethylenediamine-
tetra-acetic acid (EDTA; lO mM), potassium phosphate
(50 mm), sucrose (30 mm) and substrates (10 mM, except
fumarate mm). For measurements ofoxidative phosphoryl-
ation, glucose (60 mM), glycylglycine (16.7 mM) and hexo-
kinase (200-400 units) were added to the above-mentioned
mixture. In each case the medium was adjusted to pH 6.7-
6-8 with KOH. For either type of experiment 0*3 ml. of a
suspension of mitochondria in 0-3M-sucrose, equivalent to
150 mg. wet wt. of liver, was used. For experiments on
oxidative phosphorylation, when a range of concentrations
of barbiturate was examined (method 1), P uptake was
measured between 10 and 22 min. after placing the flasks
in the bath at 370. Uptake of02for the same period was
calculated from the slope of the best straight line through
manometer readings at 10 min., 13 min. 20 sec., 16 min.
40 sec. and 20 min. For more accurate determinations of
P/0 ratio six flasks were used for each determination
(method 2). Manometer readings were taken at 10, 14, 18,
22 and 26 min. and the reaction was stopped by the addi-
tion of 6-5 ml. ofice-cold 5% (w/w) perchloric acid at 7, 11,
15, 19, 23 and 27 min. Thus P uptake was calculated from
the slope ofthe regression line through six values. Owing to
the successive removal of flasks for the determination of
inorganic phosphate, the slope of the regression line for 02
uptake was calculated from four readings at 10 min., four
at 14 min., three at 18 min., two at 22 min. and one at
26 min. (a total of 14 readings). The standard errors ofthese
slopes were calculated (Snedecor, 1946) and thus the phos-
phorylation quotient (P/O ratio) and its standard error
could be determined (Langer, 1951).
ratios have standard errors less than 10% of the ratio, the
difference between the control and experimental values has
been tested for significance (t test) on the basis of their
being derived from a large sample (Snedecor, 1946).
Preparation of the mitochondria. Mitochondria were pre-
pared as previously described (Aldridge, 1957, 1958), with
a Potter-Elvehjem-type homogenizer with a smooth glass
tube and Perspex pestle, with a total clearance of 0-02 in.
for rat liver and 0.01 in. for rat brain.
Adenosine-triphosphatase activity. Each beaker contained
3 ml. of a solution containing ATP (3 mm), KCI 5
Since these P/O
W. N. ALDRIDiE D V. H. PARKER
,MgCl2 (14 mM), EDTA (mMJA and sucrose (30 mM). The
beakers were shaken in air at 37°.
equilibration mitochondria were added and the mixture
was incubated for 10 min. The reaction was stopped by the
addition of 0 5 ml. of ice-cold 65% (w/w) perchloric acid
and inorganic phosphate was determined.
Special chemicals and reagents. The following chemicals
were obtained from the sources indicated: adenosine 5-
phosphate, glycylglycine, sodium pyruvate (Roche Pro-
ducts Ltd.); disodium salt of ATP (Sigma Chemical Co.,
St Louis, Mo., U.S.A.); glucose, sodium fumarate, 2:4-
dinitrophenol (DNP; British Drug Houses Ltd.). Phenyl-
arsenious acid was synthesized and used as described by
Aldridge (1958). The following barbiturates have also been
used: sodium 5-ethyl-5-phenyl barbiturate (Phenobarbital:
methylbarbituric acid (Hexobarbital: Mayand Baker Ltd.);
sodium 5-ethyl-5-isoamylbarbiturate (Amytal: Eli Lilly
and Co. Ltd.); sodium 5-ethyl-5-(1-methylbutyl)-2-thio-
thal: Imperial Chemical Industries Ltd.); sodium 5-allyl-5-
isobutylthiobarbiturate (Baytenal: Farbenfabriken Bayer,
A.G., Wuppertal-Elberfeld, Germany).
Hexokinase was prepared from baker's yeast by a modi-
fication by V. H. Parker (unpublished work) of the method
of Berger, Slein, Colowick & Cori (1946). The preparation
was taken to the equivalent of step 3a and, when assayed
by their procedure at 370 (instead of 300), had an activity
of 3500 units/ml.
Potato apyrase was prepared by the
method of Lee & Eiler (1951). This preparation liberated
1100ug.atoms of inorganic phosphate/hr./ml.
Inorganic phosphate was deter-
mined by the method of Fiske & Subbarow (1925). Protein
was measured by the biuret method ofRobinson & Hogden
(1940) as modified by Ald
pressed as mg. of albumin.
Purification of barbiturat
supplied were dissolved in
acids by the addition ofHC
were washed repeatedly a
were recrystallized as fobo
ethanol, m.p. 176° (uncorre
m.p. 146-5-147-5°; Amyta
Thiopental from water-d
1620; Baytenal from benz
was recrystallized from w
forms, one melting at 1240 a
is converted into the latter
Codex, p. 394, 1953). All
stock solutions of the free a
0-03 ml. of various concent
a constant final concentra
formamide always being pr
Units. The oxidative acti
expressed aspl.of O0/m
activity asjug.atomof P li
inhibitory power of a subs'
the negative logarithm of
will produce 50% inhibitio
are expressed as mean±S.E
tions upon separate pre]
A comparison of the in]
produced by the oxyb
barbital and Phenobari
lridge (1957), and has been ex-
tes. The sodium barbiturates as
water, converted into the free
11 and the precipitated free acids
After drying they
ws: Phenobarbital from water-
ycted); Hexobarbital as supplied,
al from benzene, m.p. 156-.5;
rene, m.p. 147-148°. Kemithal
It exists in two
and the other at 140°; the former
on prolonged heating (Brit. Vet.
barbiturates were prepared as
acids in dimethylformamide and
trations was added to the flasks,
Ltion of 1% (v/v) of dimethyl-
ivity ofthe mitochondria (qo2) is
ig. of protein/hr. and ATPase
iberated/mg. of protein/hr. The
;tance is given as itspI50 value,
the molar concentration which
n. Where errors are given these
with the number ofdetermina-
,parations of mitochondria
ation by liver mitochondria.
hibition of 02 and P uptake
arbiturates Amytal, Hexo-
bital is illustrated in Fig. 1.
Inhibition of 02 uptake (%)
Fig. 1. Effect of oxybarbiturates on oxidativephosphoryl-
ation by rat-liver mitochondria withpyruvateas substrate.
All values were obtained by method 1. The broken line
shows where points should be if theP/0ratio isunchanged
and the continuous line the calculated beststraightline.
1.0 mM); *, Phenobarbital (0.5-1.0 mM). The mean qO2
was 113-2±4-6 (6) and theP/Oratio 2-67±0-08 (6).
Inhibition of 02 uptake (%6)
Fig. 2. Effect ofthiobarbiturates on oxidative phosphoryl-
ation of rat-liver mitochondria with pyruvate as substrate.
All values were obtained by method 1. The broken line
shows where points should be if the P/0 ratio is unchanged
and the continuous line the calculated best straight line.
*, Thiopental (0.06-1.0 mM); A, Baytenal (0.2-1.0 mM);
*, Kemithal (0.1-0.4 mM). The meanqO2was 107-8±3-4
(5) and the P/0 ratio 2-56±0 11 (5). When 02 uptake is
50% inhibited, P uptake is inhibited by 68-69 %.
BARBITURATES AND OXTDATIVE PHOSPHORYLATION
Table 1. Effect of barbiturate8 on pyruvate oxidation and coupled pho8phorylation of liver mitochondria
For details of technique used see method 2, described under Methods. The control and experimental values
were obtained by parallel experiments upon the same preparation of mitochondria. Results are expressed as
mean±s.E. Meanqo for controls is 90-5±1-2 (12). pC = -log conen. (M).
Oxidative phosphorylation (P/O ratio)
1*90 (P 0 06)
1-55 (P 0.13)
2-04 (P 0*05)
0-87 (P 0*4)
0-81 (P 0-41)
8.18 (P < 0 01)
2-66 (P 0-013)
5.77 (P < 0*01)
10*4 (P <0-01)
941 (P < 0.01)
4.6 (P < 0 01)
2-4 (P 0 02)
Table 2. Influence of barbiturate8 on adenosine-triphosphatase activity, 8welling
and uptake of oxygen of un8timulated mitochondria
Values for ATPase are all corrected for the activity of the mitochondria in the presence of solvent (1%, v/v,
dimethylformamide), which was 073±007(6)pg.atomofP/mg. ofprotein/hr. In the presence of0 03 mm-DNP
their activity was 11x9±0-13 (6). Swelling was measured by measurements of E at 530mpin a Unicam DG.
spectrophotometer, the mitochondria being suspended and incubated at 370 for 10 min. under conditions identical
with those used for the ATPase assay. Uptake of02was measured in the presence of pyruvate and fumarate.
Meanq°2was 22*4+0-28 (6) and, in the presence of0 03 mm-DNP, 82-9±1.6 (6), giving a stimulation of 3-7-fold.
pC = -log conen. (M).
(yg.atoms of P/mg.
The broken line shows where the points will fall
with an unchanged P/O ratio. There is clearly little
indication that the inhibition of P uptake is any
greater than that of 02 uptake. In contrast, the
thiobarbiturates Thiopental, Kemithal and Bay-
tenal impaired P uptake more than 0, uptake, so
giving a lower P/O ratio (Fig. 2). As pointed out
in the Methods section these results were obtained
with a technique when the P/O ratio for each con-
measurements of 02 uptake in one Warburg flask
only. By using more flasks for the assessment of
the rates of 02 and P uptake, the standard error of
each P/O ratio can be obtained. In Table 1 are
shown the results of such experiments with six
flasks for each concentration of barbiturate. For
Hexobarbital and Phenobarbital the difference
between the P/O ratios when
02 uptake is 40-
Uptake of 02
or decrease ( - )
60% inhibited was not significant; the significance
for Amytal wasP approx. 0 1. For thiobarbiturates
there was a significant difference between the P/O
ratios. Even with quite a small lowering of 02
uptake (cf. Kemithal 0 1 mm) there was a marked
lowering of theP/Oratio.
Adeno8ine triphosphatase of liver mitochondria.
DNP, as well as uncoupling oxidative phosphoryl-
ation, inducesATPase activity inlivermitochondtia
(Lardy & Weilman, 1953).
interest to see whether the barbiturates, and parti-
At concentrations which inhibit
uptake about 50%, Amytal, Phenobarbital and
Hexobarbital gave an increase of ATPase activity
of less than
pg.atom of P/mg. of protein/hr.
Under similar conditions the thio-
an activity of more than
It was therefore of
Bioch. 1960, 76
W. N. ALDRIDGE AND V. H. PARKER
3,ug.atomsof P/mg. of protein/hr. This activation
of ATPase was not associated with swelling of the
mitochondria (Table 2).
four times as high as than those given in Table 2 did
not produce measurable changes in the extinction
ofthe mitochondrial suspension. The stimulation of
activity by the thiobarbiturates was
associated with stimulation of 02 uptake (Table 2,
Figs. 3 and 4) with pyruvate as substrate. No
stimulation of 02 uptake was found at any concen-
tration of the oxybarbiturates (Table 2).
Since the ATPase activity elicited by DNP may
be regarded as accounting for its uncoupling action
(see Discussion), a comparison has been made
between the action of the thiobarbiturates and of
DNP in lowering the P/O ratio, by choosing con-
centrations of the different uncoupling agents
that liberated equal activities of ATPase. When 02
uptake is 50% inhibited by thiobarbiturates, P
uptake is 69 % inhibited: an increase of 19% over
that required for the maintenance of a constant
P/O ratio (Fig.
measurements of oxidative phosphorylation was in
the range pC 3 4-3-7 (Tables
ATPase liberated by these concentrations was
3-28 jg.atoms of P/mg. of protein/hr. (Table 2).
The relationship between the concentration of
DNP and the activity of ATPase which it induces
conditions. It was found that 4,uM-DNP induced
the same ATPase activity as did the thiobarbitur-
ates and inhibited P uptake by 25-2 ± 1-8%and 02
uptake by 4-0 + 1-6 0/ (mean + S.E. of three deter-
minations upon the same preparation of mito-
chondria). The difference between the percentage
inhibition ofP and 02 uptakes is 21-2, in agreement
with 19-0 obtained with the thiobarbiturates.
was concluded that the relation between uncoupling
action and ATPase production was the same for the
thiobarbiturates as for DNP.
Adeno8ine-triphosphata8e activity and uptake of
oxygen induced by dinitrophenol. Uptake of 02 in
the presence of DNP reflects the activity of the
non-phosphorylating respiratory chain and ATPase
activity induced by DNP is a measure of the
activity of the energy-transfer mechanism when it
is dissociated from the respiratory chain; hence the
use of DNP enables separate study of these two
parts of the complete mechanism (see Discussion).
A comparison has therefore been made of the
action of the various barbiturates on 02 uptake in
the presence of DNP and the action of DNP in
inducing ATPase activity. The results in Table 3
show that 02 uptake in the presence of DNP was
inhibited by concentrations of oxybarbiturates
similar to those which inhibit 02 uptake in the
presence of hexokinase and glucose. The mito-
2). The concentration of thio-
1, 3). The mean
chondria were consistently rather more sensitive to
barbiturates when in the presence of hexokinase
and glucose than with DNP though in no case was
the difference more than twofold.
ATPase activity induced by DNP is not inhibited
by the similar concentrations of oxybarbiturates
and even concentrations four times as high have no
measurable effect (Table 3).
Experiments similar to the above have been
carried out with the thiobarbiturates, but are more
difficult to interpret. The results obtained with
Kemithal illustrate the difficulties (Figs.
presence of DNP but the precise concentration at
which a 50% inhibition occurs is difficult to assess.
Taking the whole 02 uptake in the presence of
DNP, 50% inhibition
(Fig. 3). If a correction is made for the stimulation
of 02 uptake by thiobarbiturates, 50% inhibition is
at pC 3*55. Kemithal stimulates ATPase (Fig. 4)
and this increases with concentration.
concentrations of Kemithal which produce 50%
inhibition of 02 uptake (pI50 3-60, cf. Table 3) give
only a small lowering of DNP-activated ATPase,
nevertheless, unlike the behaviour of the oxy-
barbiturates, a pronounced lowering occurs with
higher concentrations. The interpretation of this
effect depends on whether or not a correction should
be made for ATPase activated by the barbiturates
themselves (cf. Fig. 4).
is obtained at pC 3-30
and activity of adenosine tripho8phata8e of liver mito-
chondria in the presence of dinitrophenol
Effect of barbiturate8 on uptake of oxygen
Uptake of 02 was measured with pyruvate as substrate.
The mean qo0 was 22-4+0-28 (6) and, in the presence of
0-03 mM-DNP, 82-9+ 1-6 (6). The values for po50in the
presence of hexokinase and glucose are the ranges obtained
from the experiments given in Table 1 and Figs. 1 and 2.
ATPase activity in the absence of DNP and barbiturate
but in the presence of solvent (1%, v/v, dimethylform-
amide) was 0-73 +0-07 (6)pg.atomofP/mg. ofprotein/hr.,
and, in the presence of 0-03 mM-DNP, 11-9+0-13 (6). For
the inhibition of ATPase in the presenceof DNP concen-
trations ofoxybarbituratesfour times as high as those in
column 2 were used.
Inhibition of 02 uptake
In the presence
BARBITURATES AND OXIDATIVE PHOSPHORYLATION
It has previously been
demonstrated that succinate oxidation is unaffected
by barbiturates (Quastel & Wheatley, 1932-33) and
more recently that phosphorylation associated with
the oxidation of succinate is likewise not inhibited
by Amytal (Eiler & McEwen, 1949; Low, Ernster &
Lindberg, 1955). In the presence of Amytal one
atom of oxygen is consumed for every molecule of
Uptake of 02 in the presence of DNP
-log concn. (M) of Kemithal (pC).
Fig. 3. Effect ofKemithal on unstimulated02uptake and
on 02uptake stimulated by 2:4-dinitrophenol. Liver mito-
chondria were used with pyruvate as substrate: 0, in the
presence of30pM-DNP;0, in the absence of DNP. The
broken line is the difference between these curves in an
attempt to correct for the stimulation of 0° uptake by
Kemithal. The arrows indicate the concentration where 02
uptake is 50% inhibited: a broken arrow forthe uncorrected
and continuous arrow for the corrected curves.
succinate oxidized (Fig. 5), indicating that other
oxidations ofthe Krebs tricarboxylic acid cycle are
suppressed. We have confirmed that phosphoryl-
ation with succinate as substrate is not affected by
Amytal. Kemithal behaves in the same way with
succinate oxidation, the oxygen consumed being
one atom for every molecule ofsuccinate. However,
in agreement with the
pyruvate as substrate
stimulates rate of 02 uptake in the presence of
In view of the effects of
barbiturates in vivo it would have been desirable to
examine their influence upon the activities ofbrain
mitochondria. However, unlike liver mitochondria,
the respiration of our preparations of brain mito-
chondria declines during the course of the experi-
ment (Aldridge, 1957). This limits the work one can
do on oxidative phosphorylation. In addition, and
phenylarsenious acid which is generally believed to
inhibit a-oxo acid oxidases (Peters, 1955), inhibits
P uptake of brain mitochondria more than 02
uptake. A comparison of the effects of phenyl-
arsenious acid with the barbiturates is shown in
Fig. 6. These results indicate that for a given in-
hibition of 03 uptake (less than 60%) Amytal,
Phenobarbital and Hexobarbital do not inhibit P
uptake more than does phenylarsenious acid. In
results obtained with
3), Kemithal also
ATPase activity in the presence of DNP
-log concn. (M) of Kemithal (pC)
Fig. 4. Effect of Kemithal on ATPase with and without
DNP. The ATPase activity in the absence of DNP and
Kemithal but in the presence of solvent (1%, v/v, di-
methylformamide) was 0-63fg.atomof P/mg. of protein/
hr. This value has been subtracted from all results.
the presence of 0 03 mM-DNP; 0, in the absence of DNP.
The broken line is the difference between these curves.
Fig. 5. Effect of Amytal on 0, uptake with succinate as
Potato apyrase (1OjLg.atoms of P/hr.) was
added to each flask to produce maximal 0, uptake. Mito-
chondria (4-1 mg. of protein) equivalent to 150 mg. wet wt.
of liver were added to each flask. The02uptake during the
10 min. equilibration period was assumed to be at the same
rate as during the following 5 min. period. Each contained
30itmolesof succinate and, on the basis of 1pg.atom of
oxygen for each f&mole of succinate, complete oxidation
would require 336pl. of 02
0, with Amytal (1.0 mM).
0, Control without Amytal;
W. N. ALDRIDGE AND V. H. PARKER
contrast, the sulphur-containing barbiturates pro-
duce a much larger depression of P uptake. The
interpretation of results upon unstable prepara-
tions is necessarily difficult but we consider that
these results do not conflict qualitatively with the
results obtained with liver mitochondria. The
ATPase activity of our brain preparation is high
without the addition of DNP; therefore studies of
the effects of barbiturates upon DNP-activated
ATPase were not carried out.
For the purposes of this paper the following
definitions have been used. The respiratory chain is
regarded as a physiological entity consisting of all
the processes involved in the oxidation of sub-
strates and the associated phosphorylation. The
involving the transfer ofelectrons from coenzyme I
through the flavin and cytochromes to oxygen.
The energy-transferring chains are the processes
whereby the energy of oxidation of electron-
transport intermediates is used for the synthesis
from inorganic phosphate of the terminal pyro-
phosphate bonds of ATP (cf. Fig. 7). Since phos-
phorylation is coupled to oxidation, certain pro-
cesses are common to both the electron-transport
and energy-transferring chains.
Recognition that the rate of oxidation by mito-
chondria is dependent upon the availability of
Inhibition Of 02 uptake (%)
Fig. 6. Effect of barbiturates and phenylarsenious acid on
oxidative phosphorylation by rat-brain mitochondria with
pyruvate as substrate. The broken line shows where points
would be if the P/O ratio were unchanged. All values were
obtained by method 2.
+, Phenylarsenious acid;
Amytal; Eo, Phenobarbital; A, Hexobarbital;
pental; *, Baytenal; A, Kemithal. The mean qo2 was
106-3±6-8 (15) and the P/O ratio 2-26+0-05 (15).
phosphate acceptors (Potter & Recknagel, 1951;
Lardy & Weilman, 1952; Siekevitz & Potter, 1953;
Chance & Williams, 1955; Aldridge, 1957) has made
it clear that, just as formation of ATP requires
oxidation, oxidation requires formation of ATP.
Hence, inhibition of an enzyme involved in the
electron-transport chain or in the energy chain
between electron transport and ATP formation
will both lead to an inhibition of oxygen uptake
(Aldridge, 1958). By measurements of oxygen and
phosphorus uptakes, three possible results can be
obtained: no inhibition ofrespirationbut alowering
of the P/O ratio, inhibition of respiration with an
unchanged P/O ratio and inhibition of respiration
with a lowered P/O ratio. An example of the first
of these is the uncoupling action of DNP. The
second and third possibilities have been obtained
with the oxy- and thio-barbiturates respectively.
Therefore the oxybarbiturates inhibit respiration
but do not uncouple, whereas the thiobarbiturates
oxidative phosphorylation. The demonstration of
such a clear difference between oxy- and thio-
barbiturates has only been made possible by the
In the presence of DNP
Fig. 7. Diagram illustrating the respiratory chain and the
actions of drugs on it. Oxybarbiturates act in the area
including flavoprotein I and coenzyme I and up to, but not
including, the DNP-sensitive site. The electron-transport
chain is taken from Slater (1958). The horizontal arrows in
the lower half of the figure represent the boundaries of the
processes defined and discussed in the text. The broken
of electron and energy
BARBITURATES AND OXIDATIVE PHOSPHORYLATION
use of a stable preparation of mitochondria. The
work with brain mitochondria illustrates the diffi-
culties arising from the use of an unstable prepara-
tion. However, the results given by the oxybarbi-
turates with brain mitochondria are in general
agreement with those with liver mitochondria and
provide no positive evidence for uncoupling.
The concentrations of thiobarbiturates which
inhibit respiration and lower the P/O ratio also
activate some ATPase. An attempt has been made
to assess the significance of this ATPase in relation
to the uncoupling of oxidative phosphorylation by
a comparison with the action of DNP. Before this
assessment can be made it is important to know
whether the ATPase activity induced by DNP is
related to the lowering by DNP of phosphorus
uptake during oxidative phosphorylation. The
mean uptake of phosphorus for all the control
experiments in this paper (Table 1, Figs. 1 and 2)
during oxidative phosphorylation is 23-6 ug.atoms/
mg. of protein/hr. This uptake is virtually elimi-
nated by 0-03 mM-DNP and this same concentra-
tion in the absence of substrate induces an ATPase
activity of (11-9-0-7) =11-2,ug.atoms of phos-
phorus/mg. of protein/hr. (Table 3). This value is
less than the uptake prevented by DNP during
oxidative phosphorylation. However, higher values
have been obtained
when the incubation time was reduced from 10 to
2 min., thus giving some indication of the initial
rates of the reaction. These range between 17 and
19ptg.atomsof phosphorus/mg. of protein/hr. Thus
there is reasonable agreement between the ATPase
activity elicited by DNP and the uptake of phos-
phorus prevented during oxidative phosphoryl-
ation. Although there is agreement this does not
indicate that the action of DNP upon oxidative
phosphorylation involves the formation of ATP
before its breakdown. The action of DNP could be
due to the breakdown of a 'high-energy' inter-
mediate in the energy-transferring chain and the
ATPase results could be an experimental demon-
stration of the reaction sequence in reverse (Lardy,
1955). Experiments described in this paper have
shown that concentrations of thiobarbiturates and
DNP which induce the same ATPase activity
cause the same depression of uptake of phosphorus
in oxidative-phosphorylation experiments. There-
fore on the basis of this comparison with DNP it is
concluded that the ATPase activity induced by the
thiobarbiturates could account for the observed
contrast with DNP, the thiobarbiturates share with
the oxybarbiturates the property of inhibiting
respiration. However, it is not known ifthe activa-
tion ofATPase and the inhibition of respiration by
the thiobarbiturates result from the same primary
It is possible that the thiobarbiturates
have two unrelated mechanisms of action, one
inhibiting respiration by a similar mechanism to
that ofthe oxybarbiturates andthe other activating
We have considered the possibility that the high
fat-solubility of the thiobarbiturates (Mark et al.
1958) will favour their concentration in the mito-
chondria, followed by swelling and activation of
ATPase. By the use of optical methods for the
measurement of swelling (Cleland, 1952; Tedeschi
& Harris, 1955), no change in extinction of the
mitochondrial suspensions is found to occur after
incubation in the medium used for ATPase assay.
Although there are conditions when changes in
extinction may not be quantitatively proportional
to changes in mitochondrial volume (Tedeschi &
Harris, 1958) it seems safe to conclude that no
change in extinction is indicative of no change in
mitochondrial volume. The dissociation constants
(pK) of Phenobarbital, Amytal and Hexobarbital
are 7-41, 7-94 and 8-4 respectively (Krahl, 1940;
Brodie & Hogben, 1957). The increasing order of
the pK values reflects a decreasing ionization at the
pH of our experiments and therefore an increased
tendency to partition into fat (cf. Brodie & Hogben,
1957; Brodie, 1952).
However, little ATPase is
activated by these oxybarbiturates; therefore fat
solubility cannot be an important factor in this
We have found that oxybarbiturates will also
depress the oxidation stimulated by DNP but have
no effect on ATPase stimulated by this drug. The
interpretation of these results depends upon the
current views of the respiratory chain, which are
for the most part dependent upon a study of sub-
stances which influence its processes.
Before we can interpret the effects of barbi-
turates upon both oxidation and ATPase activity
stimulated by DNP, it is necessary to consider the
evidence that all the effects of DNP are symptoms
of the same primary
(Hunter, 1951) that the ATPase activity in mito-
chondria induced by DNP is due to a modification
ofthe normal energy-transferringchain which links
electron transport and ATP formation has now
been accepted by many workers (Potter & Reck-
1951; Lardy & Wellman, 1953; Potter,
Siekevitz & Simonson,
1957a; Bronk & Kielley, 1958). With particles
derived from rat-liver mitochondria by treatment
with digitonin, Cooper & Lehninger (1957) have
concluded that ATPase stimulated by DNP and
the ATP-inorganic phosphate-exchange reaction
are related to the enzymic mechanism responsible
for oxidative phosphorylation. The evidence for
this view is based primarily upon the specificity
towards nucleotides ofadenine ofthe three reaction
systems, their equal sensitivity to DNP and their
1953; Myers & Slater,
W. N. ALDRIDGE AND V. H. PARKER
similar pH-activity ranges. Studies of a number of
nitro- and halo-phenols (Parker, 1958) and tri-
alkyltins (Aldridge, 1958) indicate that both ATP-
ase activity and uptake of oxygen induced in
mitochondria by DNP are modifications of the
For each of several phenols
ATPase activity and uptake of oxygen are stimu-
lated to the maximum by the concentration which
inhibits oxidative phosphorylation (Parker, 1958).
These phenols therefore have a single common
action. Studies with trialkyltin homologues have
shown that, as well as inhibiting oxidative phos-
phorylation, these compounds depress both the
ATPase activity and uptake of oxygen induced by
DNP. For each of several trialkyltins, this ATPase
activity and uptake ofoxygen are both inhibited by
that concentration which also inhibits oxidative
phosphorylation (Aldridge, 1958). This indicates
that the trialkyltins also have a single common
action. The simplest hypothesis incorporating all
these findings is that trialkyltins and DNP produce
their effects by acting in different ways upon the
same single site in the energy-transferring chain.
The results with trialkyltins therefore reinforce the
view that the observed effects ofDNP are the result
of a single primary action. Therefore studies of
ATPase activity and uptake of oxygen stimulated
by DNP are studies upon separate parts which
between them involve the whole of the respiratory
chain. If the effects of drugs upon these parts are
to be logically compared and deductions made
from them the experimental conditions must be as
nearly identical as possible. The only difference in
our procedures is the absence of inorganic phos-
phate in the assay of ATPase.
DNP uncouples oxidative phosphorylation asso-
ciated with succinate oxidation (Hunter, 1951;
Judah & Williams-Ashman, 1951).
ation associated both with the oxidation of reduced
cytochrome c (Nielsen & Lehninger, 1955) and with
the transfer of electrons from ,-hydroxybutyrate
to cytochrome c (Borgstrom, Sudduth & Lehninger,
Trialkyltin prevents oxidation with succinate as a
substrate (Aldridge, 1958).
(unpublished) have shown that tri-n-butyltin in-
hibits the one-step oxidation of succinate which
takes place in the presence of Amytal (cf. Fig. 5).
The effective concentrations of tri-n-butyltin are
the same as those which inhibit oxidation in the
presence of pyruvate. The simplest hypothesis to
explain these observations is that, at the site where
DNP and trialkyltins act, there is an intermediate
or enzyme common to all three energy-transferring
chains. Ifthis is so then it is reasonable to conclude
that, from this site at least, the three energy-
transferring chains are identical (cf. Fig. 7).
This view is not in agreement with the opinion of
is completely inhibited by 0.1 mM-DNP.
other workers (Myers & Slater, 1957a, b; Hulsmann
& Slater, 1957).
Myers & Slater (1957a) have
measured ATPase activities in liver mitochondria
with pH optima of 6-3, 7-4, 8-5 and 9 4. The activi-
ties at the first three pH values are stimulated by
DNP but only 'pH 6 3 ATPase' is clearly activated
by 0 01 mM-DNP. The 'pH 7.4 ATPase and pH 8-5
ATPase' require concentrations of 0 1 and 1-0 mM
respectively and the 'pH 8-9 ATPase' is not acti-
vated. On the basis of a demonstration of three
peaks in the curve relating P/Oratio and pH it has
been suggested that the ATPase activities with pH
optima at 6-3, 7-4 and 8-5 correspond to the three
steps of electron transport coupled to phosphoryl-
ation (Hulsmann & Slater, 1957; Myers & Slater,
1957a). The three ATPase activities with different
pH optima have been confirmed
derived from rat-liver mitochondria by treatment
with digitonin. However, the peaks in the curve
relating oxidative phosphorylation to pH have not
been found with these particles with ,B-hydroxy-
butyrate as substrate (Cooper & Lehninger, 1956;
(Chance, 1959; Chance & Conrad, 1959).
been pointed out (Chance, 1959) that the magnitude
ofthe optima (Hulsmann & Slater, 1957) is so small
that an unusually high accuracy for the determina-
tion of the P/O ratio is required. In this work we
have been surprised to find how difficult it is to
obtain P/O ratios with a low standard error (cf.
Table 1, controls; mean P/O 2-66, mean S.E. 0.10).
It seems therefore that there is no certain evidence
that the energy-transferring chains from the site of
action ofDNP and trialkyltins are not very similar
or identical. The simpler hypothesis that the chains
are identical is summarized in Fig. 7.
In the presence of Amytal (Fig. 5) or Kemithal,
one atom of oxygen is absorbed for every molecule
of succinate oxidized. The phosphorylation associ-
ated with this oxidation is not inhibited by Amytal
(Low et al. 1955; Eiler & McEwen, 1949). It has
been claimed that barbiturates in general un-
couple phosphorylation associated with succinate
oxidation (Brody, 1955) but the only published
experiments are with a thiobarbiturate, Thiopental
(Brody & Bain, 1954). This result is to be expected,
for Thiopental activates ATPase at the concentra-
tions used (Table 2). In contrast with its effects
with pyruvate as substrate Amytal does not inhibit
uptakes of oxygen and phosphorus in the presence
of succinate. This indicates that this drug acts in
the respiratory chain below cytochrome cl, i.e.
between coenzyme I and cytochrome c and includ-
ing flavoprotein (cf. Fig.
reminiscent ofthe earlier conclusions ofMichaelis &
Quastel (1941) and Grieg (1946) that barbiturates
stimulated by DNP is not influenced by oxybarbi-
7). This conclusion is
BARBITURATES AND OXIDATIVE PHOSPHORYLATION
turates, they must therefore act in the energy-
transferring chain before the DNP-sensitive site (in
the area on Fig. 7 bounded by cytochrome cl,
coenzyme I and DNP-sensitive site). This is the
area where, as defined earlier, the electron-transport
Although it seems probable that the thiobarbi-
turates also act at the same site as the oxybarbi-
turates this conclusion is not at present certain.
The activation of ATPase by the thiobarbiturates
has made the interpretation of our experiments
difficult; more information is needed on the nature
of mitochondrial ATPase.
1. The oxybarbiturates Phenobarbital, Amytal
and Hexobarbital inhibit but do not uncouple
oxidative phosphorylation of liver mitochondria
with pyruvate as substrate.
2. The thiobarbiturates Thiopental, Baytenal
and Kemithal inhibit and, to a certain extent,
coupling appears to be correlated with their ability
to activate mitochondrial adenosine triphosphatase.
with brain mitochondria
mitochondria but owing to the instability of the
preparation the interpretation of these results is
4. The oxybarbiturates inhibited oxygen uptake
2:4-dinitrophenol was not inhibited. The thio-
barbiturates inhibit the oxygen uptake and, to a
lesser extent, the adenosine triphosphatase.
5. Neither Amytal nor Kemithal inhibits oxida-
tion in the presence of succinate.
6. The interpretation of these results in the light
of current views of the respiratory chain is dis-
We are indebted to Mrs J. I. Tombs and Mr B.
for skilled technical assistance and to Dr J. 0. Irwin for
helpful,discussion on statistical maters. We also wish to
thank Dr F. Hobbiger,*Dr G. Hecht (Farbenfabriken Bayer
A.G.), Dr R. Wein (May and Baker"Ltd.) and Imperial
Chemical Industries Ltd. (Pharmaceutical Division) for
gifts of barbiturates, and Eli Lilly and Co. for a grant
towards the cost of apparatus.
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The Activation of Plasminogen by Staphylokinase:
Comparison with Streptokinase
BY FLORA M. DAVIDSON*
Department of Pathology, Institute of Orthopaedic8, Royal National Orthopaedic Ho&pital,
(Received 2 November 1959)
Plasminogen, the precursor of the proteolytic
and fibrinolytic enzyme of blood, is activated by
various substances, including the bacterial acti-
vators streptokinase (Milstone, 1941) and staphylo-
kinase (Lack, 1948; Gerheim, Ferguson, Travis,
Johnston & Boyles, 1948). Some animal plasmino-
gens which are unaffected by streptokinase alone
are activated by a mixture of streptokinase and
human globulin (Miillertz & Lassen, 1953; Sherry,
Miillertz has postulated the existence in
human serum of a proactivator which is converted
by streptokinase into an activator of plasminogen
(Miillertz & Lassen, 1953; Mullertz, 1955). Staphylo-
kinase activates a wider range of animal plasmin-
ogeus (Gerheim & Ferguson, 1949) and may there-
fore not require proactivator. The activation of
humanand dog plasminogensbystaphylokinase has
been studied by Lewis & Ferguson (1951), Hayashi
& Maekawa (1954) and Celander & Guest (1959).
This paper describes the activation by staphylo-
kinase of human, rabbit and guinea-pig plasmino-
gens. When sufficiently concentrated, staphylo-
kinase acted as rapidly as streptokinase. It did not,
however, activate ox plasminogen and it was un-
affected by the addition of proactivator.
Buffer. Palitzsch's borate buffer, pH 7.4, prepared as
described by Norman (1957), was used throughout.
Casein. Light white soluble casein (British Drug Houses
Ltd.) was used without further purification.
Human plasminogen. This was purified by the procedure
of Kline (1953). This preparation was also used as a source
Euglobulin. Human and animal euglobulins were pre-
cipitated from serum as described by Norman (1957).
They were resuspended in borate buffer in the original
serum volume or, with ox euglobulin, in 0 4 vol.
Dornokinase (Burroughs Weilcome and
Co.) was used.
Activation and assay ofplasminogen. This was carried out
by the method described by Norman (1957).
acid was used instead of trichloroacetic acid to precipitate
1955). This modification raised the
extinction at 280 mu due to the products of casein di-
gestion by 50%. Measurements of E were made in the
Unicam spectrophotometer model SP. 500. Activity was
expressed in terms of a unit which gives, under the condi-
tions of the test, an increase in E at 280 m,u of 100/min. of
Assay of staphylokinase. Several concentrations of the
staphylokinase to be assayed were incubated for 30 min. at
370 with 50 x 10-3 unit ofhuman plasminogen, in 1 1 ml. of
borate buffer, pH 7-4. A portion (1 ml.) of 4% (w/v)
casein was added and incubation continued for 30 min.
Casein was then precipitated with 3 ml. of 10% perchloric
acid and E at 280mizof the digestion products was
measured. The staphylokinase activity was determined by
interpolation. A unit of staphylokinase was defined as that
activity which gives rise, under these conditions, to an
increase in E of0-300. This unit was approximately equal ta
one-third of a Christensen unit of streptokinase (Christen-
sen, 1949) (Fig. 1).
In the experimental results to be
described, streptokinase activity also is expressed in these
*Present address: The London Chest Hospital, London,