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 %.
W. N. ALDRIDGE AND V. H. PARKER
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Parker, V. H. (1958). Biochem. J. 69, 306.
Peters, R. A. (1955). John8s Hopk. Ho8p. Bull. 97, 1.
Potter, V. R. & Recknagel, R. 0. (1951). In Phosphorus
Metabolism, vol. 1, p. 377. Ed. by McElroy, W. D. &
Glass, B. Baltimore: Johns Hopkins Press.
Potter, V. R., Siekevitz, P. & Simonson, H. C. (1953).
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Purvis, J. L. (1959). Exp. Cell Res. 16, 98.
Quastel, J. H. & Wheatley, A. H. M. (1932-33). Proc. Roy.
Soc. B, 112, 60.
Robinson, H. W. & Hogden, G. G. (1940). J. biol. Chem.
Siekevitz, P. & Potter, V. R. (1953).
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Snedecor, G. W. (1946). Statoitical Method&. Iowa: State
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Biochem. J. (1960) 76, 56
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,