564
BIOCHIMICA ET BIOPHYSIC:\ ACTA
VOL. 2 4
(1957)
DISPLACEMENT OF THIAMINE P Y R O P H O S P H A T E
FROM SWOLLEN MITOCHONDRIA
M. U. D I A N Z A N I * ArCD M. A. D I A N Z A N I MOR*
Laboratory o/Physiological Chemistry, The University, Utrecht (Netherlands)
It has been shown in previous publications that swollen mitochondria from fatty liver
contain decreased amounts of cytochrome c, of pyridine nucleotides and of adenylnucleotides 1-8. One of the most important reasons for the decrease has been found
to be the increased diffusion of these substances from the mitochondria into the
surrounding medium.
It seemed interesting to us to investigate the behaviour of another coenzyme,
thiamine pyrophosphate (TPP) in mitochondria from fatty liver. The distribution
of T P P in liver cells of normal and thiamine-deficient rats has been studied by
GOETHART4. This author reported that in the normal rat about 30 % of the T P P is
contained in the mitochondria, while the rest is present mainly in the soluble part
of the cytoplasm.
EXPERIMENTAL
W i s t a r albino rats weighing lOO-12o g fed on a s t a n d a r d diet were used. T h e y were killed by
decapitation and the livers i m m e d i a t e l y r e m o v e d and transferred to the cold r o o m at 2 ° C. lO%
h o m o g e n a t e s were p r e p a r e d in a P o t t e r E l v e h j e m homogenizer with o.25M sucrose in m o s t cases.
I n some instances, o.25M sucrose containing polyvinylpyrrolidone, p H 7.6, as described b y
NOVlKOF#, was used. I n o t h e r experiments, the h o m o g e n a t e s were made in water. Differential
centrifugation of the h o m o g e n a t e s was performed either in the Spinco preparative ultracentrifuge,
or in the Zernike refrigerated centrifuge, at 2% The nuclear fraction, which contained nuclei,
u n b r o k e n cells, red cells, tissue d6bris and a s m a l l a m o u n t of mitochondria, was sedimented at
i,ooo g for i o min. The sediment was washed thrice, in order to decrease the mitochondrial
c o n t a m i n a t i o n . Mitochondria were sedimented f r o m the combined s u p e r n a t a n t fluids at 12,000 g
for 3 ° m i n . I n order to p r e v e n t d a m a g e to the particles, the m i t o c h o n d r i a l sediment was used
w i t h o u t washing. Micros0mes were sedimented at 48,000 g for i h. Since the a m o u n t of T P P
p r e s e n t in this fraction is v e r y small 4, microsomes were n o t isolated in m o s t experiments. The
fraction consisting of the soluble p a r t of the c y t o p l a s m plus microsomes is reported in the t e x t
and in the t a b l e s as " s u p e r n a t a n t f r a c t i o n " .
T P P w a s determined b y the "one-stage m e t h o d " of WESTENBRINK AND STEYN PARVI~6. Each
d e t e r m i n a t i o n was performed in triplicate.
T h e nitrogen c o n t e n t of each h o m o g e n a t e and of each fraction was determined in small
samples b y the usual micro-kjeldahl technique.
The c o n t e n t of total lipids of each liver w a s determined b y extracting the dry powder of
the organ w i t h ether for 3-4 h o u r s in a K u m a g a w a : S u t o a p p a r a t u s and b y determining the
difference in weight of the sample before and after t h e extraction.
F a t t y infiltration of the liver was obtained: (i) by injecting daily s u b c u t a n e o u s l y 0.2 ml
of a 20% solution of CC1, in p e a n u t oil, during a period of 3 days (2) by feeding the animals
for 4 weeks on a diet deficient in choline. The composition of t h i s diet has been reported elsewhere z.
A group of r a t s fed on this diet received each day io m g choline hydrochloride intraperitoneally;
this group was used as a control. The obtained values were analysed for statistical significance
* P r e s e n t address: I n s t i t u t e of General Pathology of the University of Genoa, Genoa, Italy.
Re/erences p. 568.
VOL. 24 (1957)
DISPLACEMENTOF THIAMINE PYROPHOSPHATE
565
by calculating the "'t" values of Fisher. Only the differences with a "t" value corresponding to
a probability P d o . o i were accepted as significant. The standard deviation of the mean (a) is
reported in the tables after each average value.
RESULTS
Table I r e p o r t s t h e d a t a for the T P P distribution in the homogenate fractions of
n o r m a l rats. The o b t a i n e d values were v e r y similar to those r e p o r t e d b y GOETHART4,
when the suspension m e d i u m was o . 2 5 M sucrose. The same d i s t r i b u t i o n was found
also in the h o m o g e n a t e s p r e p a r e d with o . 8 8 M sucrose (mitochondria in this case were
s e d i m e n t e d at 48,000 g for 30 rain), or with o.25 M sucrose containing p o l y v i n y l pyrrolidone. W h e n the homogenates were p r e p a r e d w i t h distilled water, the distrib u t i o n of T P P in the c y t o p l a s m fractions was however v e r y different. I n fact, the
a m o u n t of T P P c o n t a i n e d in the m i t o c h o n d r i a was s t r o n g l y decreased u n d e r these
conditions, while a corresponding increase was observed in the s u p e r n a t a n t fraction.
Some e x p e r i m e n t s were m a d e in order to s t u d y the influence of h y p o t o n i c t r e a t m e n t of m i t o c h o n d r i a p r e p a r e d in o . 2 5 M sucrose on their T P P content. Mitochondria
isolated from i g n o r m a l liver with o . 2 5 M sucrose were suspended again in o.25 M
sucrose, d i v i d e d into two identical fractions a n d resedimented at 18,ooo g for 30 min.
The two m i t o c h o n d r i a l pellets were t h e n suspended in o . 2 5 M sucrose a n d in w a t e r
respectively, a n d the suspensions i n c u b a t e d at I 8 ° C for times v a r y i n g from o to
3 ° min. I n some e x p e r i m e n t s the i n c u b a t i o n t e m p e r a t u r e was 4 ° C.
CONTENT
AND
DISTRIBUTION
OF
TABLE I
TPP I N C Y T O P L A S M
FRACTION
HomogenaIes in 0.25 M sucrose*
I~g/g liver
Homogenate
Nuclear fraction
Mitochondria
Supernatant
Recovery %
* IO
14.o
0.9
4.85
7.58
94.2
± 1.9
:L 0.3
~: 1.14
4- 1.23
± 2.5
I*g/mg N
0.45
o.19
o.61
o.45
%
7.1 4- 2.9
36.1 ± 5.2
56.8 4- 4.1
OF
NORMAL
RATS
Homogenales in water**
ILg[g liver
13.4
0.55
2.76
8.62
89.0
4- 0.9
4- o.16
4- o.29
4- o.71
4- 2.2
Itg/mg N
0.42
o.13
0.32
0.50
%
4.6 4- 1.3
23.1 4- 2.1
72.3 ± 2.4
experiments.
** 6 experiments.
A f t e r incubation, the m i t o c h o n d r i a were s e d i m e n t e d again at 18,ooo g for 3 ° min.
T P P was then d e t e r m i n e d b o t h in the sediment a n d in the s u p e r n a t a n t fluid. As is
shown in Table I I , more t h a n 80% of the T P P was still contained in the s e d i m e n t
after IO min i n c u b a t i o n at 18 ° C when the suspension fluid was o.25 M sucrose. W h e n
the suspension fluid was water, o n l y 35 % of the T P P was still contained in the sedim e n t after this time. The t o t a l a m o u n t of T P P recovered was higher in o.25 M sucrose
t h a n in water. This means t h a t a considerable p a r t of T P P is d e s t r o y e d as a result
of i n c u b a t i o n of m i t o c h o n d r i a in water. This fact agrees with the finding b y BERTHET
et al. 7 t h a t the a c t i v i t y of the acid phosphatase, present in the lysosomes, increases
s t r o n g l y u p o n suspending in water.
W h e n the i n c u b a t i o n t i m e was more t h a n IO rain, the a m o u n t of T P P present
in the s e d i m e n t was s t r o n g l y decreased also in m i t o c h o n d r i a s u s p e n d e d in o . 2 5 M
References p. 568.
37
566
M. L~. DIANZANI, M. ,\. DIANZANI MOR
(1957)
VOL. 24
TABLE II
RELEASE OF T P P FROM MITOCHONDRIA A F T E R
INCUBATION
IN
DIFFERENT
SUSPENSION
MEDIA
The values represent the mean of 3-4 experiments.
Incubation
time
in minutes
Incubation
temperature
T P P recovered in the sediment
ttg
IO
18 ° C
20
18 ° C
3°
18 ° C
20
4° C
20
18 ° C
T P P recovered in the supernatant
% o/total
Total
recovery
ttg
14.1 5- 4.6
64.8 5- 5.8
57-3 5-4.3
83.8 5- 3.9
69.9 5- 2.7
76.2 5- 4.7
4.7 5- 1.3
60. 7 5- 2.6
20.5 5- 2.4
83.6 5- 0.9
5.82
3.18
3.25
2.46
2.73
1.57
4.3 °
3.95
3.42
2.26
Suspension medium
o.25M
water
o.25M
water
o.25M
water
o.253I
water
o.88M
water
sucrose
sucrose
sucrose
sucrose
sucrose
5.00
1.12
1.39
0.40
0.82
0.37
4 .1°
1.5.5
2.72
0.37
e l o/total
5- 0.20
5- 0.09
±o.19
zk 0.08
5- o.15
5- o.12
5- 0.23
5- 0.35
5- o.13
5- o.t i
ttg
85.9 5- 6. 3
35 .2 5- 5.5
42.7 5-4.8
16.2 5- 2. 5
3o.1 5- 2.9
23.8 5- 2.2
95.3 ± 1.4
39.3 5- 2.8
79-5 5- 2.8
16.4 5- 0.9
0.82
2.06
1.86
2.06
1.91
1.2o
0.20
2.40
0.70
1.89
5- 0.05
5- 0.42
5-o.25
-- 0.38
5- 0.24
5- 0.25
5- 0.05
:~ o.19
5- 0.23
5- 0.23
s u c r o s e , w h i l e a c o r r e s p o n d i n g i n c r e a s e w a s f o u n d in t h e s u p e r n a t a n t . A f t e r a n i n c u b a t i o n t i m e of 2o to 3o m i n u t e s , m i t o c h o n d r i a a r e c o n s i d e r a b l y s w o l l e n in o.25 M
s u c r o s e also. T h u s it s e e m s h i g h l y p r o b a b l e t h a t loss of T P P f r o m t h e p a r t i c l e s occ u r r i n g u n d e r t h e s e c o n d i t i o n s is c o n c e r n e d w i t h t h e loss of t h e i r s t r u c t u r a l i n t e g r i t y .
W h e n t h e i n c u b a t i o n t e m p e r a t u r e w a s 4 ° C, p r a c t i c a l l y n o d i f f e r e n c e in t o t a l
T P P r e c o v e r y w a s f o u n d b e t w e e n m i t o c h o n d r i a i n c u b a t e d in o.25 M s u c r o s e a n d t h o s e
s u s p e n d e d in w a t e r . T h e d i f f e r e n c e in d i s t r i b u t i o n b e t w e e n s e d i m e n t a n d s u p e r n a t a n t
r e m a i n e d , h o w e v e r , c o n s i s t e n t . T h i s m e a n s t h a t t h e d e c r e a s e of T P P in m i t o c h o n d r i a
s u s p e n d e d in w a t e r is t h e r e s u l t of a t l e a s t t w o p h e n o m e n a , viz. d e s t r u c t i o n , p r o b a b l y
t h r o u g h p h o s p h a t a s e a c t i o n , a n d d i s p l a c e m e n t b y diffusion. Since t h e t e m p e r a t u r e
d u r i n g t h e f r a c t i o n a t i o n p r o c e d u r e of t h e h o m o g e n a t e s w a s 2 ° C, it s e e m s i m p r o b a b l e
t h a t t h e a c t i v a t i o n of p h o s p h a t a s e h a s p l a y e d a role in d e c r e a s i n g t h e p e r c e n t a g e
of T P P c o n t a i n e d in t h e m i t o c h o n d r i a i s o l a t e d f r o m w a t e r h o m o g e n a t e s (Table I).
T h i s is s h o w n also b y t h e f a c t t h a t t h e % r e c o v e r y of T P P a f t e r f r a c t i o n a t i o n w a s
a b o u t t h e s a m e for b o t h t h e h o m o g e n a t e s p r e p a r e d w i t h o.25 M s u c r o s e a n d t h o s e
prepared with water.
TABLE III
CONTENT
AND
DISTRIBUTION
Homogenate
Nuclear fraction
Mitochondria
Supernatant
Recovery %
12.18
1.i6
2.41
7.90
94 .2
Number of experiments
Total fats in the liver
(nag/g)
Re[erences p. 568.
5- 3.43
5- 0.6
m 0.75
± 2.57
± 3-5
/1grog AT
%/
0.42
0.24
o.3o
0.52
lO.1 ~ 4.6
21.o ± 3.2
68.9 ± 5-3
/*g'g liver
8.23
0.47
2.04
5.36
94 -1
8
58.8
~ 6.9
TPP
IN
FATTY
LIVER
Fatty liver by/ceding on
the diet deficient in ckoline
Fatty liver by CCI 4
Ilg/g liver
OF
5- 1.92
± o.19
5- 0.53
± 1.15
± 3 .8
8
86.0
~ 8. 7
llg/mg N
%
0.31
0.09
0.27
0.39
6.0 ~= 2.3
25.8 + 1.9
68.2 4- 2.8
VOL, 24 (X957)
DISPLACEMENTOF THIAMINE PYROPHOSPHATE
567
Table I I I reports the values for T P P distribution in fatty liver. A strong decrease
of T P P content of the mitochondria as compared to the content of mitochondria
from normal livers was recorded in both types of f a t t y livers studied. The amount
contained in the supernatant was increased on a % basis. The T P P distribution in
f a t t y liver thus resembles that found for water homogenates of normal livers.
Rats fed on a diet deficient in choline, in contrast to those injected with CC14,
also showed a decrease in total T P P per g liver. This was not entirely dependent
upon partial replacement of protein by fat since the amount of T P P per mg N was
also decreased.
Rats fed on the same diet for 5 weeks, and injected with io mg choline every
day, showed neither a significant change of total T P P content per g liver, nor a
modification of the T P P distribution in the cytoplasm fractions. Total lipid content
of the livers of these animals was normal.
DISCUSSION
The experiments described in this paper show that T P P is displaced from the mitochondria into the surrounding medium as a consequence of hypotonic treatment.
This displacement is already observed with swollen mitochondria from f a t t y livers
that have not undergone hypotonic treatment. This seems to be another case of a
coenzyme that is displaced from mitochondria as a consequence of morphological
damage.
The amount of T P P contained in f a t t y liver obtained b y feeding rats on the
diet deficient in choline was considerably decreased. This result would be expected
on the basis of the existing knowledge. In fact, increase of acid phosphatase activity
occurs in fatty liverss, and it m a y produce increased destruction of TPP. In addition,
ATP concentration in fatty liver is less than normal 8, and this m a y be the cause
of decreased synthesis of T P P through the phosphorylation of thiamine.
The fact that the decrease of T P P did not occur in fatty liver produced b y CC14
is however difficult to reconcile with these hypotheses. In fact, both phosphatase
activation and decrease of ATP occur also in this type of liver injury 8. The reason
for the different behaviour of T P P in the two types of fatty liver infiltration studied,
m a y lie perhaps in the difference in the length of time
for which the treatment was applied; this was 4 weeks
in the case of rats fed on the diet deficient in choline,
and only 3 days in that of animals injected with CC14.
Rats/ed on this diet and treated
with xo nag choline daily
In addition, a considerable amount of regeneration
ttg/g liver
t~g/mg N
%
always occurs in the liver following treatment with
CC14, and this m a y have complicated the results. It is
r2.o ~ 0.80
o.41
at present impossible to say whether the displacement
o.9 ± o.2
o.15
7.8 ~ 1.5
of T P P from mitochondria cooperates in the production
4.o 4- o.2
o.52
36.2 4- o.9
6.2 4- 0. 3
o.41
56.0 4- 1.8
of decreased oxidation of pyruvate, which has been
03.3 4- 2.6
observed in fatty liver (DIANZANI ~, F!SCHER 9, FISCHER
AND RECANTI°). It is known that the pyruvate oxidizing system requires the presence of several other
40.5 4- 2.8
factors, some of which (DPN, DIANZANI2 ; CoA, SEVERI
AND FONNESUn) are also decreased in f a t t y liver. The
R e / e r e n c e s p. 568.
568
M . U . DIANZANI, M. A. DIANZANI MOR
VOL. 2 4
(1957)
addition of sufficient amounts of DPN to mitochondrial preparations restores almost
completely pyruvate oxidation under aerobic conditions 2. It m a y be remembered,
with regard to the last point, that the effect of the addition of T P P on the oxidation
of pyruvate by oxygen is also very small in homogenates of breast and heart muscles
of thiamine-deficient pigeons (FRANKEN AND STAPERT12).
ACKNOWLEDGEMENT
The authors are indebted to Professor H. G. K. V~ESTENBRINK for the stimulating
criticism and generous help which made this investigation possible.
SUMMARY
T h e d i s t r i b u t i o n of T P P in c y t o p l a s m fractions of r a t liver is studied. W h e n t h e h o m o g e n a t e
w a s p r e p a r e d w i t h o . 2 5 M sucrose, a b o u t 3o% of T P P w a s p r e s e n t in m i t o c h o n d r i a . W h e n t h e
h o m o g e n i z a t i o n m e d i u m w a s distilled water, t h e a m o u n t of T P P p r e s e n t in t h i s fraction was
m a r k e d l y decreased, while t h a t p r e s e n t in t h e s u p e r n a t a n t fluid w a s increased. M i t o c h o n d r i a
s u s p e n d e d in o . 2 5 M sucrose a n d i n c u b a t e d a t i8 ° C for io m i n release in t h e s u s p e n s i o n fluid
a b o u t 20% of t h e i r T P P ; u n d e r t h e s a m e c o n d i t i o n s , m i t o c h o n d r i a s u s p e n d e d in w a t e r release
a b o u t 65% of their T P P . A n increased d e s t r u c t i o n of T P P was o b s e r v e d as a r e s u l t of i n c u b a t i o n
of m i t o c h o n d r i a in w a t e r a t 18 ° C.
T h e d i s t r i b u t i o n of T P P in f a t t y liver h o m o g e n a t e s p r e p a r e d in 0.25 M sucrose resembles
s t r o n g l y t h a t o b s e r v e d for h o m o g e n a t e s of n o r m a l r a t liver p r e p a r e d in distilled w a t e r ; T P P w a s
decreased in t h e m i t o c h o n d r i a l fraction a n d c o r r e s p o n d i n g l y increased in t h e s u p e r n a t a n t . T o t a l
T P P is decreased in f a t t y liver o b t a i n e d b y feeding r a t s on a diet deficient in choline. I t r e m a i n s
p r a c t i c a l l y u n c h a n g e d in f a t t y liver p r o d u c e d b y CC14.
REFERENCES
1 M. U. DIANZANI AND I. VITI, Biochem. J., 59 (1955) 141.
M. U. DIANZANI, Biochim. Biophys. Acta, 17 (1955) 391.
3 M. U. DIANZANI, Biochem. J., 65 (1957) 116.
4 G. GOETHART, Biochim. Biophys. Acta, 8 (I952) 479.
5 A. B. NOVlKOFF, 3~me Congr. Intern. Biochim., Bruxelles, 1955, Res. communs., p. 315 .
6 H. G. K. WESTENBRINI~ AND E. P. STEYN PARV~, Intern. Rev. Vitamin Research, 21 (195 o) 47 j . BERTHET AND CH. DE DUVE, Biochem. J., 5 ° (1951) 174.
8 M. U. DIANZANI, Biochim. Biophys. Acre, 14 (1954) 514 .
9 C. L. FISCHER, Proc. Soc. Exptl. Biol. Med., 90 (1955) 153.
10 C. L. FISCHER AND L. RECANT, J. Lab. Clin. Med., 48 (1956) 171.
11 C. SEVERI AND A. FONNESU, Proc. Soc. Exptl. Biol. Med., 91 (1956) 368.
13 j . F. FRANKEN AND F. P. STAPERT, Biochim. Biophys. Acre, 14 (1954) 293.
Received
December
20th, 1956
THE EFFECT OF EDTA ON THE INTERACTION BE'IWEEN
ACTOMYOSIN AND ADENOSINE TRIPHOSPHATE*
YUJI TONOMURA, HIROYUKI
M A T S U M I Y A AND S H O T A R O K I T A G A W A
Research Institute for Catalysis, and Chemistry Department, Faculty o] Science,
University of Hokkaido, Sapporo (Japan)
I t h a s b e e n w e l l e s t a b l i s h e d b y FRIESS 1 a n d B O W E N .AND K E R W l N ~ t h a t E D T A is
a striking activator of myosin ATPase in the presence of high concentration of KC1.
* T h e following a b b r e v i a t i o n s will be u s e d ; A T P adenosine t r i p h o s p h a t e , A T P a s e adenosinet r i p h o s p h a t a s e , a n d E D T A e t h y l e n e d i a m i n e t e t r a a c e t i c acid.
Re[erences p. 575/576.