BIOCHEMICAL
Vol. 86, No. 2, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 293-299
January 30, 1979
THE POSSIBLE ROLE OF INTRACELLULAR POLYAMINES IN MITOCHONDRIAL METABOLIC REGULATION Rowand R.J.
Chaffee,
Department
Received
Robert
M. Arine
and Rene H. Rochelle
of Ergonomics, University Santa Barbara, California
of California 93106
November 28,1978
SUMMARY: Studies were made on the effects of very low concentrations of the polyamine, spermine, on rat liver mitochondrial metabolism associated with B-hydroxybutyrate. The respiratory control ratio and the rate of respiration during ADP-ATP conversion are significantly altered with shifts in spermine concentrations of as little as 15.7 nMoles/ml within the physiological Mgf+ concentration range. These spermine concentration changes are small compared to the estimated hepatic intracellular levels of spermine which have been reported to be between 200 and 1200 nMoles/gm wet weight under normal conditions. There is now evidence that exposure of an animal to certain environmental conditions induces changes of 164 nMoles/gm wet weight in intracellular levels of liver spermine in a few hours. Also there is evidence that the concentration of intracellular polyamines is influenced by endocrines since the levels of the enzymes responsible for their synthesis are markedly affected by hormonal changes. Therefore, alterations of polyamine levels may play a role in mitochondrial metabolic regulation in vivo. It
has long
RNA synthesis affect
and thus
mitochondrial
interesting a change did
protein
that
in mitochondrial his
assay
did
ratios.
naturally
there
The few papers
with
substrates,
respiratory
control
ratio
These
polyamine
levels
(8).
However,
the data
and therefore
the oxidation
for
the
during
spermine rate.
of studying
and after
little
information
polyamines
on these on this
mitochondrial (5-7)
and 1.25
mM spermidine
and respiration
during
the conversion
the concentration
in our previous
papers
ranges (5-7)
of ADP to
on the effects
subject
might
he
the polyamine
respiratory
1.0 mM spermine
are within
causes
However,
the conversion
on mitochondrial
we have written
indirectly
made the very
of succinate,
purpose
can affect
would
(4)
and the mean respiratory
is very
intracellular
such as spermine
Huunan-Seppala
any information
parameters. certain
during
respiration
In fact,
(l-3)
Also,
procedures
not obtain
occurring
polyamines
synthesis
swelling
on mitochondrial
ATP and thus
that
respiration.
observation
not design
effects
trol
been recognized
found well
conof
metabolic show that affect
the
of ADP to ATP. in rat
liver
be subject
to
0006-291X/79/020293-07$01.00/0 293
Copyright @ 1979 by Academrc Press, Inc. All rights ofreproduction in any form resew-ved.
Vol. 86, No. 2, 1979
critcism
because
significant
it
argue
polyamine
changes
mechanism.
that
over that
which
effects
changes
changes
the polyamines
ratio.
the data
metabolic
control as
additions
con-
the
of small
presented
is
latter
amounts
the first
can cause
very
ADP-ATP conversion
of these effects
mM
respiratory
Furthermore,
during
regulatory in this
such gross
of a polyamine
The significance
presented
requiring
as low as 0.0157
herein
respiration
have possible
the hypothesis
extra
And
B-hydroxybutyrate
conversion.
with
to cause any
substrates.
mitochondrial
in concentration
on mitochondrial
control
liver
ADP-ATP
Thus,
with
concentration
affects
range.
system
presented
a spermine
during
those
an insensitive
herein
significantly
a wide
respiratory
is
that
such small
significant
suggest
in the data
with
regulatory
represent
RESEARCH COMMUNICATIONS
of a polyamine
metabolism
at best
show incremental
of spermine
the
would
and respiration
two parameters
concentrations
any intracellular
nMoles/ml)
ratios
evidence
such high
we have found
15.7
AND BIOPHYSCAL
in mitochondrial
However,
substrate,
trol
took
changes
one might
(or
BIOCHEMICAL
findings
is
on cellular
and
that
they
respiration
paper.
METHODS Mitochondria were isolated from livers of adult male Sprague-Dawley rats by the method described by Hoch and Lipmann (9). All assays were made at 37' C, using the polarographic apparatus described by Estabrook (10). In the liver in vivo, acetoacetate is reduced to @-hydroxybutyrate (ll), but if there is an the reaction is reversed and the oxidaexcess of 8-hydroxybutyrate in vitro, tion of NADH thus generated can be followed polarographically. For assaying a modification of the reaction mixture of Harris B-hydroxybutyrate oxidation, et al (12) was used. The 3.6 ml reaction mixture contained: 900 UMoles 1.08 PMoles ADP, sucrose, 90 VMoles KCl, 18 PMoles KH2P04, 72 PMoles Tris-HCl, 90 PMoles B-hydroxybutyrate and the appropriate concentrations of spermine and Mg++. In each assay, 2-4 mg of mitochondrial protein was used. Protein concentration was determined spectrophotometrically by the method of Lowry et al (13). Spermine concentration was varied between 0.0157 and 2.0 mM whereas Mgtt used were those estimated to concentrations were 0.6 and 0.93 mM. Mg++ levels be free in rat liver cytoplasmic fluid (14-16). RESULTS AND DISCUSSION In figure centration droxybutyrate of spermine
1 is
shown the
from 0 up to 0.25
this
effects
range
of increasing
mM on the respiratory As was mentioned
as substrate. within
striking
are at or below
294
the spermine
control
previously,
ratio
with
con-
B-hy-
such concentrations
the concentration
levels
found
Vol.
86,
No.
BIOCHEMICAL
2, 1979
0.0157
0.0313
0.0625
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0.125 SPERMINE
Figure
in rat
1
Effects of increasing the concentration of spermine on the respiratory control r$$io with a-hydroxybutyrate as substrate at 0.6 and 0.93 mM Mg .
liver
trations
homogenates
of at least
influence
during
these
levels
are
metabolism
concentration
evidence
Therefore,
amounts
and how they
is particularly
that
environmental when rats
spermine weight
levels which
are
tively
0.164
is evidence
rapid their
returned
is a change
of approximately There
conditions
increase
synthesis
the
in light
Tsvetnenko
of 164 nMoles/gm mM) in a matter indicates
have remarkably
of oxidation
after
that
concen-
and therefore
1 shows how small
much in excess (20).
likely
in the cell Table
important
from 213 nMoles/gm
changes
is quite
influence
to normoxia
that
concentration
present
the rate
have been shown to fluctuate
certain
it
in vivo.
elevate
ADP-ATP conversion
The above
for
(17-20).
mitochondrial
in spermine
0.250 (mM)
increases
of @-hydroxybutyrate respiratory
control
of the fact of 0.0157
that
(ibid)
to hyperoxia,
wet weight
ratio. spermine
mM --in vivo
and Shugaley
exposure
wet weight
would
under reported
liver
up to 379 nMoles/gm (or
a concentration
wet change
of hours. that
polyamines
in the cell short
295
since
half-lives.
may be subject the enzymes
to rela-
responsible
For example,
S-adenosyl-
1.
1.; 2:o
** 0 0.0157 0.0313 0.0625 0.125 0.250 0.50
** 0 0.0157 0.0313 0.0625 0.125 0.250 0.50 1.0 1.5 2.0
Sw (mM)
on rat
59.51 66.24 68.71 70.43 73.61 75.09 78.36 79.02 79.17 83.00
54.60 60.37 63.65 66.85 69.89 74.27 82.23 82.69 84.62 85.80
during ADP-ATP Conversion
Q02*
Spermine effects B-hydroxybutyrate.
* ** ***
In: ~1 O2 consumed/ mg rat Control Not significantly different
The reaction mixture contained: 90 umoles e-hydroxybutyrate and
0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93
i*: 0:6 0.6 0.6
0.6 0.6 0.6 0.6 0.6
Mg++ (mM)
Table
0.05 0.001 0.01 0.001 0.001 0.001 0.001 0.001 0.001
-----
----0.05 0.02 0.01 0.001 0.001 0.001 0.001 0.001 0.001
from
liver
P
control.
mitochondrial
sucrose, ADP (pH
mitochondrial
900 pmoles 1.08 umoles
liver
protein/hr
90 pimoles 7.2).
31.34 30.71 31.12 25.69 25.79 24.26 23.36 24.47 25.88 27.15
38.76 42.00 38.39 29.16 26.74 24.65 23.37 24.37 26.81 29.86
Q02* after ADP-ATP Conversion
respiration
KCl,
18pmoles
NSD NSD NSD NSD 0.05 NSD NSD NSD NSD
-----
0.02 NSD 0.05 0.01 0.001 0.001 0.001 0.01 0.05
P
metabolic
-----
and
KH2P04,
0.05 0.05 0.01 0.01 0.001 0.001 0.01 0.01 0.01
----a
___-_
P
*** NSD NSD 0.02 0.001 0.001 0.001 0.001 0.001 0.001
with
Tris-HCl,
associated
72 umoles
2.00 2.35 2.41 2.76 2.91 3.13 3.41 3.31 3.12 3.14
1.44 1.47 1.76 2.30 2.62 3.03 3.61 3.50 3.24 2.97
Respiratory Control Ratio
control
BIOCHEMICAL
Vol. 86, No. 2, 1979
methionine
decarboxylase
as ornithine 23-25). lase
Furthermore,
terior
anterior
to stressful
pituitary,
influence Other drial
metabolic heat-acclimated
mine
than
are
animals
since
changes
nificantly amines
(20)
affect
the rate and since
further
From the
data
like
is
on the an-
activity
and thus
can cause
mitochon-
that
mitochondria
concentration
mitochondria elevation
from
of sperthe heat-
of the respiratory in the presence
can respond
in levels
of polyamines
would
that
of
to environcan sig-
the possible
role
seem to be an area
spermine
control
in some respects in figure
the effects
ratio
control
ratio
elevated
of poly-
which
within
certain
1, with
1.44
ratio
as little
concentration
297
whereas
is elevated
1, it
ranges
has been metabolism
is obvious
0.6 mM Mg" with
it
protein
on mitochondrial
that
the
metabolism and no spermine,
as little
to 2.30.
above that
as 0.0157
in a capacity
since
in affecting
1 and table
is considerably
with
control
of spermine
is only
control
can function
metabolic
As shown in table
significantly
and spermine
synthesis
in vitro,
control
is
Mgtt
the respiratory
the respiratory
metabolism
presented
of 8-hydroxybutyrate.
spermine,
changes
of Mg++ in mitochondrial
of Mg++ can alter
the respiratory
small
possibility
to function
levels
changes
investigation.
to that
reported
is,
from the controls
metabolic
One interesting similar
is
to a given
That
of polyamine
mitochondrial
in mitochondrial
warrants
effects
conditions
greater
mitochondria
via
of polyamines
are more sensitive (6).
physiological
decarboxylase
environmental
of controls
decarboxy-
--in vivo.
due to the action
did
can,
(1, 8,
(ibid).
Therefore, mental
synthesis
21, 22) where-
ornithine
Thus,
conditions
had a consistently
than
increase
(26-30).
in ornithine
stressful
rats
those
ratio
spermine
that
changes
from
control
of polyamine
evidence
acclimated
environmental
(1,
10 and 24 minutes
hormones
organs
changes
60 minutes
between
pituitary target
mediate
the rate
of about
has a half-life
in appropriate
in response
(3).
has a half-life
decarboxylase
activity
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
as 0.0625 At 0.93
mM Mg++,
at 0.6 mM Mg++ and
mM spermine.
Thus,
seem to be synergistic
mM
Mg++ in
BIOCHEMICAL
Vol. 86, No. 2, 1979
elevating
the
respiratory
of B-hydroxybutyrate. smaller that
than
those
the range
while
report
changes
in levels
ible
role
But the of Mg++.
ratio
with
is
interesting
cytoplasmic
0.2 and 1.2 mM spermine
metabolic
oxidation
involved
are much
because
Mg++ is very
it
narrow
more variable. in rat
such as spermine
of cellular
mitochondrial
of spermine
may be relatively
of a polyamine
in alterations
synergism
of free
concentration
between
associated
concentrations
This
of variation
the spermine
workers
control
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
liver
may well
control
than
is thought (14-16)
Different
(17-20). play
Thus,
a more flex-
do changes
in Mg++
concentration. ACKNOWLEDGMENTS This work was supported by ONR Grant and NASA-Ames NCA 2-OR-608-601.
N00014-75-C-0506,
NIH Grant
AM 18895-01
BIBLIOGRAPHY 1.
2. 3. 4. 5. 6. 7.
8.
9.
10. 11. 12. 13. 14. 15. 16.
Russell, D.H. (1973) In: Polyamines in Normal and Neoplastic Growth, (Russell, D.H,, ed) pp. l-13. Raven Press, N.Y. Bachrach, U. (1973) Function of Naturally Occurri‘ng Polyamines. 211~. Academic Press, N.Y. Cohe;il;.Sinc(1971j Introductton to the Polyamines. 179p. PrenticeCliffs. N.J. , . Enolewood Huunan-Seppala, A." (1971) J. Bioenergetics 2: 197-207. Chaffee, R.R.J., Salganicoff, L,, Arine, R.M., Rochelle, R.H. and Schultz, E.L. (1977) Biochem. Biophys. Res. Commun. 77: 1009-1016. Chaffee, R.R.J., Arine, R.M., Rochelle, R.H. and %'lker, C.D. (1978) Experientia (In Press). Chaffee, R.R.J., Arine, R.M., Rochelle, R.H. and Walker, C.D. (1978) In: Advances in Polyamine Research. (Campbell, R.A., Morris, D.R., Bartos, D., Daves, G.D. and Bartos, F., eds) Vol. 2: 123-128. Raven Press, N.Y. Canellak-is, E.S., Heller, J.S., Kyriakidis, D. and Chen, K.Y. (1978) In: Advances in Polyamine Research. (Campbell, R.A., Morris, D.R., Bartos, D., Daves, G.D. and Bartos, F., eds) Vol. -1: 17-30. Raven Press, N.Y. Hoch, F.L. and Lipmann, F. (1954) Proc. Natl. Acad. Sci. 40: 909-921, Estabrook, R.W. (1967) In: Methods in Enzymology. (Estabzok, R.W. and Pullman, M.E., eds) Vo?. X: 41-47. Academic-Press, N.Y. Newsholme, E.A. and Start. C. F.legulation in Metabolism. p. 324-~ -119731 >----I 325. John Wiley and Son. ,-- I N.Y. G. and Pressman. B.C. 11967) Binchemistrv 6: Harris, E.J., Catlin. 1360-1369. N.J., Farr, A.L. and Randall, Lowry, O.H., Rosebrough, R.J. (1951). J. Biol. Chem. -193: 265-275. M. and Veech, R.L. (1973) J. Biol. Veloso, D., Guynn, R.W., Oskarsson, Chem. 248: 4811-4819. Weisiger, J. (1978) In: Advances in Polyamine Research. (Campbell, R.A., Morris, D.R., Bartos, D Daves, G.D. and Bartos, I-., eds) Vol. 2: 133-187. Raven Press, i:Y. Veech, R.L. (1978) Personal Communication.
298
Vol. 86, No. 2, 1979
i;: 19.
20. 21.
22. z* 25: 26. 27. 23. 29. 30.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Neish, W.J.P. and Key, L. (1967) Int. J. Cancer 2: 69-75, Rosenthal, S.M. and Tabor, C.W. (1956) J. Pharmacol. Exp, Ther. -116: 131-133. Unemoto, T., Ikeda, K., Hayashi, M and Miyaki, K. (1963) Chem. Pharmacol. Bull. (Tokyo) 11: 143-151. Tsvetnenko, E.Z. and Shugaley7V.S. (1973) Byull. Exsp. Biol. Med.
35: 23-30. Russell, f??;se:l;
D.H. and Taylor, R.L. (1971) Endocrinology 33: 1397-1403. D.H: and Potyraj, J.J. (1972) Biochem. 3. E: 1109-1115. Lindsay, V.J. and Cooke, A. (1972) FEBS Letters 21: 123-126. Rusie1i;fI.H. and Snyder, S.H. (1969) Mol. Pharmacol. 5: 25%262. Russell, D.H., Snyder, S.H. and Medina, V.J. (1970) Enbcrinology 83: 1414-1419. Levi=, J.H., Learning, A.B. and Raskin, P. (1978) In: Advances in Polyamine Research. (Campbell, R.A., Morris, D.R., Bartos, D., Daves, G.D. and Bartos, F., eds) Vol. 1: 51-53. Raven Press, N.Y. Janne, J., Raina, A. and Si'imes, M. (1963r Biochim. Biophys. Acta 166: 419-426. Levi= J.H., Nicholson, W.E., Liddle, G.E. and Orth, D.N. (1973) Endocrinology 92: 1039-1095, Kobayashi, Y., Kupqian, J. and Maudsley, D.V. (1971) Science -172: 379-330. Nicholson, W.E., Levine, J.H. and Orth, D.N, (1976) Endocrfnology -98: 123-123.
299
Vol. 86, No. 2, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 293-299
January 30, 1979
THE POSSIBLE ROLE OF INTRACELLULAR POLYAMINES IN MITOCHONDRIAL METABOLIC REGULATION Rowand R.J.
Chaffee,
Department
Received
Robert
M. Arine
and Rene H. Rochelle
of Ergonomics, University Santa Barbara, California
of California 93106
November 28,1978
SUMMARY: Studies were made on the effects of very low concentrations of the polyamine, spermine, on rat liver mitochondrial metabolism associated with B-hydroxybutyrate. The respiratory control ratio and the rate of respiration during ADP-ATP conversion are significantly altered with shifts in spermine concentrations of as little as 15.7 nMoles/ml within the physiological Mgf+ concentration range. These spermine concentration changes are small compared to the estimated hepatic intracellular levels of spermine which have been reported to be between 200 and 1200 nMoles/gm wet weight under normal conditions. There is now evidence that exposure of an animal to certain environmental conditions induces changes of 164 nMoles/gm wet weight in intracellular levels of liver spermine in a few hours. Also there is evidence that the concentration of intracellular polyamines is influenced by endocrines since the levels of the enzymes responsible for their synthesis are markedly affected by hormonal changes. Therefore, alterations of polyamine levels may play a role in mitochondrial metabolic regulation in vivo. It
has long
RNA synthesis affect
and thus
mitochondrial
interesting a change did
protein
that
in mitochondrial his
assay
did
ratios.
naturally
there
The few papers
with
substrates,
respiratory
control
ratio
These
polyamine
levels
(8).
However,
the data
and therefore
the oxidation
for
the
during
spermine rate.
of studying
and after
little
information
polyamines
on these on this
mitochondrial (5-7)
and 1.25
mM spermidine
and respiration
during
the conversion
the concentration
in our previous
papers
ranges (5-7)
of ADP to
on the effects
subject
might
he
the polyamine
respiratory
1.0 mM spermine
are within
causes
However,
the conversion
on mitochondrial
we have written
indirectly
made the very
of succinate,
purpose
can affect
would
(4)
and the mean respiratory
is very
intracellular
such as spermine
Huunan-Seppala
any information
parameters. certain
during
respiration
In fact,
(l-3)
Also,
procedures
not obtain
occurring
polyamines
synthesis
swelling
on mitochondrial
ATP and thus
that
respiration.
observation
not design
effects
trol
been recognized
found well
conof
metabolic show that affect
the
of ADP to ATP. in rat
liver
be subject
to
0006-291X/79/020293-07$01.00/0 293
Copyright @ 1979 by Academrc Press, Inc. All rights ofreproduction in any form resew-ved.
Vol. 86, No. 2, 1979
critcism
because
significant
it
argue
polyamine
changes
mechanism.
that
over that
which
effects
changes
changes
the polyamines
ratio.
the data
metabolic
control as
additions
con-
the
of small
presented
is
latter
amounts
the first
can cause
very
ADP-ATP conversion
of these effects
mM
respiratory
Furthermore,
during
regulatory in this
such gross
of a polyamine
The significance
presented
requiring
as low as 0.0157
herein
respiration
have possible
the hypothesis
extra
And
B-hydroxybutyrate
conversion.
with
to cause any
substrates.
mitochondrial
in concentration
on mitochondrial
control
liver
ADP-ATP
Thus,
with
concentration
affects
range.
system
presented
a spermine
during
those
an insensitive
herein
significantly
a wide
respiratory
is
that
such small
significant
suggest
in the data
with
regulatory
represent
RESEARCH COMMUNICATIONS
of a polyamine
metabolism
at best
show incremental
of spermine
the
would
and respiration
two parameters
concentrations
any intracellular
nMoles/ml)
ratios
evidence
such high
we have found
15.7
AND BIOPHYSCAL
in mitochondrial
However,
substrate,
trol
took
changes
one might
(or
BIOCHEMICAL
findings
is
on cellular
and
that
they
respiration
paper.
METHODS Mitochondria were isolated from livers of adult male Sprague-Dawley rats by the method described by Hoch and Lipmann (9). All assays were made at 37' C, using the polarographic apparatus described by Estabrook (10). In the liver in vivo, acetoacetate is reduced to @-hydroxybutyrate (ll), but if there is an the reaction is reversed and the oxidaexcess of 8-hydroxybutyrate in vitro, tion of NADH thus generated can be followed polarographically. For assaying a modification of the reaction mixture of Harris B-hydroxybutyrate oxidation, et al (12) was used. The 3.6 ml reaction mixture contained: 900 UMoles 1.08 PMoles ADP, sucrose, 90 VMoles KCl, 18 PMoles KH2P04, 72 PMoles Tris-HCl, 90 PMoles B-hydroxybutyrate and the appropriate concentrations of spermine and Mg++. In each assay, 2-4 mg of mitochondrial protein was used. Protein concentration was determined spectrophotometrically by the method of Lowry et al (13). Spermine concentration was varied between 0.0157 and 2.0 mM whereas Mgtt used were those estimated to concentrations were 0.6 and 0.93 mM. Mg++ levels be free in rat liver cytoplasmic fluid (14-16). RESULTS AND DISCUSSION In figure centration droxybutyrate of spermine
1 is
shown the
from 0 up to 0.25
this
effects
range
of increasing
mM on the respiratory As was mentioned
as substrate. within
striking
are at or below
294
the spermine
control
previously,
ratio
with
con-
B-hy-
such concentrations
the concentration
levels
found
Vol.
86,
No.
BIOCHEMICAL
2, 1979
0.0157
0.0313
0.0625
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0.125 SPERMINE
Figure
in rat
1
Effects of increasing the concentration of spermine on the respiratory control r$$io with a-hydroxybutyrate as substrate at 0.6 and 0.93 mM Mg .
liver
trations
homogenates
of at least
influence
during
these
levels
are
metabolism
concentration
evidence
Therefore,
amounts
and how they
is particularly
that
environmental when rats
spermine weight
levels which
are
tively
0.164
is evidence
rapid their
returned
is a change
of approximately There
conditions
increase
synthesis
the
in light
Tsvetnenko
of 164 nMoles/gm mM) in a matter indicates
have remarkably
of oxidation
after
that
concen-
and therefore
1 shows how small
much in excess (20).
likely
in the cell Table
important
from 213 nMoles/gm
changes
is quite
influence
to normoxia
that
concentration
present
the rate
have been shown to fluctuate
certain
it
in vivo.
elevate
ADP-ATP conversion
The above
for
(17-20).
mitochondrial
in spermine
0.250 (mM)
increases
of @-hydroxybutyrate respiratory
control
of the fact of 0.0157
that
(ibid)
to hyperoxia,
wet weight
ratio. spermine
mM --in vivo
and Shugaley
exposure
wet weight
would
under reported
liver
up to 379 nMoles/gm (or
a concentration
wet change
of hours. that
polyamines
in the cell short
295
since
half-lives.
may be subject the enzymes
to rela-
responsible
For example,
S-adenosyl-
1.
1.; 2:o
** 0 0.0157 0.0313 0.0625 0.125 0.250 0.50
** 0 0.0157 0.0313 0.0625 0.125 0.250 0.50 1.0 1.5 2.0
Sw (mM)
on rat
59.51 66.24 68.71 70.43 73.61 75.09 78.36 79.02 79.17 83.00
54.60 60.37 63.65 66.85 69.89 74.27 82.23 82.69 84.62 85.80
during ADP-ATP Conversion
Q02*
Spermine effects B-hydroxybutyrate.
* ** ***
In: ~1 O2 consumed/ mg rat Control Not significantly different
The reaction mixture contained: 90 umoles e-hydroxybutyrate and
0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.93
i*: 0:6 0.6 0.6
0.6 0.6 0.6 0.6 0.6
Mg++ (mM)
Table
0.05 0.001 0.01 0.001 0.001 0.001 0.001 0.001 0.001
-----
----0.05 0.02 0.01 0.001 0.001 0.001 0.001 0.001 0.001
from
liver
P
control.
mitochondrial
sucrose, ADP (pH
mitochondrial
900 pmoles 1.08 umoles
liver
protein/hr
90 pimoles 7.2).
31.34 30.71 31.12 25.69 25.79 24.26 23.36 24.47 25.88 27.15
38.76 42.00 38.39 29.16 26.74 24.65 23.37 24.37 26.81 29.86
Q02* after ADP-ATP Conversion
respiration
KCl,
18pmoles
NSD NSD NSD NSD 0.05 NSD NSD NSD NSD
-----
0.02 NSD 0.05 0.01 0.001 0.001 0.001 0.01 0.05
P
metabolic
-----
and
KH2P04,
0.05 0.05 0.01 0.01 0.001 0.001 0.01 0.01 0.01
----a
___-_
P
*** NSD NSD 0.02 0.001 0.001 0.001 0.001 0.001 0.001
with
Tris-HCl,
associated
72 umoles
2.00 2.35 2.41 2.76 2.91 3.13 3.41 3.31 3.12 3.14
1.44 1.47 1.76 2.30 2.62 3.03 3.61 3.50 3.24 2.97
Respiratory Control Ratio
control
BIOCHEMICAL
Vol. 86, No. 2, 1979
methionine
decarboxylase
as ornithine 23-25). lase
Furthermore,
terior
anterior
to stressful
pituitary,
influence Other drial
metabolic heat-acclimated
mine
than
are
animals
since
changes
nificantly amines
(20)
affect
the rate and since
further
From the
data
like
is
on the an-
activity
and thus
can cause
mitochon-
that
mitochondria
concentration
mitochondria elevation
from
of sperthe heat-
of the respiratory in the presence
can respond
in levels
of polyamines
would
that
of
to environcan sig-
the possible
role
seem to be an area
spermine
control
in some respects in figure
the effects
ratio
control
ratio
elevated
of poly-
which
within
certain
1, with
1.44
ratio
as little
concentration
297
whereas
is elevated
1, it
ranges
has been metabolism
is obvious
0.6 mM Mg" with
it
protein
on mitochondrial
that
the
metabolism and no spermine,
as little
to 2.30.
above that
as 0.0157
in a capacity
since
in affecting
1 and table
is considerably
with
control
of spermine
is only
control
can function
metabolic
As shown in table
significantly
and spermine
synthesis
in vitro,
control
is
Mgtt
the respiratory
the respiratory
metabolism
presented
of 8-hydroxybutyrate.
spermine,
changes
of Mg++ in mitochondrial
of Mg++ can alter
the respiratory
small
possibility
to function
levels
changes
investigation.
to that
reported
is,
from the controls
metabolic
One interesting similar
is
to a given
That
of polyamine
mitochondrial
in mitochondrial
warrants
effects
conditions
greater
mitochondria
via
of polyamines
are more sensitive (6).
physiological
decarboxylase
environmental
of controls
decarboxy-
--in vivo.
due to the action
did
can,
(1, 8,
(ibid).
Therefore, mental
synthesis
21, 22) where-
ornithine
Thus,
conditions
had a consistently
than
increase
(26-30).
in ornithine
stressful
rats
those
ratio
spermine
that
changes
from
control
of polyamine
evidence
acclimated
environmental
(1,
10 and 24 minutes
hormones
organs
changes
60 minutes
between
pituitary target
mediate
the rate
of about
has a half-life
in appropriate
in response
(3).
has a half-life
decarboxylase
activity
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
as 0.0625 At 0.93
mM Mg++,
at 0.6 mM Mg++ and
mM spermine.
Thus,
seem to be synergistic
mM
Mg++ in
BIOCHEMICAL
Vol. 86, No. 2, 1979
elevating
the
respiratory
of B-hydroxybutyrate. smaller that
than
those
the range
while
report
changes
in levels
ible
role
But the of Mg++.
ratio
with
is
interesting
cytoplasmic
0.2 and 1.2 mM spermine
metabolic
oxidation
involved
are much
because
Mg++ is very
it
narrow
more variable. in rat
such as spermine
of cellular
mitochondrial
of spermine
may be relatively
of a polyamine
in alterations
synergism
of free
concentration
between
associated
concentrations
This
of variation
the spermine
workers
control
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
liver
may well
control
than
is thought (14-16)
Different
(17-20). play
Thus,
a more flex-
do changes
in Mg++
concentration. ACKNOWLEDGMENTS This work was supported by ONR Grant and NASA-Ames NCA 2-OR-608-601.
N00014-75-C-0506,
NIH Grant
AM 18895-01
BIBLIOGRAPHY 1.
2. 3. 4. 5. 6. 7.
8.
9.
10. 11. 12. 13. 14. 15. 16.
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