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Coexistence of isocitrate lyase and NADP-isocitrate dehydrogenase in Turbatrix aceti mitochondria
Vol. 8 1, No. 2, 1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
March 30,1978
Pages
COEXISTENCE OF ISOCITRATE LYASE AND NADP-ISOCITRATE IN TURBATRIX ACETI MITOCHONDRIA
434-438
DEHYDROGENASE
Michael P. McKinley and Richard N. Trelease Departments of Zoology and Botany-Microbiology Arizona State University Tempe, Arizona 85281 Received
January
9,197s
SUMMARY: Glyoxylate and Krebs cycle marker enzymes cobanded on sucrose gradients aftersopycnic centrifugation of mitochondrial pellets. Mitochondrial heterogeneity was investigated by treating particles with a Nitro Blue Tetrazolium reaction mixture designed to specifically increase the buoyant density of mitochondria containing the Krebs cycle enzyme isocitrate dehydrogenase. The banding of Krebs cycle enzymes at a heavier buoyant density was accompanied by the glyoxylate cycle marker isocitrate lyase. Both isocitrate-metabolizing enzymes appear to be compartmented within the same mitochondria. This strongly suggests that regulation of one or both of these enzymes is necessary for directing carbon flow through the two different cycles. The glyoxylate synthesis
of biosynthetic
ethanol
Rubin
Recent
work
(7) showed
in developing
with
non-mitochondrial
and Trelease
(9) aceti,
on sucrose determine
increasing
found
glyoxylate
and Krebs
density
here
the distribution cycle
of isocitrate
434
glyoxylate Ascaris -suggested
enzymes
cobanded
enzymes
(8).
housing
suum. a McKinley
species, with
mitochondria
was undertaken banded
How-
(6).
free-living
and another
0006-29ix/7a/0ai2-0434$0i.00/0 0 1978 by Academic Press, Inc. of reproduction in any form reserved.
of these
of mitochondria
was to compare
typically
glyoxysomes
elegans,
reported
markers
are
nematode
enzymes
the net
on acetate,
contained
in another
cycle
chondria.
Copyright All rights
termed
Caenorhabditis
groups
cycle
marker)
the buoyant
of the bypass
mitochondria
that
for
subsisting
of the parasitic
and Krebs
two separate
cycle
that
providing
organisms
organelles
The investigation
The approach
(a glyoxylate
for
variant
compartmentation
glyoxylate
whether
catalysts.
form,
subsequently
gradients.
one containing
larvae
a free-living
particulate,
Turbatrix
cycle
The enzymes
specialized
and Trelease
enzymes
a Krebs
(l-5).
within
bypass
is
intermediates
or triglycerides
compartmentalized ever,
bypass
together, Krebs
dehydrogenase-containing
i.e.,
cycle
of isocitrate on sucrose
to
lyase
gradients
after mito-
Vol. 81, No. 2, 1978
8lOCHEMlCAL
MATERIALS
AND 8lOPHYSlCAL
RESEARCH COMMUNICATIONS
AND METHODS
Axenic cultures of x. aceti were grown in a basal medium described by Bothstein (10). Fourteen-day-old worm cultures were harvested, cleaned and homogenized as previously described (9), except that the grinding medium contained 250 mM sucrose and 1 mM EDTA in 50 mM Tris-HCl, pH 8.5. Differential fractions were collected and handled as before (91, except that mitochondrial pellets were collected at 10,200 x g for 10 min. This pellet was either treated with Nitro Blue Tetrazolium prior to gradient application or it was layered untreated onto a 46-ml sucrose gradient (38 to 51% w/w) buffered with 50 mM Tris-HCl, pH 7.5. The particles were centrifuged to equilibrium in a Beckman SW-25.2 rotor for 13 hr at 29,000 x g. Two-ml fractions were collected dropwise from the bottom of cellulose nitrate tubes. Activities of the following enzymes were determined as described in the accompanying references: isocitrate lyase (EC 4.1.3.1) (9); succinate dehydrogenase (EC 1.3.99.1) (11); NAD-isocitrate dehydrogenase (EC 1.1.1.41) and NADP-isocitrate dehydrogenase (EC 1.1.1.42) (12). Treatment of mitochondrial samples for NADP-isocitrate dehydrogenase reactivity was accomplished by incubating the particles for 20 min in the following Nitro Blue Tetrazolium reaction mixture: 50 'mg Na+-isocitrate, 5 mg Nitro Blue Tetrazolium, 5 mg NADP, 0.6 mg phenazine methosulfate, 1 ml 10 mM manganous chloride, 15 ml 500 mW Tris-HCl, pH 7.5 containing 250 mW sucrose, and 7.8 ml distilled water. The reaction mixture was prepared iznnediately prior to incubation of the organelles. Following incubation, particles were pelleted and resuspended in gradient buffer prior to application to the sucrose gradient. Polyacrylamide gel electrophoresis was done according to Volk --et al. Isocitrate dehydrogenase reactivity was detected on gels by the method (13). of Reeves -et al. (14).
RBSULTS AND DISCUSSION Isopycnic
centrifugation
of untreated
profiles
of glyoxylate
(isocitrate
similar isocitrate
dehydrogenase
together showed Erebs
at 1.204 a similar cycle
(1.204 isocitrate
g/cm31
mixture.
show that
at a higher (cf.
The reaction
lower
dehydrogenase
density the Nitro
density
(1.214
panels
to upper
activity
Fumarase
treated
with
produces of the Blue
g/cm31 panels).
at the higher
435
cycle
are not plotted.
were
yielded (NAD-,
To select the Nitro
organelles.
the untreated The banding
for
Blue
fonnazan Figures
Tetrazolium-treated
density
peaking
activities
an insoluble
stained
than
NADP-
enzyme activities
panels).
the data
pellets
and Krebs
dehydrogenase)
particles
the buoyant
panel)
lyase)
1 and 2, upper but
only,
increase
1 and 2 (lower
(Figs.
distribution,
reaction
should
equilibrated
g/cm3
mitochondria
Tetrazolium which
, and succinate
mitochondrial
particles controls of NADP-
was coincident
with
(15)
Vol. 8 1, No. 2, 1978
8lOCHEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
02
121 DENSITY
1.211 1.121
(g/cm3)
1. Distribution of isocitrate lyase and NADP-isocitrate dehydrogenase activities on equilibrium sucrose density gradients under three experimental conditions: UNTREATED - organelles applied to gradient immediately following isolation from organism, TREATED - organelles applied to gradient following incubation with Nitro Blue Tetrazolium reaction mixture, UNTREATED plus TREATED - a mixture of organelles from both of the above conditions applied to the gradient. 2. Distribution of succinate dehydrogenase and NAD-isocitrate dehydrogenase activities on equilibrium sucrose density gradients under the experimental conditions as described in Fig. 1: UNTREATED, TREATED and UNTREATED plus TREATED.
other
Krebs-cycle
citrate
lyase;
panel).
That
enzymes. it
also
peaked
at the higher
Blue
Tetrazolium-treated
the Nitro
influencing
the equilibration
ments
treated
where
(Figs. in both
1.204
Polyacrylamide specificity
panels).
and 1.214 gel
of the Nitro
was the distribution buoyant
density
mitochondria
of unreacted
and untreated
1 and 2, middle the
Of significance
mitochondria
mitochondria Glyoxylate
were
of iso(Fig.
1, lower not
were
was shown in experimixed
and Erebs
and centrifuged
cycle
enzymes
co-banded
g/cm3 peaks. electrophoresis
Blue
was
Tetrazolium
436
used
treatment
to demonstrate for
the
NADP-isocitrate
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 81, No. 2, 1978
3. Staining of NADP-isocitrate dehydrogenase activity gels. Electrophoresis and staining were as referenced complete reaction medium, gel B - NADP omitted, gel C A single band showing enzyme reactivity occurs at the
dehydrogenase. reaction
Figure
mixture),
substrate,
3 shows a single
while
the control
respectively)
genase aid not migrate (16) reported Therefore,
a similar
gels B and C (lacking
with
this
isocitrate
citrate
accompanied this
Seelig
enzyme extracted
of mitochondria
have been due to their
cofactor
dehydroana Colman
from heart
at the heavier
dehydrogenase shift,
and
That NADP-isocitrate
the gels was not unexpected;
result
the equilibration
lyase activity
band at the top of gel A (complete
remained unstained. into
on polyacrylamide disc Gel A in the Methods. - isocitrate omitted. top of gel A only.
density
tissue. must
reactivity,
end since
one is left
to conclude
isothat
both enzymes are in the same mitochondria. Reiss isozymes
and Rothstein which
migrate
(17) found that 2 to 3 cm into
scanned at 324 nm to detect lyase
activities.
isocitrate
pdyacrylamide
the phenylhydrazone
We have confirmed
lyase
these results
437
gels.
produced
exists
as five
The gels were by the isocitrate
using the system described
Vol. 81, No. 2, 1978
for
BIOCHEMICAL
electrophoresing
The different that
the
within
of these
two enzymes
the
perhaps
the NADP-isocitrate
migration
and therefore
to elucidate
a0 not
are probably two cycles
involving this
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
two enzymes
exist not
five
in
shown in Figure
the same gel
system
as an isocitrate-metabolizing
regulated
is more likely
the
dehydrogenase
isocitrate
as such.
regulated lyase
indicates
complex,
Isocitric
by another isozymes.
3.
acid
catalysis
mechanism(s),
Work is
in progress
problem.
ACKNOWLEDGEMENTS The authors manuscript ported
thank
and suggestions
by NSF Grant
Dr.
William
on the gel
PCM74-01442
F. Burke
for
electrophoresis.
his
critical This
review work
of the
was sup-
A04.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Kornberg, H. and Krebs, H. (1957) Nature 179, 988-991. (1969) Ann. N.Y. Acad. Sci. 168, 211-369. Hogg, J.F. D. (1970) Comp. Biochem. Physiol. Barrett, J., Ward, C. and Fairbairn, 35, 577-586. Rothstein, M. and Mayoh, H. (1965) Comp. Biochem. Physiol. 16, 361-365. Rothstein, M. and Mayoh, H. (1966) Comp. Biochem. Physiol. 17, 1181-1188. (1973) Biochem. 2, 237-285. Vigil, E. Rubin, H. and Trelease, R. (1976) J. Cell Biol. 70, 374-383. (1977) Arch. Biochem. Biophys. 183, 24-30. Patel, T. and McFadden, B. Protoplasma (in press). McKinley, M. and Trelease, R. (1978) Rothstein, M. (1974) Comp. Biochem. Physiol. 49, 669-678. G. and Hogue, P. (1973) Plant Physiol. 52, Singer, T., Oestreicher, 616-621. (1967) Comp. Biochem. Physiol. 23, 335-359. ward, c. and Schofield, P. (1974) Anal. Biochem. 58, 315-321. Volk, M., Trelease, R. and Reeves, H. Biochem. Biophys. M. (1972) Reeves, H., Daumy, C., Lin, C. end Houston, Acta 258, 27-39. A. (1957) Nachlas, M., TSOU, K., Sousa, E. De, Cheng, C. and Seligman, J. Histochem. Cytochem. 5, 420-436. (1977) J. Biol. Chem. 252, 3671-3678. Seelig, G. and Colman, R. (1974) Biochem. 13, 1796-1800. miss, U. end Rothstein, M.
438
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
March 30,1978
Pages
COEXISTENCE OF ISOCITRATE LYASE AND NADP-ISOCITRATE IN TURBATRIX ACETI MITOCHONDRIA
434-438
DEHYDROGENASE
Michael P. McKinley and Richard N. Trelease Departments of Zoology and Botany-Microbiology Arizona State University Tempe, Arizona 85281 Received
January
9,197s
SUMMARY: Glyoxylate and Krebs cycle marker enzymes cobanded on sucrose gradients aftersopycnic centrifugation of mitochondrial pellets. Mitochondrial heterogeneity was investigated by treating particles with a Nitro Blue Tetrazolium reaction mixture designed to specifically increase the buoyant density of mitochondria containing the Krebs cycle enzyme isocitrate dehydrogenase. The banding of Krebs cycle enzymes at a heavier buoyant density was accompanied by the glyoxylate cycle marker isocitrate lyase. Both isocitrate-metabolizing enzymes appear to be compartmented within the same mitochondria. This strongly suggests that regulation of one or both of these enzymes is necessary for directing carbon flow through the two different cycles. The glyoxylate synthesis
of biosynthetic
ethanol
Rubin
Recent
work
(7) showed
in developing
with
non-mitochondrial
and Trelease
(9) aceti,
on sucrose determine
increasing
found
glyoxylate
and Krebs
density
here
the distribution cycle
of isocitrate
434
glyoxylate Ascaris -suggested
enzymes
cobanded
enzymes
(8).
housing
suum. a McKinley
species, with
mitochondria
was undertaken banded
How-
(6).
free-living
and another
0006-29ix/7a/0ai2-0434$0i.00/0 0 1978 by Academic Press, Inc. of reproduction in any form reserved.
of these
of mitochondria
was to compare
typically
glyoxysomes
elegans,
reported
markers
are
nematode
enzymes
the net
on acetate,
contained
in another
cycle
chondria.
Copyright All rights
termed
Caenorhabditis
groups
cycle
marker)
the buoyant
of the bypass
mitochondria
that
for
subsisting
of the parasitic
and Krebs
two separate
cycle
that
providing
organisms
organelles
The investigation
The approach
(a glyoxylate
for
variant
compartmentation
glyoxylate
whether
catalysts.
form,
subsequently
gradients.
one containing
larvae
a free-living
particulate,
Turbatrix
cycle
The enzymes
specialized
and Trelease
enzymes
a Krebs
(l-5).
within
bypass
is
intermediates
or triglycerides
compartmentalized ever,
bypass
together, Krebs
dehydrogenase-containing
i.e.,
cycle
of isocitrate on sucrose
to
lyase
gradients
after mito-
Vol. 81, No. 2, 1978
8lOCHEMlCAL
MATERIALS
AND 8lOPHYSlCAL
RESEARCH COMMUNICATIONS
AND METHODS
Axenic cultures of x. aceti were grown in a basal medium described by Bothstein (10). Fourteen-day-old worm cultures were harvested, cleaned and homogenized as previously described (9), except that the grinding medium contained 250 mM sucrose and 1 mM EDTA in 50 mM Tris-HCl, pH 8.5. Differential fractions were collected and handled as before (91, except that mitochondrial pellets were collected at 10,200 x g for 10 min. This pellet was either treated with Nitro Blue Tetrazolium prior to gradient application or it was layered untreated onto a 46-ml sucrose gradient (38 to 51% w/w) buffered with 50 mM Tris-HCl, pH 7.5. The particles were centrifuged to equilibrium in a Beckman SW-25.2 rotor for 13 hr at 29,000 x g. Two-ml fractions were collected dropwise from the bottom of cellulose nitrate tubes. Activities of the following enzymes were determined as described in the accompanying references: isocitrate lyase (EC 4.1.3.1) (9); succinate dehydrogenase (EC 1.3.99.1) (11); NAD-isocitrate dehydrogenase (EC 1.1.1.41) and NADP-isocitrate dehydrogenase (EC 1.1.1.42) (12). Treatment of mitochondrial samples for NADP-isocitrate dehydrogenase reactivity was accomplished by incubating the particles for 20 min in the following Nitro Blue Tetrazolium reaction mixture: 50 'mg Na+-isocitrate, 5 mg Nitro Blue Tetrazolium, 5 mg NADP, 0.6 mg phenazine methosulfate, 1 ml 10 mM manganous chloride, 15 ml 500 mW Tris-HCl, pH 7.5 containing 250 mW sucrose, and 7.8 ml distilled water. The reaction mixture was prepared iznnediately prior to incubation of the organelles. Following incubation, particles were pelleted and resuspended in gradient buffer prior to application to the sucrose gradient. Polyacrylamide gel electrophoresis was done according to Volk --et al. Isocitrate dehydrogenase reactivity was detected on gels by the method (13). of Reeves -et al. (14).
RBSULTS AND DISCUSSION Isopycnic
centrifugation
of untreated
profiles
of glyoxylate
(isocitrate
similar isocitrate
dehydrogenase
together showed Erebs
at 1.204 a similar cycle
(1.204 isocitrate
g/cm31
mixture.
show that
at a higher (cf.
The reaction
lower
dehydrogenase
density the Nitro
density
(1.214
panels
to upper
activity
Fumarase
treated
with
produces of the Blue
g/cm31 panels).
at the higher
435
cycle
are not plotted.
were
yielded (NAD-,
To select the Nitro
organelles.
the untreated The banding
for
Blue
fonnazan Figures
Tetrazolium-treated
density
peaking
activities
an insoluble
stained
than
NADP-
enzyme activities
panels).
the data
pellets
and Krebs
dehydrogenase)
particles
the buoyant
panel)
lyase)
1 and 2, upper but
only,
increase
1 and 2 (lower
(Figs.
distribution,
reaction
should
equilibrated
g/cm3
mitochondria
Tetrazolium which
, and succinate
mitochondrial
particles controls of NADP-
was coincident
with
(15)
Vol. 8 1, No. 2, 1978
8lOCHEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
02
121 DENSITY
1.211 1.121
(g/cm3)
1. Distribution of isocitrate lyase and NADP-isocitrate dehydrogenase activities on equilibrium sucrose density gradients under three experimental conditions: UNTREATED - organelles applied to gradient immediately following isolation from organism, TREATED - organelles applied to gradient following incubation with Nitro Blue Tetrazolium reaction mixture, UNTREATED plus TREATED - a mixture of organelles from both of the above conditions applied to the gradient. 2. Distribution of succinate dehydrogenase and NAD-isocitrate dehydrogenase activities on equilibrium sucrose density gradients under the experimental conditions as described in Fig. 1: UNTREATED, TREATED and UNTREATED plus TREATED.
other
Krebs-cycle
citrate
lyase;
panel).
That
enzymes. it
also
peaked
at the higher
Blue
Tetrazolium-treated
the Nitro
influencing
the equilibration
ments
treated
where
(Figs. in both
1.204
Polyacrylamide specificity
panels).
and 1.214 gel
of the Nitro
was the distribution buoyant
density
mitochondria
of unreacted
and untreated
1 and 2, middle the
Of significance
mitochondria
mitochondria Glyoxylate
were
of iso(Fig.
1, lower not
were
was shown in experimixed
and Erebs
and centrifuged
cycle
enzymes
co-banded
g/cm3 peaks. electrophoresis
Blue
was
Tetrazolium
436
used
treatment
to demonstrate for
the
NADP-isocitrate
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 81, No. 2, 1978
3. Staining of NADP-isocitrate dehydrogenase activity gels. Electrophoresis and staining were as referenced complete reaction medium, gel B - NADP omitted, gel C A single band showing enzyme reactivity occurs at the
dehydrogenase. reaction
Figure
mixture),
substrate,
3 shows a single
while
the control
respectively)
genase aid not migrate (16) reported Therefore,
a similar
gels B and C (lacking
with
this
isocitrate
citrate
accompanied this
Seelig
enzyme extracted
of mitochondria
have been due to their
cofactor
dehydroana Colman
from heart
at the heavier
dehydrogenase shift,
and
That NADP-isocitrate
the gels was not unexpected;
result
the equilibration
lyase activity
band at the top of gel A (complete
remained unstained. into
on polyacrylamide disc Gel A in the Methods. - isocitrate omitted. top of gel A only.
density
tissue. must
reactivity,
end since
one is left
to conclude
isothat
both enzymes are in the same mitochondria. Reiss isozymes
and Rothstein which
migrate
(17) found that 2 to 3 cm into
scanned at 324 nm to detect lyase
activities.
isocitrate
pdyacrylamide
the phenylhydrazone
We have confirmed
lyase
these results
437
gels.
produced
exists
as five
The gels were by the isocitrate
using the system described
Vol. 81, No. 2, 1978
for
BIOCHEMICAL
electrophoresing
The different that
the
within
of these
two enzymes
the
perhaps
the NADP-isocitrate
migration
and therefore
to elucidate
a0 not
are probably two cycles
involving this
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
two enzymes
exist not
five
in
shown in Figure
the same gel
system
as an isocitrate-metabolizing
regulated
is more likely
the
dehydrogenase
isocitrate
as such.
regulated lyase
indicates
complex,
Isocitric
by another isozymes.
3.
acid
catalysis
mechanism(s),
Work is
in progress
problem.
ACKNOWLEDGEMENTS The authors manuscript ported
thank
and suggestions
by NSF Grant
Dr.
William
on the gel
PCM74-01442
F. Burke
for
electrophoresis.
his
critical This
review work
of the
was sup-
A04.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Kornberg, H. and Krebs, H. (1957) Nature 179, 988-991. (1969) Ann. N.Y. Acad. Sci. 168, 211-369. Hogg, J.F. D. (1970) Comp. Biochem. Physiol. Barrett, J., Ward, C. and Fairbairn, 35, 577-586. Rothstein, M. and Mayoh, H. (1965) Comp. Biochem. Physiol. 16, 361-365. Rothstein, M. and Mayoh, H. (1966) Comp. Biochem. Physiol. 17, 1181-1188. (1973) Biochem. 2, 237-285. Vigil, E. Rubin, H. and Trelease, R. (1976) J. Cell Biol. 70, 374-383. (1977) Arch. Biochem. Biophys. 183, 24-30. Patel, T. and McFadden, B. Protoplasma (in press). McKinley, M. and Trelease, R. (1978) Rothstein, M. (1974) Comp. Biochem. Physiol. 49, 669-678. G. and Hogue, P. (1973) Plant Physiol. 52, Singer, T., Oestreicher, 616-621. (1967) Comp. Biochem. Physiol. 23, 335-359. ward, c. and Schofield, P. (1974) Anal. Biochem. 58, 315-321. Volk, M., Trelease, R. and Reeves, H. Biochem. Biophys. M. (1972) Reeves, H., Daumy, C., Lin, C. end Houston, Acta 258, 27-39. A. (1957) Nachlas, M., TSOU, K., Sousa, E. De, Cheng, C. and Seligman, J. Histochem. Cytochem. 5, 420-436. (1977) J. Biol. Chem. 252, 3671-3678. Seelig, G. and Colman, R. (1974) Biochem. 13, 1796-1800. miss, U. end Rothstein, M.
438