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Possible isozyme structures in malate dehydrogenase from Bacillus subtilis
Vol. 39, No. 2, 1970
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
POSSIBLE ISOZYME STRUCTURES IN MAIATE
DEHYDROGENASE
FROM BACILLUS SUBTILIS
S. Antohi,
I. Moraru,
D. Cotae
Babes Institute of Pathology and Genetics. Laboratory of Genetics. Bucharest. 35, Romania.
Received February 23, 1970 It was reported that NAD dependentmalate dehydrogenase (MDH) from crude extracts of Bacillus subtilis, as well as the crystalized enzyme, showed a single band of activity in polyacrylamide gel electrophoresis (Yoshida, 1965). Murphey et al. (1967) and Murphey & Kaplan (1967) have found the same homogeneity of MDH activity in starch gel electrophoresis, but they have reported a heterogeneity of the subunits isolated from MDH tetrametric molecules with respect to their molecular weights. The present report illustrates the followings:
i) MDH
in crude extracts from Bacillus subtilis exhibits two isozymes which are different with respect to electrophoretic running, substrate afinity and sensitivity to heat treatment and to 4-chloromercuri benzoate. ii) The existence of two MDH isozymes in Bacillus subtilis, appears to account for the unexplained heterogeneity of the MDH subunits. Material and Method Bacillus subtilis WT strain kindly provided by Anagno3 was used. The origin of the WT strain was explained stopoulos by Barat et al. (1965) . Fresh culture of bacteria was inoculated in concentration of lo7 cells/ml in Penassay Broth (Difco Antibiotic Medium No 3) and was incubated with aeration at 37’ for 16 hours. The cells were harvested by centrifugation and washed once in phosphatebuffer 226
BIOCHEMICAL
Vol. 39, No. 2, 1970
(0.01
M Na2HP04,
o. 002 M KH2P04.
resuspended
in the same buffer
(Mann,Res.
Lab. N. Y) 2oo/pg/ml
incubated
at 37’C
lO/pg/ml
10 min at 37’C
the product
g for 1 hour.
lysozyme
high viscous
l/pg/ml
were
The clear
suspension,
and ribonuclease
added to remove
was centrifuged
at 55’C,
to chloromercuri romercuri
the viscosity.
After
with refrigeration
yellowish
supernatant
(Fluka
(treated
were
from
was tested by adding AG,
Buchs
or untreated)
immediately
(+Z’C)
was used
crude
The sensibility
10W3M Sodium
in polyacrylamide (1964) and Davis
(1964).
Soft and
separation
gel the pH was 9. o and for the soft gel was 6.8. (hydroxymethyl)
respectively.
aminomethane
The electrophoretic
a cell constructed The running
3 and 7 % acrylamide
gel was carried
gels were
pH 9.3.
From
of 40 ~1 in electrophoretic
separation
Germany)
4-chlo-
used.
by Ornstein
was glycine-tris
extract
SG) in the same extract.
samples
Disc electrophoresis out as described
aliquots
60°C and 70°C for 30 minutes.
benzoate
benzoate
each aliquot
Jena,
was
cell free extract.
heated
analysis
was
in 5 ml),
In the clearer,
To test the heat inactivation, were
The pellet
was added and the suspension
(NBC-Cleveland)
(NRC-Cleveland)
as crude
pH ‘7.5).
(1 g wet pellet
for 1 hour.
desoxyribonuclease
at 12,000
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in this laboratory
time was 3-4 hours
For the
(Merck-Darmstadt-
running
was carried
after that of Langvad
at a current
The buffer
out in (1968 a and b).
of 5 mA (source
Karl
Zeiss
Germany). The MDH were
Res. Laboratories and Apella, sodium
L-malate
tris
(N. B. C. Cleveland blue (2 mg/ml)
o. 30 ml,
staining
(0.05
M) -HCl
Ohio)
7. o ml,
6 H20 (1M) o. 1 ml.
phenasine
The gels were
at 37’C
for 60 min and at 24’C in ‘7.5 % acetic
The quantitative
for 30 min.
227
mixture buffer
(Mann.
(Markert pH 7.5-15
ml,
methosulphate
Ohio) 20 mg, incubated
in this mixture
The gel columns
acid (Langvad
measurements
blue
(0.3 M) pH 7. 5-6 ml,
NAD (N. B. C. Cleveland
MgS04.
fixed and stored
by p-nitrotetrazolium
N. Y. ) using a modified
1961) as following:
nitrotetrazoliutn (2 mg/ml)
stained
were
then
a,b)
of the MDH activities
were
Vol. 39, No. 2, 1970
carried (Karl
BIOCHEMICAL
out by densitometry Zeiss,
Jena,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in a Karl
Zeiss
type ERIIO microdensitometer
Germany).
Results In the crude activity there
were were
anodal
found,
of Bacillus
With respect
subtilis
to their
four bands of MDH
electrophoretic
two fast bands and two slow bands
(thefastest)
activity
extracts
velocity
(fig. 1.1. ).
band had 82-85 per cent from
The most
the total MDH
(fig. 1.1. ).
c
+
I
Fig. 1
Different
electrophoretic
The explanations
For control, crude
extracts
were
incubated.
slow bands. intensity control
were
gel columns
carried
This
The presence
between required
the electrophoresis
resulted
free
in a poor (L-malate)
staining staining
mixture of the two
increased
not stained
of
their
at all in the
mixture.
exhibiting
a distance
these bands there for staining:
(fig. 1.3) in the first the other
(in which
of substrate
The two fast bands another,
of MDH.
are in the text.
The fast bands were
of staining. staining
patterns
out) in a substrate
incubation
+
3
2
appeared
(fig. 1.1. ) were of running
was a marked
whereas 10 min.
the faster
difference
one
about the time
one appeared
at 40-50 mm of incubation
228
from
of o. 3-o. 8 cm. Moreover,
of incubation
(fig. 1.3. )
very distinct
c
fast early lfast
earlier
(fe) band I late (fl) band]
Vol. 39, No. 2, 1970
BIOCHEMICAL
The heat treatment in a more
marked
inactivated of more
of extracts
inactivation
in a proportion
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in a complete
of lo-15
inactived
per cent the fl band in a percentage
The fe band was completly
The 4-chloromercuri
inactivation
resulted
of the fl band (fig. 1.2) If the fe band was
than 90 was inactivated.
at 70°C for 30 min.
at 60°C for 30 min.
of fl band,
benzoate whereas
inactivated
treatment
resulted
the fe band was not
at all.
Discussion MDH from electrophoretically of specific entire
crude
extracts
heterogenous,
activity:
pattern,
exhibiting
fast early
at a longer
of Bacillus
subtilis
was
a zymogram
of two bands
(fe) band and fast late (fl) band.
incubation
time
ir the staining
The
mixture
could be visualized. The two slow bands which period,
were
not considered
nitrotetrazolium
MDH isozymes
blue conversion,
substrate.
This
suggested Their
oxydoreductases. was explained
more
in the crude
the difference between well
the incubation
as the different
benzoate
for MDH of Bacillus
(Murphey
free of
tetrazolium
staining
in presence
remained
of malate
at the level
running
the existence
of Bacillus
afinity
time periods
the single
(infered
of
Moreover,
the inequality
of the two bands),
to heat inactivation the concept
of the two fe
of two MDH
subtilis. from
for staining
band pattern
of two types of subunits
et al. 1967),
is in agreement
mixture
as
and 4-chloromercuri
of the two isozymes
pattern
subtilis.
Whereas with the existence
indicates
homogenates
support
staining
columns.
sensitivities
inactivation,
the incubation
since they gave the
in the electrophoretic
in the substrate
during
nitroblue
MDH residues
and fl bands of MDH activity, isozymes
intensive
in the running
The difference
later
in a control
that they were
by the small
these reductases,
appeared
the existence
with the molecular
was in marked in MDH tetramers
of two isozymes weight
229
contrast
heterogeneity
for this enzyme of the subunits
Vol. 39, No. 2, 1970
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
isolated by Murphey et al (1967) from MDH of Bacillus subtilis. However the two MDH isozymes appear to be encodedby a single gene, because the both isozymes were missing in the MDH defective mutants obtained in this laboratory (Antohi et al. 1970). It would be possible to suggest that MDH gene would encode for the subunit of 31,500 Daltons (Murphey et al. 1967), which can partly be deleted into subunits of 15,000 Daltons as in the case of hexokinases (Kaji 1965, Gazith et al. 1968). In the resulted subunit pool, in which the subunit of 15,000 Daltons is a minor component (Murphey et al. 1967), two tetrameric forms would be established: the major fe MDH isozyme and the minor fl MDH isozyme.
We are indebted to Dr. Langvad (Fibiger Laboratory Lyngby Denmark) for his theretical counselling in disc electrophoresis and to Mrs. Adriana Cost&e for her technical assistance. REFERENCES Dr. C. Anagnostopoulos, Centre de G&&ique Moleculaire, 91 Gif sur Yvette, France. Antohi, S.,
Lengyel, Z.,
Cotae, D.,
(in Preparation)
Barat, M., Anagnostopoulos, C., Schneider, A.M., 357 (1965)
J. Bact., 90
Davis, B. J. , Ann. N. Y. Acad. Sci., 121, 404 (1964) Gazith, I., Schulze, I,, Goading, R., Womack, F., & Collowick, S Ann. N. Y. Acad. Sci., 151, 307 (1968) Kaji, A., Arch. Biochem. Biophys., 12, Langvad, E.,
Int. J. Cancer, s
Langvad, E.,
Europ. J. Cancer, s
Markert,
C. L.,
54 (1965)
17 (1968) 107 (1968)
Apella, E., Ann.N. Y. Acad. Sci., &
678 (1961)
Murphey, H. W., Barnaby, C., Lin, F. J., Kaplan, N. 0.) J. Biol. Chem., 242, 1548 (1967) Murphey, H. W., Kaplan, N. 0, J. Biol. Chem., 242, 1561 (1967) Ornstein, L., Ann. N. Y. Acad. Sci-2 121 321 (1964) Yoshida, A., J. Biol. Chem.I -2 240 1113 (1965) 230
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
POSSIBLE ISOZYME STRUCTURES IN MAIATE
DEHYDROGENASE
FROM BACILLUS SUBTILIS
S. Antohi,
I. Moraru,
D. Cotae
Babes Institute of Pathology and Genetics. Laboratory of Genetics. Bucharest. 35, Romania.
Received February 23, 1970 It was reported that NAD dependentmalate dehydrogenase (MDH) from crude extracts of Bacillus subtilis, as well as the crystalized enzyme, showed a single band of activity in polyacrylamide gel electrophoresis (Yoshida, 1965). Murphey et al. (1967) and Murphey & Kaplan (1967) have found the same homogeneity of MDH activity in starch gel electrophoresis, but they have reported a heterogeneity of the subunits isolated from MDH tetrametric molecules with respect to their molecular weights. The present report illustrates the followings:
i) MDH
in crude extracts from Bacillus subtilis exhibits two isozymes which are different with respect to electrophoretic running, substrate afinity and sensitivity to heat treatment and to 4-chloromercuri benzoate. ii) The existence of two MDH isozymes in Bacillus subtilis, appears to account for the unexplained heterogeneity of the MDH subunits. Material and Method Bacillus subtilis WT strain kindly provided by Anagno3 was used. The origin of the WT strain was explained stopoulos by Barat et al. (1965) . Fresh culture of bacteria was inoculated in concentration of lo7 cells/ml in Penassay Broth (Difco Antibiotic Medium No 3) and was incubated with aeration at 37’ for 16 hours. The cells were harvested by centrifugation and washed once in phosphatebuffer 226
BIOCHEMICAL
Vol. 39, No. 2, 1970
(0.01
M Na2HP04,
o. 002 M KH2P04.
resuspended
in the same buffer
(Mann,Res.
Lab. N. Y) 2oo/pg/ml
incubated
at 37’C
lO/pg/ml
10 min at 37’C
the product
g for 1 hour.
lysozyme
high viscous
l/pg/ml
were
The clear
suspension,
and ribonuclease
added to remove
was centrifuged
at 55’C,
to chloromercuri romercuri
the viscosity.
After
with refrigeration
yellowish
supernatant
(Fluka
(treated
were
from
was tested by adding AG,
Buchs
or untreated)
immediately
(+Z’C)
was used
crude
The sensibility
10W3M Sodium
in polyacrylamide (1964) and Davis
(1964).
Soft and
separation
gel the pH was 9. o and for the soft gel was 6.8. (hydroxymethyl)
respectively.
aminomethane
The electrophoretic
a cell constructed The running
3 and 7 % acrylamide
gel was carried
gels were
pH 9.3.
From
of 40 ~1 in electrophoretic
separation
Germany)
4-chlo-
used.
by Ornstein
was glycine-tris
extract
SG) in the same extract.
samples
Disc electrophoresis out as described
aliquots
60°C and 70°C for 30 minutes.
benzoate
benzoate
each aliquot
Jena,
was
cell free extract.
heated
analysis
was
in 5 ml),
In the clearer,
To test the heat inactivation, were
The pellet
was added and the suspension
(NBC-Cleveland)
(NRC-Cleveland)
as crude
pH ‘7.5).
(1 g wet pellet
for 1 hour.
desoxyribonuclease
at 12,000
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in this laboratory
time was 3-4 hours
For the
(Merck-Darmstadt-
running
was carried
after that of Langvad
at a current
The buffer
out in (1968 a and b).
of 5 mA (source
Karl
Zeiss
Germany). The MDH were
Res. Laboratories and Apella, sodium
L-malate
tris
(N. B. C. Cleveland blue (2 mg/ml)
o. 30 ml,
staining
(0.05
M) -HCl
Ohio)
7. o ml,
6 H20 (1M) o. 1 ml.
phenasine
The gels were
at 37’C
for 60 min and at 24’C in ‘7.5 % acetic
The quantitative
for 30 min.
227
mixture buffer
(Mann.
(Markert pH 7.5-15
ml,
methosulphate
Ohio) 20 mg, incubated
in this mixture
The gel columns
acid (Langvad
measurements
blue
(0.3 M) pH 7. 5-6 ml,
NAD (N. B. C. Cleveland
MgS04.
fixed and stored
by p-nitrotetrazolium
N. Y. ) using a modified
1961) as following:
nitrotetrazoliutn (2 mg/ml)
stained
were
then
a,b)
of the MDH activities
were
Vol. 39, No. 2, 1970
carried (Karl
BIOCHEMICAL
out by densitometry Zeiss,
Jena,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in a Karl
Zeiss
type ERIIO microdensitometer
Germany).
Results In the crude activity there
were were
anodal
found,
of Bacillus
With respect
subtilis
to their
four bands of MDH
electrophoretic
two fast bands and two slow bands
(thefastest)
activity
extracts
velocity
(fig. 1.1. ).
band had 82-85 per cent from
The most
the total MDH
(fig. 1.1. ).
c
+
I
Fig. 1
Different
electrophoretic
The explanations
For control, crude
extracts
were
incubated.
slow bands. intensity control
were
gel columns
carried
This
The presence
between required
the electrophoresis
resulted
free
in a poor (L-malate)
staining staining
mixture of the two
increased
not stained
of
their
at all in the
mixture.
exhibiting
a distance
these bands there for staining:
(fig. 1.3) in the first the other
(in which
of substrate
The two fast bands another,
of MDH.
are in the text.
The fast bands were
of staining. staining
patterns
out) in a substrate
incubation
+
3
2
appeared
(fig. 1.1. ) were of running
was a marked
whereas 10 min.
the faster
difference
one
about the time
one appeared
at 40-50 mm of incubation
228
from
of o. 3-o. 8 cm. Moreover,
of incubation
(fig. 1.3. )
very distinct
c
fast early lfast
earlier
(fe) band I late (fl) band]
Vol. 39, No. 2, 1970
BIOCHEMICAL
The heat treatment in a more
marked
inactivated of more
of extracts
inactivation
in a proportion
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in a complete
of lo-15
inactived
per cent the fl band in a percentage
The fe band was completly
The 4-chloromercuri
inactivation
resulted
of the fl band (fig. 1.2) If the fe band was
than 90 was inactivated.
at 70°C for 30 min.
at 60°C for 30 min.
of fl band,
benzoate whereas
inactivated
treatment
resulted
the fe band was not
at all.
Discussion MDH from electrophoretically of specific entire
crude
extracts
heterogenous,
activity:
pattern,
exhibiting
fast early
at a longer
of Bacillus
subtilis
was
a zymogram
of two bands
(fe) band and fast late (fl) band.
incubation
time
ir the staining
The
mixture
could be visualized. The two slow bands which period,
were
not considered
nitrotetrazolium
MDH isozymes
blue conversion,
substrate.
This
suggested Their
oxydoreductases. was explained
more
in the crude
the difference between well
the incubation
as the different
benzoate
for MDH of Bacillus
(Murphey
free of
tetrazolium
staining
in presence
remained
of malate
at the level
running
the existence
of Bacillus
afinity
time periods
the single
(infered
of
Moreover,
the inequality
of the two bands),
to heat inactivation the concept
of the two fe
of two MDH
subtilis. from
for staining
band pattern
of two types of subunits
et al. 1967),
is in agreement
mixture
as
and 4-chloromercuri
of the two isozymes
pattern
subtilis.
Whereas with the existence
indicates
homogenates
support
staining
columns.
sensitivities
inactivation,
the incubation
since they gave the
in the electrophoretic
in the substrate
during
nitroblue
MDH residues
and fl bands of MDH activity, isozymes
intensive
in the running
The difference
later
in a control
that they were
by the small
these reductases,
appeared
the existence
with the molecular
was in marked in MDH tetramers
of two isozymes weight
229
contrast
heterogeneity
for this enzyme of the subunits
Vol. 39, No. 2, 1970
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
isolated by Murphey et al (1967) from MDH of Bacillus subtilis. However the two MDH isozymes appear to be encodedby a single gene, because the both isozymes were missing in the MDH defective mutants obtained in this laboratory (Antohi et al. 1970). It would be possible to suggest that MDH gene would encode for the subunit of 31,500 Daltons (Murphey et al. 1967), which can partly be deleted into subunits of 15,000 Daltons as in the case of hexokinases (Kaji 1965, Gazith et al. 1968). In the resulted subunit pool, in which the subunit of 15,000 Daltons is a minor component (Murphey et al. 1967), two tetrameric forms would be established: the major fe MDH isozyme and the minor fl MDH isozyme.
We are indebted to Dr. Langvad (Fibiger Laboratory Lyngby Denmark) for his theretical counselling in disc electrophoresis and to Mrs. Adriana Cost&e for her technical assistance. REFERENCES Dr. C. Anagnostopoulos, Centre de G&&ique Moleculaire, 91 Gif sur Yvette, France. Antohi, S.,
Lengyel, Z.,
Cotae, D.,
(in Preparation)
Barat, M., Anagnostopoulos, C., Schneider, A.M., 357 (1965)
J. Bact., 90
Davis, B. J. , Ann. N. Y. Acad. Sci., 121, 404 (1964) Gazith, I., Schulze, I,, Goading, R., Womack, F., & Collowick, S Ann. N. Y. Acad. Sci., 151, 307 (1968) Kaji, A., Arch. Biochem. Biophys., 12, Langvad, E.,
Int. J. Cancer, s
Langvad, E.,
Europ. J. Cancer, s
Markert,
C. L.,
54 (1965)
17 (1968) 107 (1968)
Apella, E., Ann.N. Y. Acad. Sci., &
678 (1961)
Murphey, H. W., Barnaby, C., Lin, F. J., Kaplan, N. 0.) J. Biol. Chem., 242, 1548 (1967) Murphey, H. W., Kaplan, N. 0, J. Biol. Chem., 242, 1561 (1967) Ornstein, L., Ann. N. Y. Acad. Sci-2 121 321 (1964) Yoshida, A., J. Biol. Chem.I -2 240 1113 (1965) 230