Vol. 90, No. 1, 1979 September
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
12, 1979
Pages 36-91
NITRATE
REDUCTASE CHLORATE
AND
RESISTANT H.C.
Grupo
Received
MEMBRANE
MUTANTS
Grassi,
de Biologia Universidad
July
THE
E.
OF
CDMPOSITION
OF
ESCHERICHIA
COLI
Andrade”
and
Experimental, de Los Andes,
J.
Facultad MBrida,
PLEIOTROPIC K-12
Puig de Ciencias, Venezuela
6,1979
SUMMARY The protein composition of the membrane fraction of pleiotropic chlorate resistant mutants of --E. co1 i K-12 was analyzed as a function of Nitrate Reductase and other components. The results for all pleiotropic chlorate resistant mutants show: the presence of the Nitrate Reductase structural protein, an increase in the relative protein content in an inactive complex of this enzyme, and the disappearance of a low molecular weight protein. The later protein is lost from the wild type membrane fraction by treatment at ~o”c, 20 min. It is concluded that Nitrate Reductase is present in the cytoplasmic membrane of all pleiotropic chlorate resistant mutants in a form structurally and functionally different from the wild respiratory enzyme coupled to the electron transport chain.
INTRODUCTION Nitrate
Reductase
enzyme
of
rating
energy
which
Escherichia
is
two
were
named
and
-chlC
(3). and
CA,
B,
troling FDH,
thesis”
most of
those controling
(5). -chlD effects.
chlE
Azoulay
hypothesis”
et al -& (lo,11
* To whom reprint requests it?: Abreviations: NR, Nitrate type strain; PAGE-SDS, Dodecy 1 Sulfate; MVH,
(6),
Another
NR activity.
which
and
-chlG
mutant,
Pleiotropy
of
which (see
have
8 and
been 9)
of
and
the
have
to
lost
(7)
“particle
shown has
These
belong
to
affect
NR
FDH
activity
of
mutants,
category were
been
(3).
which
also
gene-
chlorite
mutants
shown
first
chlF has been the
nitrite,
chlorate
were
(7)
respiratory
to
mutations have
the
as
loci
completely
explained
in integrity
conlost
several hypo-
“Molybdenum-cofactor
pro-
,12). must be sent. Reductase; FDH, Formate Dehydrogenase; WT, polyacrylamide gel electrophoresis containing reduced Methyl Viologen.
0006-291X/79/170086-06$01.00/0 Copyright All rights
presence and
to
chlorate
NR-less
for
bound
nitrate
reduces (3).
(chl’), specific
locus
a membrane
reduces
the
mutants, only
important
in
or
is
also
products
resistant
pleiotropic
the
partially
the
cesing
(4))
pleiotropic and
ways,
is
It It
conditions
pleiotropic
(4)
.);‘=k
(1,2).
toxic
chlorate
non
1 .9.6.1
growth. to
anaerobic
categories:
activity,
K-12
anaerobic reduced
under
E.C.
coli
for
further
obtained mutants
(NR,
@ 1979 by Academic Press, Inc. of reproduction in any form reserved.
86
wild Sodium
Vol.
No. 1, 1979
90,
The
membrane
termine:
i)
ferent
and
of
E.
of
NR structural
and
composition
presence
chlr
tivity,
loci
in role
In
absence
or
iii)
coli.
stud
ponents
of
studied
(=A,
construction of
previous
five
in
the
in
In
this
are D and
NR has paper
present E)
in
an
been
membrane
inactive
and in that
of
de-
of
dif-
in
its
ac-
membrane
and
chlE
(14,15)
(16), -chlD our laboratory all
all
to
results:absence
chlD
chlA
reported
demonstrate
and
cytoplasmic
(13),
the
role
contradictory
of
we in
ii)
the
chlA
membranes
order
protein,
been
of
in
protein,
of
have
RESEARCH COMMUNICATIONS
important
NR structural
membrane
of
is
construction there
polypeptides
(12).
B,
of the
component
ied
AND
in
studies
NR protein
MATERIALS
NR
AND BIOPHYSICAL
mutants -chl r of NR structural
absence
the
structural
mutants
of
protein of
inactive
BIOCHEMICAL
while
an in
structural
all
com-
pleiotropic
mutants
form.
METHODS
The following strains were used: PA601 (356 in our co1 lection) --E. co1 i K-12 and its pleiotropic chlorate resistant mutants: chlA, chlB (4); chlD (unpublishand chl E (17). Cel Is of each strain were grown anaerobical lyt 32°C in 21 ed) of nutrient broth, buffered with Na-K phosphate 25mM, pH 7,2 containing 0.2 % glucose, 0.2% KN03 and O.lmM Na-azide. After 15h, the cells were harvested by centrifugation and washed 3 times with K-phosphate buffer 66mM pH 7.2 containing 5m~ MgS04, and disrupted by passage through a Yeda pressure cell with 1500psi. The crude extract was ultracentrifuged at 95,OOOxg for 75 min and the membrane fraction was pel leted. This fraction was suspended in Tris-HCI buffer O.lM pH 8,3 at 5-7.6 mg protein/ml. A part of this fraction was heated to 60”~ for 20 min (l), cooled rapidly, and left overnight at 4°C. The heated membrane fraction was recovered by another ultracentrifugation, aa above, Heated and unheated membrane fractions were dissociated with 1% SDS, 50mM B-mercaptoethanol and 5OmM EDTA at 20°C during lh, as in (12). Samples of O.lml of each strain and each treatment (except W-T-unheated for which 0.05ml were used), were applied on top of an SDS containig gel that had been prepared as in (12). The electrophoretic run was at 5mA/gel and lasted until the tracking dye (Brcmophenol blue) reached the bottom of the gel, NR activity was assayed in all gels by a procedure previously described (12), and then stained with 1% Amido black in 7% acetic acid. The colored gels were scanned at 600nm in a gel scanner. Purified NR was obtained by the method of Lund & DeMoss (18) sl ightly modified. This protein was treated with dissociating agents in the same manner as the membrane fractions. 51 ug of treated and untreated samples were applied on SDS gels, as above. RESULTS
a)
In
order
dissociates,
native
PAGE-SDS
system.
In
this
sociate over, (nr,),
of
in in
the
and
pretreated
corresponds
identify and
The system,
ta
0.26
which
the “r-1
are
band
it
is
shown
in
later
is
the
band
of
of
activity,
their which
only
it ions
previous
NR activities
the
used.
There and
87
is
NR protein
most
in
treatment
our
dis-
activity.
More-
migrate
important
activity is
run
1.
maintain
The
cond
purified NR were
Fig.
NR without
partially three
the
pretreated
NR and and
(nrl), and
under the
in
is
pretreated
there
NR activity, NR,
pattern
bands
case, and
components
SDS-mercaptoethanol-EDTA resulting
protein
later (nr2)
the
both,
several
0.13
content
to
at in
present
a protein be denominated
Rf protein
in band
0.08
the
which BmA.
gel
Bcl
Vol. 90, No. 1, 1979
0
033
BIOCHEMICAL
0.5
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0
10
0.25
0.50
0.75
1.0
I I “‘2 \ a
0
05
01
0
Fig.
1:
2:
Gel profiles for PAGE-SDS SDS-B-mercaptoethanol-EDTA. (60”~. 20 min.). membrane dicates NR actjvity.
important
, upon
The
that
except
The
other
b)
Fig.
1.0
NR and b, Methods).
the WT-membrane fraction pretreated a, unheated membrane fraction and fraction (see Materials and Methods),
is
Bc I , which
concentra
t ion. its
migrates This
protein
content
does
“‘1,
with b, heated nr in-
variably
band
SDS-
between not
increases
Rf
have
activity,
while
that
1). migrate
band
a, untreated Materials and
of
band
protein
075
Rf
treatment,
(Fig.
bands
9~2)
on
dissociating
BmA decreases
and
protein
depending
however
0.50
Gel profiles for PAGE-SDS of purified NR. B-mercaptoethanol-EDTA pretreated NR (see nr2 and nrj indicate NR activities.
Another 0.12-0.19,
0.25
02
Rf Fig.
of
1.0
0.33
faster
than
which
BmA are
appeared
NR activities
(nr2
and
only nr3)
probably in
the
subunits first
correspond
to
of
NR
(Bsl
run. bands
with
low
protein
contents.
ning
later will
the
a WT membrane
fraction
(Fig.
there
fraction for
2 shows
the
2a)
heat-treated case,
be
distribution in are
24
WT membrane
which
contains
denominated
BpP.
twice
of
our
PAGE-SDS
discrete
Band
much
88
bands
system. bands.
fraction. as
protein
protein
which I n the
Fig. 25
2b is
aa
results unheated
shows
evidently the
former.
from
run-
membrane
the
band lost
profile in
This
the band
Vol. 90, No. 1, 1979
Table
I:
WT
1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
17 18 19
20 21 22 23 24 25 26
h
0.01 ---
+
0.09 0.10 0.12 0.19
+ + + + + + + + + + + + + + + + + + + + +
in
the
corresponds we l),
it to
cl
Table
of
WT and
Results
Bsl
heated
genera
1,
is
quite
irregular, by
fractions
2.5 Bands
for 15
and
our
+
the
unheated
activity
uh-
fraction
and
corresponds
to
“t-1
h
+
+
+ + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + +
+ +
+ +
+
+
there and
is
no
therefore,
the
t ed
PAGE-SDS
,
Band
samples
there
for
17
are
present
also
12.4
(Fig.
2)
and
15 and
and
bands
in
membrane
in
all
such
in
Bcl
89
as
in
all to
17
membrane
6 of
mutant BmA,
except
in
Wf which
in
of
11.0 unheated
6)
membrane in how-
membranes, and
to
inactive
examined,
unheated
-chlD the gel
is
(1 clearly
unheated but
strains
for
fractions. bands
the and
fractions system.
migrating
heated
10.8
CB,
cases
all slow
PAGE-SDS
concentrations, in
for
same
for of
present
similar
present
quantity -chlA,
shown
bands
is
in
the
pattern
are
1 1 (BmA)
is
protein
Bcl
pattern in
are the
mutants
of
distribution (A,B,D,E.)
system,
however
7.6
fractions
tn
7 corresponds
band
unhea
WT,
WT membrane
respectively.
mutants
7(Bcl)
of
band
chlr -
pleiotropic
ratio the
and
+
patterns that
Bs2
heating.
Band
the
ever,
in
of
cases.
protein
evident and
h
+
+ +
of
indicate
chlE
uh-
+ + + + + + + + + + + + + + + + + + + + +
in
This
1 summarizes
In
all
is
11
-
BmA.
the
pleiotropic of
diminish
sample, to
compare
h
+ + + +
band
fractions
chlD
uh-
+ + + + + + + + + + + + + + +
in
heated
If
correspond
h
0.07 0.08 + + + + + + + + + + + + + + + + + +
migrates
11
(Fig.
RESEARCH COMMUNICATIONS
chlB
uh-
+
0.91
NR activity
NR
chlA
uh
0.20 0.22 0.25 0.28 0.31 0.34 0.36 0.40 0.44 0.47 0.52 0.56 0.62 0.65 0.68 0.73 0.76 0.81
activity
AND BIOPHYSICAL
Band distribution for heated (h) and unheated (uh) membrane WT, *A, EB, chlD and chlE. Numbers indicate Rf. + and presence and absence of anyband, respectively.
Band N”
band
BIOCHEMICAL
for
is chlE. membrane
Vol. 90, No. 1, 1979
fraction in
of
gels
Band
chl
of
22
heating
BIOCHEMICAL
B,
The
heated
most
and
has
a singular
the
samples,
AND BIOPHYSICAL
conspicuous
unheated
result
membrane
behaviour
in
is
the
fractions
most
RESEARCH COMMUNICATIONS
absence
of
mutants
all
of
band
25
pleiotropic
since
it
(BpP)
mutants,
appears
only
after
DISCUSSION Under In
the
the
the
conditions
presence
active
of
ied
. on
ferences
in
electrophoret
four
major
the
electrophoretic
WT and
the
supported fractions when
three
of
pleiotropic
think
that
Bcl
thought
of to
be
BpP must pleiotropic sary
subunits be
component system,
same This
NR structural
fractions, ferences
the
if
they
fall
results
to
the
in and
presence
of
conclusions D,
and
E is
the
membrane to
be
of
activity and/or
with
dif-
in
in
and
sized This
the
content third,
of
each
is
membrane of
that
(unpublished
component of
of
minimal
protein
NR, gel
evident
fractions
the
Bcl
increase
the
become
NR protein.
of
purified in
in
all
Bcl
results). NR,
NR.
with
the
but
it
Bsl
and
absent
in
cases,
as
if
may Bs2
be
are
the
the
major
of
al I pleiotropic
inactive
in
of
NR components we
conclude altered
90
in
in that
the and
each
loci,
our
have
The
two
dif-
ways: methods
and,
second,
(-21)” mutants,and
cytoplasmic the
pre-
membrane
different
used
that
to
(12). in
of
treat-
mutants
by
all
heat
mutant
explained
analysis
necesa
component
Contrary
all
selected
groups
is
SDS or
reported be
not
another
(20).
method
fundamentally
or
studies
were
al1
b which
upon
may they
complementation on
chain,
previously
results
therefore
cytochrome
NR complex
fractions is
is
NR,
the
previous
BmA
it
purified
transport
previous
depend
since
be
from
(8,9,13), not
of
activities
the
increase
could
be comparable
may
bands
which
complex
present BpP
membrane
different
obtained
previous B,
in
not
Although
not
previously
associations
Bs2)
thought
NR system
NR electron
the
these may
the
agrees
present
between mutants
From
chlA,
is
is
of
dissociates
protein.
It
in
characteristic
is
the
(13-161,
results
the
Bsl,
a physiological
of
interpretation
vious
BmA
assay
Thus
of
of
NR,
it
which
in
inactive
activity
activity.
the
or
but
resulting
present
second,
a constituent
MVH-NR
necessary
first,
of
of
are
dissociation
be
detection
been
three
a relative
not
up
had
to
BmA,
NR,
a physiological
mutants
for
ment,
may
This
the
activity
(19).
be an
the
its
permits
product
(Bcl,
mutants.
after
it
the
be
thus
migration
first,
during
may
NR,
mutants,
BmA decreases
representative
of
of
retains
NR exhibits bands
possibly
facts:
MVH
gel.
bands,
chlr
by
accumulates We
ic
may
SDS-containing
form
run
Bcl
since
purified
protein
experiments,NR
noteworthy
These
gel.
pleiotropic
NR activity.
an
a unique
The upon
in
these
is
Moreover,
such
of
for
This
band
(12,lg) appl
dissociations
Bcl
SDS.
protein
observed when
used
contrary
membrane pleiotropic
of chlr
Vol.
90,
loci of
No.
1, 1979
studied
may
a common
anything
play
do
mutants
could
in
in
the
is
structurally
which
functional
of
of
is
that
the
is
another that
pleiotropic
pleiotropic
NR respiratory
-chl
anaerobic r
(12)
protein
or
and
the
increase
type
of
structural
NR structural
of
the
FDH
in
loci
the
are
of
than
having
Bcl
in
the
alteration,
and
is in an
such present a form
physical This
chain.
involved
that
oligo-
without
transport
indirectly
membrane
rather
protein
WT (22)
electron
the
a respiratory
mutants (A,B,D,E,) -chlr to the WT enzyme, i.e.
different
with
NR and
BpP
differenttiomthat
association
mean
sembly
of
COMMUNICATIONS
incorporation
NR structural
We propose
functionally
RESEARCH
and
activates
of
there
membrane and
structure
would
disappearance
structure.
cytoplasmic
merit
the
BIOPHYSICAL
processing
that
synthesis
that
AND
the
the
indicate
quaternary
in
cofactor
with
However,
as
a role
Molybdenum
to
particle.
and
BIOCHEMICAL
in
the
as-
particle.
ACKNOWLEDGEMENTS We wish
to
thank
discussion
and
IJLA’
a Grant
, and
Drs.
criticisms. from
M.
Dubourdieu
This C.D.C.H.
work
and was
G.N.
Cohen
supported
by
for ‘Facultad
their
helpful de
Ciencias,
N”C-94-77.
REFERENCES
:: 4. 5. 6. ;: 9.
S. and E. ltagaki. (1959). Biochim. Biophys. Acta, 31, 294-295. Taniguchi, Iida, K. and S. Taniguchi. (1959). J. Biochem., 46, 1041-1055. Piichaud, M., J. Puig, F. Pichinoty, E. Azoulay and L. LeMinor. (1967). Ann. Inst. Pasteur, Paris, 112, 24-37. Puig, J. and E. Azoulay. (1967). C.R. Acad. Sci. Paris, 264, 1916-1918. Adhya, S., P. Cleary, and A. Campbell. (1968). Proc. Nat. Acad. Sci. 61,
956-962. Venables, Glaser, Azoulay, Acta, Azoulay,
389, 10. 11. 12.
13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
W.A. and J.R. Guest. (1968). Molec. Gen. Genet., 103, J.H. and J.A. DeMoss. (1972). Molec. Gen. Genet., 116, J. Puig and P. Couchoud-Beaumont. (1969). Biochem. E., 171, 238-252. J. Pommier and C. Riviere. (1975). Biochim. Biophys. E.,
127-149. l-10. Biophys. Acta,
236-250.
Glaser, J.H. and J.A. DeMoss. (1971). J. Bacterial., 108, 854-860. MacGregor, C.H. (1975). J. Bacterial., 121, 1117-1121. Dubourdieu, M., E. Andrade and J. Puig. (1976). Biochem. Biophys. Res. Commun., 70, 766-773. Azoulay, E., J. Puig and F. Pichinoty. (1967). Biochem. Biophys. Res. Commun . , 27, 270-274. MaGregor, C.H. and C.A. Schnaitman. (1971). J. Bacterial., 108, 564-570. Sperl, G.T. and J.A. DeMoss. (1975). J. Bacterial., 122, 1230-1238. Rolfe, B. and K. Onedera. (1972). J. Membrane Biol ., 9, 195-207. Puig, J. E. Azoulay, F. Pichinoty and J. Gendre. (1969). Biochem. Biophys. Res. Commun., 35, 659-662. Lund, K. and J.A. DeMoss. (1976). J. Biol. Chem., 251, 2207-2216. Ruiz-Herrera, J. (1978) in Mechanisms of Oxidizing Enzymes, T.P. Singer, R.N. Ondarza, eds., Elsevier North-Holland, Inc., New York, pp. 155-164. Schna i tman, C.A. (1969). Biochem. Biophys. Res. Commun., 37, l-5. Dassa, E., G. Frelat and P.-L. Boquet. (1,978). Biochem. Biophys. Res. Commun. 81, 616-622. Lewis, N.J. and C. Scazzochio. (1977). Eur. J. Biochem., 76, 441-446.
91
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
12, 1979
Pages 36-91
NITRATE
REDUCTASE CHLORATE
AND
RESISTANT H.C.
Grupo
Received
MEMBRANE
MUTANTS
Grassi,
de Biologia Universidad
July
THE
E.
OF
CDMPOSITION
OF
ESCHERICHIA
COLI
Andrade”
and
Experimental, de Los Andes,
J.
Facultad MBrida,
PLEIOTROPIC K-12
Puig de Ciencias, Venezuela
6,1979
SUMMARY The protein composition of the membrane fraction of pleiotropic chlorate resistant mutants of --E. co1 i K-12 was analyzed as a function of Nitrate Reductase and other components. The results for all pleiotropic chlorate resistant mutants show: the presence of the Nitrate Reductase structural protein, an increase in the relative protein content in an inactive complex of this enzyme, and the disappearance of a low molecular weight protein. The later protein is lost from the wild type membrane fraction by treatment at ~o”c, 20 min. It is concluded that Nitrate Reductase is present in the cytoplasmic membrane of all pleiotropic chlorate resistant mutants in a form structurally and functionally different from the wild respiratory enzyme coupled to the electron transport chain.
INTRODUCTION Nitrate
Reductase
enzyme
of
rating
energy
which
Escherichia
is
two
were
named
and
-chlC
(3). and
CA,
B,
troling FDH,
thesis”
most of
those controling
(5). -chlD effects.
chlE
Azoulay
hypothesis”
et al -& (lo,11
* To whom reprint requests it?: Abreviations: NR, Nitrate type strain; PAGE-SDS, Dodecy 1 Sulfate; MVH,
(6),
Another
NR activity.
which
and
-chlG
mutant,
Pleiotropy
of
which (see
have
8 and
been 9)
of
and
the
have
to
lost
(7)
“particle
shown has
These
belong
to
affect
NR
FDH
activity
of
mutants,
category were
been
(3).
which
also
gene-
chlorite
mutants
shown
first
chlF has been the
nitrite,
chlorate
were
(7)
respiratory
to
mutations have
the
as
loci
completely
explained
in integrity
conlost
several hypo-
“Molybdenum-cofactor
pro-
,12). must be sent. Reductase; FDH, Formate Dehydrogenase; WT, polyacrylamide gel electrophoresis containing reduced Methyl Viologen.
0006-291X/79/170086-06$01.00/0 Copyright All rights
presence and
to
chlorate
NR-less
for
bound
nitrate
reduces (3).
(chl’), specific
locus
a membrane
reduces
the
mutants, only
important
in
or
is
also
products
resistant
pleiotropic
the
partially
the
cesing
(4))
pleiotropic and
ways,
is
It It
conditions
pleiotropic
(4)
.);‘=k
(1,2).
toxic
chlorate
non
1 .9.6.1
growth. to
anaerobic
categories:
activity,
K-12
anaerobic reduced
under
E.C.
coli
for
further
obtained mutants
(NR,
@ 1979 by Academic Press, Inc. of reproduction in any form reserved.
86
wild Sodium
Vol.
No. 1, 1979
90,
The
membrane
termine:
i)
ferent
and
of
E.
of
NR structural
and
composition
presence
chlr
tivity,
loci
in role
In
absence
or
iii)
coli.
stud
ponents
of
studied
(=A,
construction of
previous
five
in
the
in
In
this
are D and
NR has paper
present E)
in
an
been
membrane
inactive
and in that
of
de-
of
dif-
in
its
ac-
membrane
and
chlE
(14,15)
(16), -chlD our laboratory all
all
to
results:absence
chlD
chlA
reported
demonstrate
and
cytoplasmic
(13),
the
role
contradictory
of
we in
ii)
the
chlA
membranes
order
protein,
been
of
in
protein,
of
have
RESEARCH COMMUNICATIONS
important
NR structural
membrane
of
is
construction there
polypeptides
(12).
B,
of the
component
ied
AND
in
studies
NR protein
MATERIALS
NR
AND BIOPHYSICAL
mutants -chl r of NR structural
absence
the
structural
mutants
of
protein of
inactive
BIOCHEMICAL
while
an in
structural
all
com-
pleiotropic
mutants
form.
METHODS
The following strains were used: PA601 (356 in our co1 lection) --E. co1 i K-12 and its pleiotropic chlorate resistant mutants: chlA, chlB (4); chlD (unpublishand chl E (17). Cel Is of each strain were grown anaerobical lyt 32°C in 21 ed) of nutrient broth, buffered with Na-K phosphate 25mM, pH 7,2 containing 0.2 % glucose, 0.2% KN03 and O.lmM Na-azide. After 15h, the cells were harvested by centrifugation and washed 3 times with K-phosphate buffer 66mM pH 7.2 containing 5m~ MgS04, and disrupted by passage through a Yeda pressure cell with 1500psi. The crude extract was ultracentrifuged at 95,OOOxg for 75 min and the membrane fraction was pel leted. This fraction was suspended in Tris-HCI buffer O.lM pH 8,3 at 5-7.6 mg protein/ml. A part of this fraction was heated to 60”~ for 20 min (l), cooled rapidly, and left overnight at 4°C. The heated membrane fraction was recovered by another ultracentrifugation, aa above, Heated and unheated membrane fractions were dissociated with 1% SDS, 50mM B-mercaptoethanol and 5OmM EDTA at 20°C during lh, as in (12). Samples of O.lml of each strain and each treatment (except W-T-unheated for which 0.05ml were used), were applied on top of an SDS containig gel that had been prepared as in (12). The electrophoretic run was at 5mA/gel and lasted until the tracking dye (Brcmophenol blue) reached the bottom of the gel, NR activity was assayed in all gels by a procedure previously described (12), and then stained with 1% Amido black in 7% acetic acid. The colored gels were scanned at 600nm in a gel scanner. Purified NR was obtained by the method of Lund & DeMoss (18) sl ightly modified. This protein was treated with dissociating agents in the same manner as the membrane fractions. 51 ug of treated and untreated samples were applied on SDS gels, as above. RESULTS
a)
In
order
dissociates,
native
PAGE-SDS
system.
In
this
sociate over, (nr,),
of
in in
the
and
pretreated
corresponds
identify and
The system,
ta
0.26
which
the “r-1
are
band
it
is
shown
in
later
is
the
band
of
of
activity,
their which
only
it ions
previous
NR activities
the
used.
There and
87
is
NR protein
most
in
treatment
our
dis-
activity.
More-
migrate
important
activity is
run
1.
maintain
The
cond
purified NR were
Fig.
NR without
partially three
the
pretreated
NR and and
(nrl), and
under the
in
is
pretreated
there
NR activity, NR,
pattern
bands
case, and
components
SDS-mercaptoethanol-EDTA resulting
protein
later (nr2)
the
both,
several
0.13
content
to
at in
present
a protein be denominated
Rf protein
in band
0.08
the
which BmA.
gel
Bcl
Vol. 90, No. 1, 1979
0
033
BIOCHEMICAL
0.5
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0
10
0.25
0.50
0.75
1.0
I I “‘2 \ a
0
05
01
0
Fig.
1:
2:
Gel profiles for PAGE-SDS SDS-B-mercaptoethanol-EDTA. (60”~. 20 min.). membrane dicates NR actjvity.
important
, upon
The
that
except
The
other
b)
Fig.
1.0
NR and b, Methods).
the WT-membrane fraction pretreated a, unheated membrane fraction and fraction (see Materials and Methods),
is
Bc I , which
concentra
t ion. its
migrates This
protein
content
does
“‘1,
with b, heated nr in-
variably
band
SDS-
between not
increases
Rf
have
activity,
while
that
1). migrate
band
a, untreated Materials and
of
band
protein
075
Rf
treatment,
(Fig.
bands
9~2)
on
dissociating
BmA decreases
and
protein
depending
however
0.50
Gel profiles for PAGE-SDS of purified NR. B-mercaptoethanol-EDTA pretreated NR (see nr2 and nrj indicate NR activities.
Another 0.12-0.19,
0.25
02
Rf Fig.
of
1.0
0.33
faster
than
which
BmA are
appeared
NR activities
(nr2
and
only nr3)
probably in
the
subunits first
correspond
to
of
NR
(Bsl
run. bands
with
low
protein
contents.
ning
later will
the
a WT membrane
fraction
(Fig.
there
fraction for
2 shows
the
2a)
heat-treated case,
be
distribution in are
24
WT membrane
which
contains
denominated
BpP.
twice
of
our
PAGE-SDS
discrete
Band
much
88
bands
system. bands.
fraction. as
protein
protein
which I n the
Fig. 25
2b is
aa
results unheated
shows
evidently the
former.
from
run-
membrane
the
band lost
profile in
This
the band
Vol. 90, No. 1, 1979
Table
I:
WT
1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
17 18 19
20 21 22 23 24 25 26
h
0.01 ---
+
0.09 0.10 0.12 0.19
+ + + + + + + + + + + + + + + + + + + + +
in
the
corresponds we l),
it to
cl
Table
of
WT and
Results
Bsl
heated
genera
1,
is
quite
irregular, by
fractions
2.5 Bands
for 15
and
our
+
the
unheated
activity
uh-
fraction
and
corresponds
to
“t-1
h
+
+
+ + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + +
+ +
+ +
+
+
there and
is
no
therefore,
the
t ed
PAGE-SDS
,
Band
samples
there
for
17
are
present
also
12.4
(Fig.
2)
and
15 and
and
bands
in
membrane
in
all
such
in
Bcl
89
as
in
all to
17
membrane
6 of
mutant BmA,
except
in
Wf which
in
of
11.0 unheated
6)
membrane in how-
membranes, and
to
inactive
examined,
unheated
-chlD the gel
is
(1 clearly
unheated but
strains
for
fractions. bands
the and
fractions system.
migrating
heated
10.8
CB,
cases
all slow
PAGE-SDS
concentrations, in
for
same
for of
present
similar
present
quantity -chlA,
shown
bands
is
in
the
pattern
are
1 1 (BmA)
is
protein
Bcl
pattern in
are the
mutants
of
distribution (A,B,D,E.)
system,
however
7.6
fractions
tn
7 corresponds
band
unhea
WT,
WT membrane
respectively.
mutants
7(Bcl)
of
band
chlr -
pleiotropic
ratio the
and
+
patterns that
Bs2
heating.
Band
the
ever,
in
of
cases.
protein
evident and
h
+
+ +
of
indicate
chlE
uh-
+ + + + + + + + + + + + + + + + + + + + +
in
This
1 summarizes
In
all
is
11
-
BmA.
the
pleiotropic of
diminish
sample, to
compare
h
+ + + +
band
fractions
chlD
uh-
+ + + + + + + + + + + + + + +
in
heated
If
correspond
h
0.07 0.08 + + + + + + + + + + + + + + + + + +
migrates
11
(Fig.
RESEARCH COMMUNICATIONS
chlB
uh-
+
0.91
NR activity
NR
chlA
uh
0.20 0.22 0.25 0.28 0.31 0.34 0.36 0.40 0.44 0.47 0.52 0.56 0.62 0.65 0.68 0.73 0.76 0.81
activity
AND BIOPHYSICAL
Band distribution for heated (h) and unheated (uh) membrane WT, *A, EB, chlD and chlE. Numbers indicate Rf. + and presence and absence of anyband, respectively.
Band N”
band
BIOCHEMICAL
for
is chlE. membrane
Vol. 90, No. 1, 1979
fraction in
of
gels
Band
chl
of
22
heating
BIOCHEMICAL
B,
The
heated
most
and
has
a singular
the
samples,
AND BIOPHYSICAL
conspicuous
unheated
result
membrane
behaviour
in
is
the
fractions
most
RESEARCH COMMUNICATIONS
absence
of
mutants
all
of
band
25
pleiotropic
since
it
(BpP)
mutants,
appears
only
after
DISCUSSION Under In
the
the
the
conditions
presence
active
of
ied
. on
ferences
in
electrophoret
four
major
the
electrophoretic
WT and
the
supported fractions when
three
of
pleiotropic
think
that
Bcl
thought
of to
be
BpP must pleiotropic sary
subunits be
component system,
same This
NR structural
fractions, ferences
the
if
they
fall
results
to
the
in and
presence
of
conclusions D,
and
E is
the
membrane to
be
of
activity and/or
with
dif-
in
in
and
sized This
the
content third,
of
each
is
membrane of
that
(unpublished
component of
of
minimal
protein
NR, gel
evident
fractions
the
Bcl
increase
the
become
NR protein.
of
purified in
in
all
Bcl
results). NR,
NR.
with
the
but
it
Bsl
and
absent
in
cases,
as
if
may Bs2
be
are
the
the
major
of
al I pleiotropic
inactive
in
of
NR components we
conclude altered
90
in
in that
the and
each
loci,
our
have
The
two
dif-
ways: methods
and,
second,
(-21)” mutants,and
cytoplasmic the
pre-
membrane
different
used
that
to
(12). in
of
treat-
mutants
by
all
heat
mutant
explained
analysis
necesa
component
Contrary
all
selected
groups
is
SDS or
reported be
not
another
(20).
method
fundamentally
or
studies
were
al1
b which
upon
may they
complementation on
chain,
previously
results
therefore
cytochrome
NR complex
fractions is
is
NR,
the
previous
BmA
it
purified
transport
previous
depend
since
be
from
(8,9,13), not
of
activities
the
increase
could
be comparable
may
bands
which
complex
present BpP
membrane
different
obtained
previous B,
in
not
Although
not
previously
associations
Bs2)
thought
NR system
NR electron
the
these may
the
agrees
present
between mutants
From
chlA,
is
is
of
dissociates
protein.
It
in
characteristic
is
the
(13-161,
results
the
Bsl,
a physiological
of
interpretation
vious
BmA
assay
Thus
of
of
NR,
it
which
in
inactive
activity
activity.
the
or
but
resulting
present
second,
a constituent
MVH-NR
necessary
first,
of
of
are
dissociation
be
detection
been
three
a relative
not
up
had
to
BmA,
NR,
a physiological
mutants
for
ment,
may
This
the
activity
(19).
be an
the
its
permits
product
(Bcl,
mutants.
after
it
the
be
thus
migration
first,
during
may
NR,
mutants,
BmA decreases
representative
of
of
retains
NR exhibits bands
possibly
facts:
MVH
gel.
bands,
chlr
by
accumulates We
ic
may
SDS-containing
form
run
Bcl
since
purified
protein
experiments,NR
noteworthy
These
gel.
pleiotropic
NR activity.
an
a unique
The upon
in
these
is
Moreover,
such
of
for
This
band
(12,lg) appl
dissociations
Bcl
SDS.
protein
observed when
used
contrary
membrane pleiotropic
of chlr
Vol.
90,
loci of
No.
1, 1979
studied
may
a common
anything
play
do
mutants
could
in
in
the
is
structurally
which
functional
of
of
is
that
the
is
another that
pleiotropic
pleiotropic
NR respiratory
-chl
anaerobic r
(12)
protein
or
and
the
increase
type
of
structural
NR structural
of
the
FDH
in
loci
the
are
of
than
having
Bcl
in
the
alteration,
and
is in an
such present a form
physical This
chain.
involved
that
oligo-
without
transport
indirectly
membrane
rather
protein
WT (22)
electron
the
a respiratory
mutants (A,B,D,E,) -chlr to the WT enzyme, i.e.
different
with
NR and
BpP
differenttiomthat
association
mean
sembly
of
COMMUNICATIONS
incorporation
NR structural
We propose
functionally
RESEARCH
and
activates
of
there
membrane and
structure
would
disappearance
structure.
cytoplasmic
merit
the
BIOPHYSICAL
processing
that
synthesis
that
AND
the
the
indicate
quaternary
in
cofactor
with
However,
as
a role
Molybdenum
to
particle.
and
BIOCHEMICAL
in
the
as-
particle.
ACKNOWLEDGEMENTS We wish
to
thank
discussion
and
IJLA’
a Grant
, and
Drs.
criticisms. from
M.
Dubourdieu
This C.D.C.H.
work
and was
G.N.
Cohen
supported
by
for ‘Facultad
their
helpful de
Ciencias,
N”C-94-77.
REFERENCES
:: 4. 5. 6. ;: 9.
S. and E. ltagaki. (1959). Biochim. Biophys. Acta, 31, 294-295. Taniguchi, Iida, K. and S. Taniguchi. (1959). J. Biochem., 46, 1041-1055. Piichaud, M., J. Puig, F. Pichinoty, E. Azoulay and L. LeMinor. (1967). Ann. Inst. Pasteur, Paris, 112, 24-37. Puig, J. and E. Azoulay. (1967). C.R. Acad. Sci. Paris, 264, 1916-1918. Adhya, S., P. Cleary, and A. Campbell. (1968). Proc. Nat. Acad. Sci. 61,
956-962. Venables, Glaser, Azoulay, Acta, Azoulay,
389, 10. 11. 12.
13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
W.A. and J.R. Guest. (1968). Molec. Gen. Genet., 103, J.H. and J.A. DeMoss. (1972). Molec. Gen. Genet., 116, J. Puig and P. Couchoud-Beaumont. (1969). Biochem. E., 171, 238-252. J. Pommier and C. Riviere. (1975). Biochim. Biophys. E.,
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236-250.
Glaser, J.H. and J.A. DeMoss. (1971). J. Bacterial., 108, 854-860. MacGregor, C.H. (1975). J. Bacterial., 121, 1117-1121. Dubourdieu, M., E. Andrade and J. Puig. (1976). Biochem. Biophys. Res. Commun., 70, 766-773. Azoulay, E., J. Puig and F. Pichinoty. (1967). Biochem. Biophys. Res. Commun . , 27, 270-274. MaGregor, C.H. and C.A. Schnaitman. (1971). J. Bacterial., 108, 564-570. Sperl, G.T. and J.A. DeMoss. (1975). J. Bacterial., 122, 1230-1238. Rolfe, B. and K. Onedera. (1972). J. Membrane Biol ., 9, 195-207. Puig, J. E. Azoulay, F. Pichinoty and J. Gendre. (1969). Biochem. Biophys. Res. Commun., 35, 659-662. Lund, K. and J.A. DeMoss. (1976). J. Biol. Chem., 251, 2207-2216. Ruiz-Herrera, J. (1978) in Mechanisms of Oxidizing Enzymes, T.P. Singer, R.N. Ondarza, eds., Elsevier North-Holland, Inc., New York, pp. 155-164. Schna i tman, C.A. (1969). Biochem. Biophys. Res. Commun., 37, l-5. Dassa, E., G. Frelat and P.-L. Boquet. (1,978). Biochem. Biophys. Res. Commun. 81, 616-622. Lewis, N.J. and C. Scazzochio. (1977). Eur. J. Biochem., 76, 441-446.
91
