Vol. 67, No. 3, 1975
BIOCHEMICAL
EVIDENCE
FOR
THE
ACTIVITY
TRIOSE David
AND BIOPHYSICAL
A. Fell
OF
RESEARCH COMMUNICATIONS
IMMOBILISED
PHOSPHATE
MONOMERS OF
ISOMERASE
and Christopher
J. Branford
White
Department of Biology, Oxford Polytechnic, Headington, Oxford. OX3 OBP, U.K.
Received
September
28,1975
SUMMARY: Chicken muscle triose phosphate isomerase was immobilised by attachment to Sepharose 4B. The immobilised dimeric enzyme was dissociated with guanidinium chloride to yield bound monomeric triose phosphate isomerase. This regained activity on removal of the denaturant, showing that isolated monomers possess activity; the apparent Km of the immobilised subunits was the same as that of the immobilised dimers. Under appropriate conditions, it was possible to rehybridise the immobilised monomers to native dimers, and also to form a hybrid dimer from the chicken muscle and rabbit muscle enzymes. INTRODUCTION: structure
In the
association attach
the
under
conditions
describe
the
isomerase
for
where
are
activity. support
the properties
via
of the
reassociation
application
(EC 5.3.1.1)
cyanogen
method
is
of interest
active,
One approach
to this
a single
subunit,
isolated
subunit
possible
to chicken
we attached
it
enzymically
is not
of this which
activity,
the quaternary
or whether problem
to
and to remove may be studied
(1,2,3). muscle
to Sepharose
is
Here we triose
phosphate
4B activated
by
bromide.
Triose
phosphate
isomerase
are dissociated
greater
than
0.7M chains
guanidinium
concentration
phosphate
is
composed
by guanidinium
chloride
(4,5).
polypeptide
triose
subunits
the enzyme to a solid so that
between
enzyme and its
isolated
is necessary
others
which
of the relationship
of a multi-subunit
to know whether
the
study
McVittie
had also
isomerase
et al
unfolded is
(5)
in these
lowered
regains
of two identical
by dilution its
activity
solutions showed
subunits
at concentrations that
the dissociated
conditions.
If
or dialysis, (4,6)
although
the denatured the percentage
Vol. 67, No. 3, 1975
recovery
BIOCHEMICAL
of activity
concentrations
where
unpublished
it
state
main pathway
is
site
polypeptide MATERIALS prepared
of recovery
involves
on each subunit from
is
the other
AND METHODS: by extracting
Chicken
in part
muscle
triose
chicken
out according
activity
Boehringer
isomerase
was measured
ions
the enzyme.
U.mg-l.
All
of both
soluble
against
in
other
that
of
with
was
IOmM Tris-HCl,
(5).
steps
were
The preparation pH 8.5,
enzymes
were
gave
and had a
purchased
and immobilised
(EC 1.1.1.8),
50% glycerol/50% to enhance
of Porath
A number concentrations
and 0.1 M phosphate, excess
activity
et al
its
triose
described
the
from
in
secondary
phosphate (5).
enzyme in the
0.1 M triethanolamine, stability
(9) was followed
of samples
washed
of Sepharose
of cyanogen pH 8.0,
with
bromide,
and stirred
(up to 5mg) of soluble
enzyme was then further
suggested
pH 7.4,
and to remove
contaminating
(8).
The method
large
is
The remaining
at 25O by the assay method
1% 2-mercaptoethanol
different
and although
isomerase
in tris-glycine,
dehydrogenase
was dialysed
sulphate
muscle
by a length
muscle
described
electrophoresis
activity
a-glycerophosphate assay,
it
of
(London).
The enzymic
plus
to the method
of 7,200
(7),
phosphate
breast
carried
specific
the
subunit.
macerated
in gel
that
of chicken
bounded
the
association
yet been mapped,
in 0.5mM EDTA and 0.2% Z-mercaptoethanol.
band
from
suggesting
published
pH 7.0,
a single
of activity
structure
has not
and White,
a rate-limiting
has been recently
complex
chain
(4 and Fell
process‘(4,6),
state
isomerase
at low protein
75%
The crystallographic
phosphate
active
exceeds
order
to the native
the enzyme-substrate the
particularly
The rate
a second
subunits.
triose
rarely
observations).
denatured
inactive
is variable,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
for 4B were were
The gel
0.1 M triethanolamine-HCl,
1014
immobilisation
activated
washed
overnight
enzyme.
was eluted.
the
with
with 0.5M
at 4OC together bearing pH 7.4,
of
the
NaHC03 with
a
immobilised
until
no
BIOCHEMICAL
Vol. 67, No. 3, 1975
The denaturation carried
out
minutes
of immobilised
at room temperature
guanidinium
chloride
(Sigma);
these
conditions
under
accomplished
by washing
triethanolamine-HCl, Rehybridisation isomerase
formed
of the bound
a packed
to Sepharose,
the amount activity
bromide
ml of packed
gel;
the column
monomeric
2 M
less
than
triose
with
5
0.1 M
phosphate
out by passing
a linear
in 0.1 M triethanolamine-HCl,
containing
20 Units
and washing
with
In the preparation
we found
that
bromide
the product
of cyanogen
with
of enzyme per ml
renaturing
buffer
until
was eluted.
of cyanogen of
chloride
column,
RESULTS AND DISCUSSION: bound
from
carried
in
was
of the enzyme was
studies
IOOmM 2-mercatoethanol)
activity
Renaturation
were
of gel
complete
IOOmM 2-mercaptoethanol.
pH 7.4,
no further
is
of the gel
by denaturation
isomerase
a column
denaturation (6).
RESEARCH COMMUNICATIONS
phosphate
pH 7.4,
(2 - 0 M guanidinium
through
triose
by eluting
a sample
gradient
of gel
AND BIOPHYSICAL
used
there
of triose
was a linear
used to activate
within
in these
at the upper
experiments end of this
gel
between
and the enzymic
(Fig.
between range,
isomerase
relationship
the
any one experiment
phosphate
1). 0.5
The amounts and 5 mg per
Chan and Mawer
(I)
m g cyanogen bromfde per ml.gel
Figure
1 - Immobilised triose phosphate degrees of cyanogen bromide
1015
isomerase activation.
activity
at varying
Vol. 67, No. 3, 1975
found
evidence
however,
it
BIOCHEMICAL
for
is
some degree
clear
immobilised
per unit
of cyanogen
bromide
coupling (of
phosphate protein estimated reported
their
weight
results
in
methods,
from
dehydrogenase
further,
of protein
(below
probably
of the order
activity
they
attachment
the
they amount
of their immobilised of triose
the sensitivity
of
of 0.1 - 10 ug.ml-',
of the enzyme).
of multi-site
as the amount
efficiency which
to the gel
but
although
the greater
we coupled
being
of activity
was decreasing
than
the specific
no evidence
bromide
the amount
of aldolase;
the amount
much greater
of 100 ng.ml-'1 which
attachment
that
of cyanogen
resulted
isomerase assay
of multi-point
was increased;
conditions
the order
from
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Nagradova
et al
(3)
of glyceraldehyde-3-phosphate
used 20 mg of cyanogen
bromide
per ml of
Sepharose. The kinetic immobilisation
properties on Sepharose;
glyceraldehyde-3-phosphate, soluble
enzyme,
of triose
phosphate
the apparent measured
was 1.7mM, which
is
isomerase
are
Km of innnobilised
at the optimal significantly
by
enzyme for
conditions higher
altered
than
for
the
our value
oov
0 06 -
Figure
2 - Lineweaver-Burk plot showing the dependence of immobilised triose phosphate isomerase activity on concentrations of glyceraldehyde-3-phosphate. A - Native enzyme B - Denaturedlrenatured
enzyme.
1016
BIOCHEMICAL
Vol. 67, No. 3, 1975
of 0.35mM for
the Km of the
of immobilised
2 M guanidinium
chloride,
at the solvent underwent
degree
interface;
the
total
although
with
similar
monomers, to that
of the
gradient
of the gel
phosphate
isomerase.
After
was free
of enzymic
activity,
bound
monomers
dimers.
In eight
(+ 0.25)
times
samples
from
was run
without
the
with
soluble
renaturing
under
renatured
in the absence
activities
In two experiments, rabbit
muscle
triose
until
the eluate
buffer
experiment
whether
That is
enzyme monomers
and dimers
are
the denatured
gel
isomerase;
1017
the gradient
the gradient washing
these
to twice suggests
method
of denatured
guanidinium
the activity
close
enzyme was
by the renatured
that
the
the
to form bound
in which
showed
and that
conditions
phosphate
triose
in solution
that
of free
of soluble
to detect
This
rehybridising
of the monomers
with
as the direct
conditions.
of
a decreasing
recovered
rehybridising
renatured
with
activity
of subunits,
variable,
2).
the
confirms
cases,
that
of the rehybridised
the enzyme gel
suitable
(Fig.
same activity
buffer.
the column
was again
activity
enzyme present
the
in all
the
A control
possible
glyceraldehyde-3-phosphate,
was assayed monomers
from
represents
was washed
the column
with
than
regained
in the presence
the gel
same column.
gave the
has depleted under
higher
the
of renaturation gel
washing
it
because
removed
which
column
the eluate
was not
of activity
dimers
chloride
experiments,
recovery
native
had rehybridised
the gel
activity
in the
of guanidinium
from
Km of 2mM for
immobilised
in
it
of gel
activity,
has an apparent
The remainder linear
showed
was eluted
However,
eluted Samples
The renatured
isomerase
enzyme was denatured,
cuvette.
of activity
10).
was detected
eluted
of the original
20% and 60%.
the bound
the
buffer
0.47mM,
activity
was unknown.
renaturing
(cf.
RESEARCH COMMUNICATIONS
phosphate
enzymic
amount
the percentage
between
triose
in the assay
of renaturation
and washed
enzyme
although
renaturation
to estimate
1.8
soluble
When a column with
AND BIOPHYSICAL
chloride
can be replaced of the gel that that
of the gel the
specific
similar. was rehybridised in both
cases,
with
soluble
an increase
in
Vol. 67, No. 3, 1975
activity
BIOCHEMICAL
compared
with
renatured
gel
was observed.
We conclude
that
of the chicken
and rabbit
muscle
32 differences
in amino
not
significantly
altered
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
acid the
it
enzyme
(2.8
is possible enzymes,
which
sequence
between
subunit
interface
and 1.5 times to form
active
demonstrates these
higher) hybrids that
two enzymes
the (11)
has
region.
ACKNOWLEDGEMENTS: We would like to thank Drs. J.D. McVittie and M.P. Esnouf for helpful discussions in the early stages of this work. REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8. 9. IO. 11.
Chan, W. W-C and Mawer, H.M., (1972) Arch. Biochem. Biophys., 2, 136-145. Chan, W. W-C, Schutt, H. and Brand, K., (1973) Eur. J. Biochem., $, 533-541. T.O. and Mevkh, A.T. (1974) FEBS Letters, Nagradova, N.K., Golovina, 2, 242-245. Waley, S.G. (1973) Biochem. J. 135, 165-172. McVittie, J.D., Esnouf, M.P. and Peacocke, A.R. (1972) Eur. J. Biochem. 29, 67-73. Mczttie, J.D. (1973) D.Phil. thesis, Oxford. Banner, D.W., Bloomer, A.C., Petsko, G.A., Phillips, D.C., Pogson, C.I. and Wilson, I.A. (1975) Nature, 255, 609-614. Turner, D.H., Blanch, E.S., Gibbs, M. and Turner, J.F. (1965) Plant Physiol., 40, 1146-1151. H. and Axen, R. (1973) J. Chromatog., Porath, J., Aspberg, K., Drevin, 86, 53-56. Coulson, A.F.W., Farley, I.R.T., Riddleston, B. and Putman, S.J., Knowles, J.R. (1972) Biochem. J. 129, 301-310. Corran, P.H. and Waley, S.G. (1975)Biochem. J. 145, 335-344.
1018
BIOCHEMICAL
EVIDENCE
FOR
THE
ACTIVITY
TRIOSE David
AND BIOPHYSICAL
A. Fell
OF
RESEARCH COMMUNICATIONS
IMMOBILISED
PHOSPHATE
MONOMERS OF
ISOMERASE
and Christopher
J. Branford
White
Department of Biology, Oxford Polytechnic, Headington, Oxford. OX3 OBP, U.K.
Received
September
28,1975
SUMMARY: Chicken muscle triose phosphate isomerase was immobilised by attachment to Sepharose 4B. The immobilised dimeric enzyme was dissociated with guanidinium chloride to yield bound monomeric triose phosphate isomerase. This regained activity on removal of the denaturant, showing that isolated monomers possess activity; the apparent Km of the immobilised subunits was the same as that of the immobilised dimers. Under appropriate conditions, it was possible to rehybridise the immobilised monomers to native dimers, and also to form a hybrid dimer from the chicken muscle and rabbit muscle enzymes. INTRODUCTION: structure
In the
association attach
the
under
conditions
describe
the
isomerase
for
where
are
activity. support
the properties
via
of the
reassociation
application
(EC 5.3.1.1)
cyanogen
method
is
of interest
active,
One approach
to this
a single
subunit,
isolated
subunit
possible
to chicken
we attached
it
enzymically
is not
of this which
activity,
the quaternary
or whether problem
to
and to remove may be studied
(1,2,3). muscle
to Sepharose
is
Here we triose
phosphate
4B activated
by
bromide.
Triose
phosphate
isomerase
are dissociated
greater
than
0.7M chains
guanidinium
concentration
phosphate
is
composed
by guanidinium
chloride
(4,5).
polypeptide
triose
subunits
the enzyme to a solid so that
between
enzyme and its
isolated
is necessary
others
which
of the relationship
of a multi-subunit
to know whether
the
study
McVittie
had also
isomerase
et al
unfolded is
(5)
in these
lowered
regains
of two identical
by dilution its
activity
solutions showed
subunits
at concentrations that
the dissociated
conditions.
If
or dialysis, (4,6)
although
the denatured the percentage
Vol. 67, No. 3, 1975
recovery
BIOCHEMICAL
of activity
concentrations
where
unpublished
it
state
main pathway
is
site
polypeptide MATERIALS prepared
of recovery
involves
on each subunit from
is
the other
AND METHODS: by extracting
Chicken
in part
muscle
triose
chicken
out according
activity
Boehringer
isomerase
was measured
ions
the enzyme.
U.mg-l.
All
of both
soluble
against
in
other
that
of
with
was
IOmM Tris-HCl,
(5).
steps
were
The preparation pH 8.5,
enzymes
were
gave
and had a
purchased
and immobilised
(EC 1.1.1.8),
50% glycerol/50% to enhance
of Porath
A number concentrations
and 0.1 M phosphate, excess
activity
et al
its
triose
described
the
from
in
secondary
phosphate (5).
enzyme in the
0.1 M triethanolamine, stability
(9) was followed
of samples
washed
of Sepharose
of cyanogen pH 8.0,
with
bromide,
and stirred
(up to 5mg) of soluble
enzyme was then further
suggested
pH 7.4,
and to remove
contaminating
(8).
The method
large
is
The remaining
at 25O by the assay method
1% 2-mercaptoethanol
different
and although
isomerase
in tris-glycine,
dehydrogenase
was dialysed
sulphate
muscle
by a length
muscle
described
electrophoresis
activity
a-glycerophosphate assay,
it
of
(London).
The enzymic
plus
to the method
of 7,200
(7),
phosphate
breast
carried
specific
the
subunit.
macerated
in gel
that
of chicken
bounded
the
association
yet been mapped,
in 0.5mM EDTA and 0.2% Z-mercaptoethanol.
band
from
suggesting
published
pH 7.0,
a single
of activity
structure
has not
and White,
a rate-limiting
has been recently
complex
chain
(4 and Fell
process‘(4,6),
state
isomerase
at low protein
75%
The crystallographic
phosphate
active
exceeds
order
to the native
the enzyme-substrate the
particularly
The rate
a second
subunits.
triose
rarely
observations).
denatured
inactive
is variable,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
for 4B were were
The gel
0.1 M triethanolamine-HCl,
1014
immobilisation
activated
washed
overnight
enzyme.
was eluted.
the
with
with 0.5M
at 4OC together bearing pH 7.4,
of
the
NaHC03 with
a
immobilised
until
no
BIOCHEMICAL
Vol. 67, No. 3, 1975
The denaturation carried
out
minutes
of immobilised
at room temperature
guanidinium
chloride
(Sigma);
these
conditions
under
accomplished
by washing
triethanolamine-HCl, Rehybridisation isomerase
formed
of the bound
a packed
to Sepharose,
the amount activity
bromide
ml of packed
gel;
the column
monomeric
2 M
less
than
triose
with
5
0.1 M
phosphate
out by passing
a linear
in 0.1 M triethanolamine-HCl,
containing
20 Units
and washing
with
In the preparation
we found
that
bromide
the product
of cyanogen
with
of enzyme per ml
renaturing
buffer
until
was eluted.
of cyanogen of
chloride
column,
RESULTS AND DISCUSSION: bound
from
carried
in
was
of the enzyme was
studies
IOOmM 2-mercatoethanol)
activity
Renaturation
were
of gel
complete
IOOmM 2-mercaptoethanol.
pH 7.4,
no further
is
of the gel
by denaturation
isomerase
a column
denaturation (6).
RESEARCH COMMUNICATIONS
phosphate
pH 7.4,
(2 - 0 M guanidinium
through
triose
by eluting
a sample
gradient
of gel
AND BIOPHYSICAL
used
there
of triose
was a linear
used to activate
within
in these
at the upper
experiments end of this
gel
between
and the enzymic
(Fig.
between range,
isomerase
relationship
the
any one experiment
phosphate
1). 0.5
The amounts and 5 mg per
Chan and Mawer
(I)
m g cyanogen bromfde per ml.gel
Figure
1 - Immobilised triose phosphate degrees of cyanogen bromide
1015
isomerase activation.
activity
at varying
Vol. 67, No. 3, 1975
found
evidence
however,
it
BIOCHEMICAL
for
is
some degree
clear
immobilised
per unit
of cyanogen
bromide
coupling (of
phosphate protein estimated reported
their
weight
results
in
methods,
from
dehydrogenase
further,
of protein
(below
probably
of the order
activity
they
attachment
the
they amount
of their immobilised of triose
the sensitivity
of
of 0.1 - 10 ug.ml-',
of the enzyme).
of multi-site
as the amount
efficiency which
to the gel
but
although
the greater
we coupled
being
of activity
was decreasing
than
the specific
no evidence
bromide
the amount
of aldolase;
the amount
much greater
of 100 ng.ml-'1 which
attachment
that
of cyanogen
resulted
isomerase assay
of multi-point
was increased;
conditions
the order
from
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Nagradova
et al
(3)
of glyceraldehyde-3-phosphate
used 20 mg of cyanogen
bromide
per ml of
Sepharose. The kinetic immobilisation
properties on Sepharose;
glyceraldehyde-3-phosphate, soluble
enzyme,
of triose
phosphate
the apparent measured
was 1.7mM, which
is
isomerase
are
Km of innnobilised
at the optimal significantly
by
enzyme for
conditions higher
altered
than
for
the
our value
oov
0 06 -
Figure
2 - Lineweaver-Burk plot showing the dependence of immobilised triose phosphate isomerase activity on concentrations of glyceraldehyde-3-phosphate. A - Native enzyme B - Denaturedlrenatured
enzyme.
1016
BIOCHEMICAL
Vol. 67, No. 3, 1975
of 0.35mM for
the Km of the
of immobilised
2 M guanidinium
chloride,
at the solvent underwent
degree
interface;
the
total
although
with
similar
monomers, to that
of the
gradient
of the gel
phosphate
isomerase.
After
was free
of enzymic
activity,
bound
monomers
dimers.
In eight
(+ 0.25)
times
samples
from
was run
without
the
with
soluble
renaturing
under
renatured
in the absence
activities
In two experiments, rabbit
muscle
triose
until
the eluate
buffer
experiment
whether
That is
enzyme monomers
and dimers
are
the denatured
gel
isomerase;
1017
the gradient
the gradient washing
these
to twice suggests
method
of denatured
guanidinium
the activity
close
enzyme was
by the renatured
that
the
the
to form bound
in which
showed
and that
conditions
phosphate
triose
in solution
that
of free
of soluble
to detect
This
rehybridising
of the monomers
with
as the direct
conditions.
of
a decreasing
recovered
rehybridising
renatured
with
activity
of subunits,
variable,
2).
the
confirms
cases,
that
of the rehybridised
the enzyme gel
suitable
(Fig.
same activity
buffer.
the column
was again
activity
enzyme present
the
in all
the
A control
possible
glyceraldehyde-3-phosphate,
was assayed monomers
from
represents
was washed
the column
with
than
regained
in the presence
the gel
same column.
gave the
has depleted under
higher
the
of renaturation gel
washing
it
because
removed
which
column
the eluate
was not
of activity
dimers
chloride
experiments,
recovery
native
had rehybridised
the gel
activity
in the
of guanidinium
from
Km of 2mM for
immobilised
in
it
of gel
activity,
has an apparent
The remainder linear
showed
was eluted
However,
eluted Samples
The renatured
isomerase
enzyme was denatured,
cuvette.
of activity
10).
was detected
eluted
of the original
20% and 60%.
the bound
the
buffer
0.47mM,
activity
was unknown.
renaturing
(cf.
RESEARCH COMMUNICATIONS
phosphate
enzymic
amount
the percentage
between
triose
in the assay
of renaturation
and washed
enzyme
although
renaturation
to estimate
1.8
soluble
When a column with
AND BIOPHYSICAL
chloride
can be replaced of the gel that that
of the gel the
specific
similar. was rehybridised in both
cases,
with
soluble
an increase
in
Vol. 67, No. 3, 1975
activity
BIOCHEMICAL
compared
with
renatured
gel
was observed.
We conclude
that
of the chicken
and rabbit
muscle
32 differences
in amino
not
significantly
altered
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
acid the
it
enzyme
(2.8
is possible enzymes,
which
sequence
between
subunit
interface
and 1.5 times to form
active
demonstrates these
higher) hybrids that
two enzymes
the (11)
has
region.
ACKNOWLEDGEMENTS: We would like to thank Drs. J.D. McVittie and M.P. Esnouf for helpful discussions in the early stages of this work. REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8. 9. IO. 11.
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