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A conformational change in concanavalin A detected by a calorimetric study of the binding of methyl α-D-mannopyranoside
Vol. 84, No. 3, 1978 October
BIOCHEMICAL
AND 8ldPHYSlCAL
RESEARCH COMMUNICATIONS
16,1978
Pages
684-690
A CONFOFMATIONAL CHANGE IN CONCANAVALIN A DETECTED BY A CALORIMETRIC
STUDY OF THE BINDING OF
METHYL a-D-MANNOPYRANOSIDE
G. R. Munske,
Program
Received
August
22,
J. A. Magnuson
and H. Krakauer
in Biochemistry and Biophysics Washington State University Pullman, WA 99164
1978 SUMMARY
The binding of methyl a-D-mannopyranoside to the lectin concanavalin A was studied by means of calorimetry. An apparent enthalpy of binding was also calculated from the variation of the equilibrium constant with temperature (van't Hoff AH). The AH measured directly was-30 to -38 kJ/mole indicating that the binding is driven by the AH change. In contrast, the van't Hoff AH was substantially smaller, about zero at pH 5.2. The difference in the AH measured directly and the van't Hoff AH implies that the conformation of concanavalin A undergoes a temperature dependent change at both pH's but most predominantly at pH 5.2. The existence of this conformational change was verified by difference absorption spectroscopy. Concanavalin from the
jack
A (Con A) is
bean.
It
a-D-mannopyranoside, property
is
to induce
the
of Con A is
(7.2),
ability
high
temperature
predominates
tertiary
structure
as detected
sites
to which
specifically
(1,2).
class
(3).
by measurement
In addition,
of
salt
most notably
Associated and other
the circular
684
is
this
cell
types
The quarternary
favored,
concentration
ion
while
undergoes
structure
at high
pH
M) the changes
binding
We report
and
At low
(1.0
and sugar
dichroism.
form
with
concentration.
the protein
of metal
000629ur/78/0843-0684$oi.o0/0
Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
sugars,
and salt a dimer
and high
as a consequence
in pure
of lymphocytes.
concentration (37°C)
obtainable
erythrocytes
on pH, temperature
and salt
tetramar
readily
to agglutinate
in a certain
dependent
pH, temperature
possesses
can bind
mitosis
a lectin
(4,5), herein
in
Vol. 84, No. 3, 1978
a conformational temperature
BIOCHEMICAL
change and perhaps
binding
of the sugar,
course
of measurements
AND BIdPHYSICAL
not
previously
observed
also
on salt
concentration,
methyl
which
a-D-mannopyranoside
of the heat
RESEARCH COMMUNICATIONS
is
and which It
(aMM).
of binding
dependent
of that
on
perturbs
the
was detected
in the
sugar.
MATERIALS AND METHODS Con A was prepared from jack beans (Sigma) by the method of Agrawal and Goldstein (6) and gave a single band on electrophoresis on 5% polyacrylamide disc gels at pH 4.3 (7). Before use, the Con A wa.s2demetallized+$y acid treatment followed by remetallization with 2 mM Ca and 2 mM Mn (8). The Con A concentrations were determined by absorbance at 280 nm using = 12.4 at pH 5.2 and 13.7 at pH 7.2 for a 1% solution (9). The buffers A280 used at pH 5.2 and 7.2 were 0.05 M acetate and 0.05 M MOPS (Sigma), respectively. The sugar aMM was purchased from Sigma (Grade III) and used without further purification. Calorimetric measurements were carried out at constant temperature (+ 0.001 C) using a modified Beckman 19OC flow microcalorimeter (10,ll). The calorimeter was calibrated by both the reaction of Tris with HCl (12) and by the heat of dilution of aqueous sucrose (13). The Con A solutions were equilibrated by dialysis against the buffer solution at the temperature The ctMM solutions were of the measurement of binding for at least 24 hours. The two reacting prepared by dissolution of aMM in the dialysis buffer. The components were mixed at a constant Con A to aMM volume ratio of 1:4. desired mole ratios of protein to aMM wzy obtained bz4use of initial Con A concentrations in the range of 1.5 x 10 to3.0xlO M. The heats of dilution of Con A and aMM were obtained by mixing buffer with the protein or sugar solutions. The heats of the reaction were much greater than the heats of dilution for I, values less than 0.9. These heats, which represented at most 8% of the observed total for both Con A and for aMM, were subtracted from the total observed heats. All measurements were made in triplicate. The value of AH was determined by saturating the available Con A with heat of aMM at initial concentrations of 0.2, 0.1 and 0.05 M. The observed reaction may be used to calculate the equilibrium constant for binding of aMM when saturation is not attained, according to equation 1, the Scatchard when expressed in terms of the experimental parameters, equation (14), which, is converted to equation 2. v/L = K - vK
Q AH[aMM1total
where
the symbols v = the L = free
are
fraction
defined
heat
A1total
Q.K
(2)
"Con A AH
below:
of ligand
concentration
Q = observed
=K-
nCon A - 'Icon
(1)
bound
to total
Con A monomers.
of aMM of reaction
at fixed
685
concentration
of Con A
Vol. 84, No. 3, 1978
BIOCHEMICAL AND BIdPHYSICAL RESEARCH COMMUNICATIONS
AH = enthalpy
change
K = association
per
mole aMM bound
constant
= total concentration of crMM total = total number of moles of Con A monomers nCon A [Con A] = total concentration of Con A, in terms total Ial
Ultraviolet spectrophotometer holders.
of monomers
difference spectra of Con A were obtained using with an O-O.1 slide wire and temperature-controlled
a Cary 14 cuvette
RESULTS Heats
of binding
specified
in Table
determinations. pendent based
to aMM to Con A were I.
Each entry
The reaction
of temperature, on equation
predicts of 15'
under
observed
the
by contributions
of binding
of crMM measured
saturation
the
conditions
of two or more
with
concentration.
salt
the loo-25'
complicated
are
the average
a AH nearly
Typical
constant
for
concentration,
inde-
Scatchard
A AH of approximately
1.
of the measurements.
at either
under
exothermic
of the association
at pH 5.2
kJ/mol)
clearly
shown in Figure
conditions
in 1.0 M NaCl in
represents
pH or salt
1 are
a doubling
is
measured
plots -33 kJ/mole
a decrease
in temperature
Such changes
in K are not
and only
partially
at pH 7.2
interval.
The caloriuuatric
from heats
of ionization
of the buffers.
the absence
of MOPS (AH = -39.3
at pH 7.2 in
indistinguishable
from
of Con A with
aMM under
those
obtained
similar
in
conditions
measurements
its
are
Heats
presence.
results
In addition,
in no change
in pH. The effect evident
of salt
at pH 5.2,
concentration
25',
where
the NaCl concentration It
is
interesting
clMM binding of the
Because
in
(see
that
standard
indicate
Discussion
a doubling
of Con A for
in K results
from
aMM is lowering
0.5 M to 0.1 M.
be accounted
the molar
the data
nearly
to note
can nearly
sugar
at pH 5.2
from
on the affinity
below),
the decrease for
by the
in entropy loss
consequent
of the entropy
to
of mixing
state.
a temperature-dependent difference
686
absorption
conformation spectra
not
change were
taken
'Listed d Errors
aListed b Listed
pH 7.2 1 M NaCl
are
errors
37.0
6,080
25.0
range
the standard
the
errors
of
error the errors
by saturation. in AG and AH divided
by the
and lower
of Scatchard
-38.6 (.04)
-41.6 C.92)
C.88)
-34.1
-38.5 C-29)
-35.6 C.29)
-35.2 C.29)
-31.7 (.96>'
in K.
to a straight
temperature.
values
fitted
56.5 (1.26)
-66.9 (2.93)
-43.5 (2.93)
-54.0 C.96)
-48.1 (3.81)
-41.7 (1.26)
absolute
error
plots
deg
-35.7 (3.47)d
J/mole
AS cal
BINDING TO CONCANAVALIN A
AHcal kJ/mole
the upper
the slope
from
in
C.42)
-20.9
t.08)
-21.6
-21.1 (.17)
-21.8 (.04)
-21.3 (.13)
C.25)
-22.8
-21.6 (.13)b
AG kJ/xmle
of AH obtained
of AG calculated
standard
3,300 (500)
(210)
9,820 (730)
10.1
the root-mean-squares
are
are half
errors
4,790
37.0
(8’3)
5,300 (290)
25.0
the calculated
are
errors
pH 5.2 0.5 M NaCl
9,740
25.0
(880)
9,640 (580)a
10.1
pH 5.2 0.1 M NaCl
Ka
Temperature OC
Experimental Conditions
SUMMARY OF RESULTS OF METHYL a-D-MANNOPYRANOSIDE
TABLE I
-29.2
-6.3
line
0.9
AHV.H. kJ/mole
(18).
BIOCHEMICAL
Vol. 84, No. 3, 1978
Fig.
AND BIdPHYSICAL
RESEARCH COMMUNICATIONS
1.
(A) Scatchard plot s%wing binding of methyl a-D;ynnopyranoside M, in 0.1 M NaCl, 2 mM Ca , and 2 ti (Con A) = 1.54 x 10 t"+zon AThe free aMM concentration is denoted pH 5.2 and 25'C. Ml-l , 0.05 M acetate and the fraction of sites occupied per Con A monomer is v. (B) by Lfree Scatchard plot showing binding of methy:2a-D-mannopyr$!oside to Con A (Con and2mMMn , 0.05 M MOPS at pH A) = 1.40 x lO-4 M, 1.0 M NaCl, 2 mM Ca 7.2 and 25°C.
at various
temperatures
Differences
are
and referenced
at this
observed
pH.
when the
to the spectrum
The results
temperature
are
presented
in Figure
of one Con A sample
of a second
Con A sample
is
maintained
2.
varied at 23.5"C.
DISCUSSION Calorimetry
has been
and conformation analysis
artifacts
for
AH.
change
changes
the
alters
aMM, to compensate
measured the
above is
of measurement,
for
temperature. is
that
is
the
feature
observed
AH and
In the absence
changing
temperature
at
the
effect
of temperature
on K predicted
from
change
with
on K is also
consequent
structure
at pH 5.2. of proteins
688
of Con A, and thereby
of
conformation
A conformational
tertiary
the
binding
example
The remarkable
between
of K with
of ligand
A recent
(16).
the difference
implication
analysis
the
in NaCl concentration
in the
(15).
interaction
from the variation
sufficiently
in the
in biochemistry
ricin-saccharide
presented
AH calculated
pH 5.2
equilibria
of the
of the data
used extensively
an effect
Such temperature are well
known
its
affinity
and salt (17).
the to
dependent
BIOCHEMICAL
Vol. 84, No. 3, 1978
I 270
Fig. 2. referent5 2mMMn
shown that
to the direct
calorimetry
and eliminates present
of other
290 Wavelength,
300
RESEARCH COMMUNICATIONS
I 310
m-n
Difference absorption spectra of Con A5at various temperatures Con A at 23.5'C. (Con A) = 5.4 x 10 , 0.5 M NaCl, 2 mM Ca , 0.05 M acetate, pH 5.2.
In addition
This
280
AND BIdPHYSICAL
allows
the need study
saccharides
determination
is
for
the direct special
now being
ligands
extended
of enthalpy determination
of binding, of binding
in competitive to investigations
binding
with ,
we have constants assays.
on the binding
to Con A.
Acknowledgements This Grant
research
was supported
in part
by United
States
Public
Health
Service
CA 14496.
REFERENCES 1. 2. 3.
Bessler, W., Shafer, J. A., and Golstein, I. J. (1974) J. Biol. Chem., 2, 2819-2822. Loontiens, F. G., Clegg, R. M., and Jovin, T. M. (1977) Biochemistry, 16, 159-166. Huet, M., and Claverie, J. M. (1978) Biochemistry, 17, 236-241.
689
Vol. 84, No. 3, 1978
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
BIOCHEMICAL
AND EIOPHYSICAL RESEARCH COMMUNICATIONS
McCubbin, W. D., Oikawa, K., and Kay, C. M. (1971) Biochem. Biophys. Res. Cormnun., 42, 666-674. Pflumm, M. N., Wang, .I. L., and Edelman, G. M. (1971) J. Biol. Chem., 246, 4369-70. Agrawal, B. B. L., and Goldstein, I. J. (1976) Biochem. Biophys. Acta, 133, 376-379. Shephard, G. R., and Gurley, L. R. (1966) Anal. Biochem., 14, 356-363. Kalb, A. J., and Levitzki, A. (1968) Biochem. .I., 109, 669-672. Yariv, J., Kalb, A. J., and Levitzki, A. (1968) Biochem. Biophys. Acta, 165, 303-305. Krakauer, H. (1972) Biopolymers, 11, 811-828. Velick,_~S. F., Baggott, J. P., and Sturtevant, J. M. (1971) Biochemistry, 10, 779-786. Felund, G., and Wads;, I. (1969) Acta Chem. Stand., 22, 2691-2699. Gucker, F. T., Pickard, H. B., and Planck, R. W. (1939) J. Am. Chem. sot., 61, 459-470. Scatchard, G. (1949) Ann. N. Y. Acad. Sci., 5l, 660-672. Sturtevant, J. W. (1974) Ann. Rev. Biophy. and Bioeng., 3, 35-51. Zentz, C., Frenoy, J-P., and Bourrillon, R. (1977) FEBS Letters, 8& 23-27. Zav'yalov, V. P., Tetin, S. Y., Abramov, V. M., and Troitsky, G. Y. (1978) Biochim. Biophys. Acta, 533, 496-503. Fischer, 0. (1969) in Survey of Applicable Mathematics (Rektorys, K., ed.) p. 1285-1321, M.I.T. Press, Cambridge.
690
BIOCHEMICAL
AND 8ldPHYSlCAL
RESEARCH COMMUNICATIONS
16,1978
Pages
684-690
A CONFOFMATIONAL CHANGE IN CONCANAVALIN A DETECTED BY A CALORIMETRIC
STUDY OF THE BINDING OF
METHYL a-D-MANNOPYRANOSIDE
G. R. Munske,
Program
Received
August
22,
J. A. Magnuson
and H. Krakauer
in Biochemistry and Biophysics Washington State University Pullman, WA 99164
1978 SUMMARY
The binding of methyl a-D-mannopyranoside to the lectin concanavalin A was studied by means of calorimetry. An apparent enthalpy of binding was also calculated from the variation of the equilibrium constant with temperature (van't Hoff AH). The AH measured directly was-30 to -38 kJ/mole indicating that the binding is driven by the AH change. In contrast, the van't Hoff AH was substantially smaller, about zero at pH 5.2. The difference in the AH measured directly and the van't Hoff AH implies that the conformation of concanavalin A undergoes a temperature dependent change at both pH's but most predominantly at pH 5.2. The existence of this conformational change was verified by difference absorption spectroscopy. Concanavalin from the
jack
A (Con A) is
bean.
It
a-D-mannopyranoside, property
is
to induce
the
of Con A is
(7.2),
ability
high
temperature
predominates
tertiary
structure
as detected
sites
to which
specifically
(1,2).
class
(3).
by measurement
In addition,
of
salt
most notably
Associated and other
the circular
684
is
this
cell
types
The quarternary
favored,
concentration
ion
while
undergoes
structure
at high
pH
M) the changes
binding
We report
and
At low
(1.0
and sugar
dichroism.
form
with
concentration.
the protein
of metal
000629ur/78/0843-0684$oi.o0/0
Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
sugars,
and salt a dimer
and high
as a consequence
in pure
of lymphocytes.
concentration (37°C)
obtainable
erythrocytes
on pH, temperature
and salt
tetramar
readily
to agglutinate
in a certain
dependent
pH, temperature
possesses
can bind
mitosis
a lectin
(4,5), herein
in
Vol. 84, No. 3, 1978
a conformational temperature
BIOCHEMICAL
change and perhaps
binding
of the sugar,
course
of measurements
AND BIdPHYSICAL
not
previously
observed
also
on salt
concentration,
methyl
which
a-D-mannopyranoside
of the heat
RESEARCH COMMUNICATIONS
is
and which It
(aMM).
of binding
dependent
of that
on
perturbs
the
was detected
in the
sugar.
MATERIALS AND METHODS Con A was prepared from jack beans (Sigma) by the method of Agrawal and Goldstein (6) and gave a single band on electrophoresis on 5% polyacrylamide disc gels at pH 4.3 (7). Before use, the Con A wa.s2demetallized+$y acid treatment followed by remetallization with 2 mM Ca and 2 mM Mn (8). The Con A concentrations were determined by absorbance at 280 nm using = 12.4 at pH 5.2 and 13.7 at pH 7.2 for a 1% solution (9). The buffers A280 used at pH 5.2 and 7.2 were 0.05 M acetate and 0.05 M MOPS (Sigma), respectively. The sugar aMM was purchased from Sigma (Grade III) and used without further purification. Calorimetric measurements were carried out at constant temperature (+ 0.001 C) using a modified Beckman 19OC flow microcalorimeter (10,ll). The calorimeter was calibrated by both the reaction of Tris with HCl (12) and by the heat of dilution of aqueous sucrose (13). The Con A solutions were equilibrated by dialysis against the buffer solution at the temperature The ctMM solutions were of the measurement of binding for at least 24 hours. The two reacting prepared by dissolution of aMM in the dialysis buffer. The components were mixed at a constant Con A to aMM volume ratio of 1:4. desired mole ratios of protein to aMM wzy obtained bz4use of initial Con A concentrations in the range of 1.5 x 10 to3.0xlO M. The heats of dilution of Con A and aMM were obtained by mixing buffer with the protein or sugar solutions. The heats of the reaction were much greater than the heats of dilution for I, values less than 0.9. These heats, which represented at most 8% of the observed total for both Con A and for aMM, were subtracted from the total observed heats. All measurements were made in triplicate. The value of AH was determined by saturating the available Con A with heat of aMM at initial concentrations of 0.2, 0.1 and 0.05 M. The observed reaction may be used to calculate the equilibrium constant for binding of aMM when saturation is not attained, according to equation 1, the Scatchard when expressed in terms of the experimental parameters, equation (14), which, is converted to equation 2. v/L = K - vK
Q AH[aMM1total
where
the symbols v = the L = free
are
fraction
defined
heat
A1total
Q.K
(2)
"Con A AH
below:
of ligand
concentration
Q = observed
=K-
nCon A - 'Icon
(1)
bound
to total
Con A monomers.
of aMM of reaction
at fixed
685
concentration
of Con A
Vol. 84, No. 3, 1978
BIOCHEMICAL AND BIdPHYSICAL RESEARCH COMMUNICATIONS
AH = enthalpy
change
K = association
per
mole aMM bound
constant
= total concentration of crMM total = total number of moles of Con A monomers nCon A [Con A] = total concentration of Con A, in terms total Ial
Ultraviolet spectrophotometer holders.
of monomers
difference spectra of Con A were obtained using with an O-O.1 slide wire and temperature-controlled
a Cary 14 cuvette
RESULTS Heats
of binding
specified
in Table
determinations. pendent based
to aMM to Con A were I.
Each entry
The reaction
of temperature, on equation
predicts of 15'
under
observed
the
by contributions
of binding
of crMM measured
saturation
the
conditions
of two or more
with
concentration.
salt
the loo-25'
complicated
are
the average
a AH nearly
Typical
constant
for
concentration,
inde-
Scatchard
A AH of approximately
1.
of the measurements.
at either
under
exothermic
of the association
at pH 5.2
kJ/mol)
clearly
shown in Figure
conditions
in 1.0 M NaCl in
represents
pH or salt
1 are
a doubling
is
measured
plots -33 kJ/mole
a decrease
in temperature
Such changes
in K are not
and only
partially
at pH 7.2
interval.
The caloriuuatric
from heats
of ionization
of the buffers.
the absence
of MOPS (AH = -39.3
at pH 7.2 in
indistinguishable
from
of Con A with
aMM under
those
obtained
similar
in
conditions
measurements
its
are
Heats
presence.
results
In addition,
in no change
in pH. The effect evident
of salt
at pH 5.2,
concentration
25',
where
the NaCl concentration It
is
interesting
clMM binding of the
Because
in
(see
that
standard
indicate
Discussion
a doubling
of Con A for
in K results
from
aMM is lowering
0.5 M to 0.1 M.
be accounted
the molar
the data
nearly
to note
can nearly
sugar
at pH 5.2
from
on the affinity
below),
the decrease for
by the
in entropy loss
consequent
of the entropy
to
of mixing
state.
a temperature-dependent difference
686
absorption
conformation spectra
not
change were
taken
'Listed d Errors
aListed b Listed
pH 7.2 1 M NaCl
are
errors
37.0
6,080
25.0
range
the standard
the
errors
of
error the errors
by saturation. in AG and AH divided
by the
and lower
of Scatchard
-38.6 (.04)
-41.6 C.92)
C.88)
-34.1
-38.5 C-29)
-35.6 C.29)
-35.2 C.29)
-31.7 (.96>'
in K.
to a straight
temperature.
values
fitted
56.5 (1.26)
-66.9 (2.93)
-43.5 (2.93)
-54.0 C.96)
-48.1 (3.81)
-41.7 (1.26)
absolute
error
plots
deg
-35.7 (3.47)d
J/mole
AS cal
BINDING TO CONCANAVALIN A
AHcal kJ/mole
the upper
the slope
from
in
C.42)
-20.9
t.08)
-21.6
-21.1 (.17)
-21.8 (.04)
-21.3 (.13)
C.25)
-22.8
-21.6 (.13)b
AG kJ/xmle
of AH obtained
of AG calculated
standard
3,300 (500)
(210)
9,820 (730)
10.1
the root-mean-squares
are
are half
errors
4,790
37.0
(8’3)
5,300 (290)
25.0
the calculated
are
errors
pH 5.2 0.5 M NaCl
9,740
25.0
(880)
9,640 (580)a
10.1
pH 5.2 0.1 M NaCl
Ka
Temperature OC
Experimental Conditions
SUMMARY OF RESULTS OF METHYL a-D-MANNOPYRANOSIDE
TABLE I
-29.2
-6.3
line
0.9
AHV.H. kJ/mole
(18).
BIOCHEMICAL
Vol. 84, No. 3, 1978
Fig.
AND BIdPHYSICAL
RESEARCH COMMUNICATIONS
1.
(A) Scatchard plot s%wing binding of methyl a-D;ynnopyranoside M, in 0.1 M NaCl, 2 mM Ca , and 2 ti (Con A) = 1.54 x 10 t"+zon AThe free aMM concentration is denoted pH 5.2 and 25'C. Ml-l , 0.05 M acetate and the fraction of sites occupied per Con A monomer is v. (B) by Lfree Scatchard plot showing binding of methy:2a-D-mannopyr$!oside to Con A (Con and2mMMn , 0.05 M MOPS at pH A) = 1.40 x lO-4 M, 1.0 M NaCl, 2 mM Ca 7.2 and 25°C.
at various
temperatures
Differences
are
and referenced
at this
observed
pH.
when the
to the spectrum
The results
temperature
are
presented
in Figure
of one Con A sample
of a second
Con A sample
is
maintained
2.
varied at 23.5"C.
DISCUSSION Calorimetry
has been
and conformation analysis
artifacts
for
AH.
change
changes
the
alters
aMM, to compensate
measured the
above is
of measurement,
for
temperature. is
that
is
the
feature
observed
AH and
In the absence
changing
temperature
at
the
effect
of temperature
on K predicted
from
change
with
on K is also
consequent
structure
at pH 5.2. of proteins
688
of Con A, and thereby
of
conformation
A conformational
tertiary
the
binding
example
The remarkable
between
of K with
of ligand
A recent
(16).
the difference
implication
analysis
the
in NaCl concentration
in the
(15).
interaction
from the variation
sufficiently
in the
in biochemistry
ricin-saccharide
presented
AH calculated
pH 5.2
equilibria
of the
of the data
used extensively
an effect
Such temperature are well
known
its
affinity
and salt (17).
the to
dependent
BIOCHEMICAL
Vol. 84, No. 3, 1978
I 270
Fig. 2. referent5 2mMMn
shown that
to the direct
calorimetry
and eliminates present
of other
290 Wavelength,
300
RESEARCH COMMUNICATIONS
I 310
m-n
Difference absorption spectra of Con A5at various temperatures Con A at 23.5'C. (Con A) = 5.4 x 10 , 0.5 M NaCl, 2 mM Ca , 0.05 M acetate, pH 5.2.
In addition
This
280
AND BIdPHYSICAL
allows
the need study
saccharides
determination
is
for
the direct special
now being
ligands
extended
of enthalpy determination
of binding, of binding
in competitive to investigations
binding
with ,
we have constants assays.
on the binding
to Con A.
Acknowledgements This Grant
research
was supported
in part
by United
States
Public
Health
Service
CA 14496.
REFERENCES 1. 2. 3.
Bessler, W., Shafer, J. A., and Golstein, I. J. (1974) J. Biol. Chem., 2, 2819-2822. Loontiens, F. G., Clegg, R. M., and Jovin, T. M. (1977) Biochemistry, 16, 159-166. Huet, M., and Claverie, J. M. (1978) Biochemistry, 17, 236-241.
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4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
BIOCHEMICAL
AND EIOPHYSICAL RESEARCH COMMUNICATIONS
McCubbin, W. D., Oikawa, K., and Kay, C. M. (1971) Biochem. Biophys. Res. Cormnun., 42, 666-674. Pflumm, M. N., Wang, .I. L., and Edelman, G. M. (1971) J. Biol. Chem., 246, 4369-70. Agrawal, B. B. L., and Goldstein, I. J. (1976) Biochem. Biophys. Acta, 133, 376-379. Shephard, G. R., and Gurley, L. R. (1966) Anal. Biochem., 14, 356-363. Kalb, A. J., and Levitzki, A. (1968) Biochem. .I., 109, 669-672. Yariv, J., Kalb, A. J., and Levitzki, A. (1968) Biochem. Biophys. Acta, 165, 303-305. Krakauer, H. (1972) Biopolymers, 11, 811-828. Velick,_~S. F., Baggott, J. P., and Sturtevant, J. M. (1971) Biochemistry, 10, 779-786. Felund, G., and Wads;, I. (1969) Acta Chem. Stand., 22, 2691-2699. Gucker, F. T., Pickard, H. B., and Planck, R. W. (1939) J. Am. Chem. sot., 61, 459-470. Scatchard, G. (1949) Ann. N. Y. Acad. Sci., 5l, 660-672. Sturtevant, J. W. (1974) Ann. Rev. Biophy. and Bioeng., 3, 35-51. Zentz, C., Frenoy, J-P., and Bourrillon, R. (1977) FEBS Letters, 8& 23-27. Zav'yalov, V. P., Tetin, S. Y., Abramov, V. M., and Troitsky, G. Y. (1978) Biochim. Biophys. Acta, 533, 496-503. Fischer, 0. (1969) in Survey of Applicable Mathematics (Rektorys, K., ed.) p. 1285-1321, M.I.T. Press, Cambridge.
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