Vol. 71, No. 3,1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MECHANISM OF ACTION OF CESALIN, J. Elting
AN ANTIT'UMOR PROTEIN*
and R. Montgomery
Department of Biochemistry University of Iowa Iowa City, Iowa 52242 Received
June
8.1976 SUMMARY
A protein, cesalin, isolated from Caesalpinia gilliesii is cytotoxic to KB cells in tissue culture. It has been shown to bind to the plasma membrane of this cell line and to inhibit Na+,K+-ATPase (ATP Phosphohydrolase EC 3.6.1.3). Similar studies with HTC cells show no cytotoxicity or inhibition of plasma membrane Na+,K+-ATPase. The Na+,K+-ATPase of human erythrocytes and rat brain and kidney tissues are not inhibited. 5'-Nucleotidase and Mg*-ATPase are not inhibited by cesalin in any cells tested. INTRODUCTION It
is well
membranes peptide
toxin
may alter hormones
plasma with
known
membrane
cell
secreted
after
penetration of elongation
of chick
lectin
a protein,
into
cesalin,
possesses GM-14013
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
and
the cells
GM-550
from
activity.
(2).
The receptors
inhibits
of which
protein
Varied concern
eftrans-
of uridine
into
with
concanavalin
of thymidine
into
(5). the seeds It
appears
from USPHS National
871
interacts
2 (3).
fibroblasts
incorporation
purified
antitumor
some
that
of the
cholerae
the cell, factor
surface shown
surface
ganglioside
transport
embryo
inhibits
transport
by grants
into
cell have
cyclase
to specific
stimulates
treatment
inhibiting
adenylate
binds
with
studies
by Vibrio
and stimulates
Phytohemagglutinin
which
Supported
on the exterior
have been observed,
pseudoacacia
We report
receptors
modification
(4) while
DNA without
gilliesii,
and,
interaction
processes.
A or Robinia
Copyright All rights
surface
of lectin
Numerous
diphtheriae
by enzymatic
lymphocytes
*
membranes
of some proteins
processes.
The enterotoxin
of Corynebacterium
synthesis
port
to specific
(1).
eucaryotic
the interaction
intracellular bind
on the membrane
fects
that
of Caesalpinia to act
in
Institutes
tissue
of Health.
Vol. 71, No. 3,1976
culture bind is
BIOCHEMICAL
by directly to plasma
altering
membranes
AND BIOPHYSICAL
transport
functions.
and to affect
RESEARCH COMMUNICATIONS
Cesalin
the Na+,K+-ATPase
has been shown of cells
for
to
which
it
cytotoxic. MATERIALS
ANDMETHODS
KB (human epidermoid carcinoma derived) cells are routinely subcultured by seeding at 4 x lo4 cells/cm2 in Falcon monolayer flasks in Eagle's minimum essential medium supplemented with 10% heat-inactivated fetal calf serum and 100 units/ml penicillin and 100 ug ml streptomycin. HTC (rat hepatoma derived) cells (6) are seeded at 2-2.5 x 10 4 cells/ml in spinner flasks with Swim's S-77 medium supplemented with cystine, glutamine, penicillin, streptomycin, 5% fetal calf serum and 5% calf serum. All media and supplements were purchased from Grand Island Biological Company. Ouabain was purchased from Sigma Chemical Co. FITC-conjugated IgG from goat anti-rabbit IgG serum was purchased from Miles Laboratories. Protein concentrations of solutions were determined by the method of Lowry (7) using crystalline bovine serum albumin as standard. Due to the presence of substances interfering with the Lowry method, total KB cell protein was determined by the microbiuret method of Goa (8). 5'-Nucleotidase was assayed by the method of Michell and Hawthorne (9) and released phosphate measured by the method of Lowry (10). Phosphate released during ATPase assays was determined by the method of Marsh (11) due to the instability of ATP in the system of Lowry. Growth inhibition was determined by adding cesalin at various concentrations in phosphate buffered saline (PBS) (10 mM Na phosphate, 0.14 M NaCl, 4 mM KCl, pH 7.5) to KB monolayer cultures according to the procedure of Smith, cultures. Growth of cesalin-treated cultures --et al. (12) or to HTC spinner and PBS-treated controls was monitored by hemocytometer counting of HTC cells or, for KB cells, determination of total cell protein after 4 days growth. Cesalin was prepared from the meal of Caesalpinia seeds (13). Na+,K+ ATPase was partially purified by preparation of a plasma membrane-enriched fraction from KB and HTC cells using the method of Butters and Hughes (14). Plasma membrane-enriched microsomes from rat brain and kidney were prepared according to the method described by Dorling and LePage (15) for rat liver plasma membranes. Na+,K+-ATPase was determined as ouabain-sensitive activity by incubation of the enzyme in buffer containing 25 mM HEPES, 3 mM MgC12, 0.13 M NaCl, 20 mM KCl, 3 n&l ATP (final concentrations) in the presence and absence of 10-3 M ouabain (16). The incubation was for 20 min. at 37' with enzyme concentrations at which the reaction rate was linear for at least 30 min. Reactions were started by the addition of ATP. RESULTS AND DlSCUSSION The growth shown
in Figure
(ED50)
is 2.0
centrations
response
of HTC and KB cells
1.
The concentration -2 - 5.0 x 10 ug/ml.
up to 11 pglml.
that Growth
Cesalin-treated
872
to various inhibits
growth
of HTC cells KB cells
levels
of cesalin
of KB cells
was unaffected appear
by phase
are by 50%
by concontrast
BIOCHEMICAL
Vol. 71, No. 3,1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1
1 CESALIN
of growth 1 Inhibition procedure see Materials
Figure
2 Effect of cesalin (lmg/ml) cells that have spontaneously of cells that exclude trypan
microscopic
examination
12-16
they
all
released. blue
of the
cells
Figure
2.
(0.25%
in Earle's
37"
begin
dye until still
C) during
to a new culture cesalin
first flask
treatment. of further
the
cesalin
continuous 30 minutes, death with
contact
by either Earle's Assay
balanced
This
exposure
cesalin.
tested,
could
or EDTA treatment salts
of the membrane
solution phosphatases
or complete showed
873
themselves
reattached
cesalin
be rescued
12-16
that
for from
cesalin
of in
hours left
after in
as little eventual
or multiple
culture
trypsin
mM in PBS, 7 min.,
as do the cells
(see above)
with
have
to detach
50%
shown in
7 hours
begin
not
is
after
that
have
24 hours
reattach
cells
with
they
to exclude
events
will
was lost
treated
After
ability
of the flask
treatment
just
of percentage
After
of these
thrs)
experimental HTC cells.
hrs.
C) or EDTA (0.5
to die
Cells
their
the surface
will
l-10
40
ADDN.
and by 24 hours in
course
property
and begin
time
30 CESALIN
for
addition.
37'
of cesalin
for
substratum
cesalin-treated
addition
trypsin
from 5 min.,
surface.
the shortest
normal
cesalin
The time
salts,
cesalin
with
the
after
are harvested
However,
the absence initial
hours
6 hours
by cesalin; KB cells -
show no reduction
dye.
balanced
the
from
cells
14-16
which
20 AFTER
on KB cells; w percentage released from substratum;blue.
to detach
exclude
Cells
of KB and HTC cells and Methods. O---O
to be morphologically
The treated
trypan
10 TIME
Figure
hours
%
lug/d
washings
medium. inhibits
ouabain-
as
I
BIOCHEMICAL
Vol. 71, No. 3,1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Inhibition
1
of Na+,K+-ATPase
Na+ K+-ATPase Altivity (umoles Pi/mg*hr)'
Cesalin Added (ug)
% Inhibition
0
4.98
+ 0.962
1
3.54
+ 0.83
29
10
3.09 + 0.69
38
100
3.53 + 1.0
29
' Na+,I?-ATPase
activity
by 1O-3 M ouabain. 6X by density
determined
as that
Enzyme source
gradient
is
ATPase
plasma
activity
membrane
inhibited
of KB cells
purified
centrifugation.
2 S.E.M.
sensitive
Na+,K+-ATPase
membranes. cesalin
not
The degree
is
was never
shown
Similarly, not
than further
is unaffected
not
1.
that
Na+,K+-ATPases
of Na+,K+ -ATPase
The total inhibited
by adding
by cesalin,
or Mg+I- -ATPase
5'-nucleotidase
of inhibition
in Table
greater
be increased
growth
but
ATPase
by various
activity
inhibited
by 10B3 M ouabain, cesalin.
is not
which
Na+,K+-ATPase
inhibited
at all
from human erythrocytes
levels
of
by cesalin inhibition
could
from HTC cells, under
and rat
in KB plasma
brain
these
whose
conditions.
and kidney
were
inhibited. Studieswith[l25Ij
of KB cells cesalin
acts
and not
cesalin to HTC cell
at the cell
surface
indicated
that
it
plasma
membranes.
comes from indirect
874
binds
to the plasma
membrane
Corroborative
evidence
fluorescent
antibody
that
Vol. 71, No. 3,1976
staining
BIOCHEMICAL
of cesalin-treated
washed
and incubated
again
washed
gated
goat
treated that toxic
cesalin
is known
be produced
by cesalin-treated
sizing
without
on the
shows
products
interacts
with
against
cesalin.
The cells
fluorescein membrane
fluorescence
membrane
of buffer-
antiserum.
after
were
was observed
staining
cesalin-specific
cell
cesalin,
isothiocyanate-conju-
or no non-specific
would
be expected
transport
Similar
of other
in preliminary changes
addition
of ouabain.
KB cells
is
that
60 min with
1 hour,
It
follows
by which
time
cyto-
in dysfunction
of
to be irreversible.
has been shown
by the
incubated
with
minimal
and ion-coupled
transport.
were
Intense
of Na+,K+-ATPase
ment of KB cells
lin
with
or controls
transport
nucleoside
antiserum
y-globulin.
was present
action
molecular
rabbit
Cells
and incubated
cells
controls
membrane
with
anti-rabbit
Inhibition ion
KB cells.
extensively
on cesalin-treated
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the cells' is nearly the plasma
in
metabolites. studies
While
uptake
membrane
This without
and
materials
of thymidine rupturing
to incorporate
treat-
glucose
of these
reduced,
unaffected.
Cesalin
to alter
the transport
significantly ability
to result
can
and uridine the cell
nucleosides
into
supports
the proposal
inhibiting
nucleic
macrothat
acid
cesa-
synthe-
enzymes.
ACKNOWLRDGMRNTS The authors cell
cultures
this
study.
wish and Mrs.
Mr.
Dennis
H. Jane Mead for
KB and HTC cells
ment of Microbiology, ment,
to thank
University
were
Drews
for
preparation
gifts
of Dr.
of Iowa,and
Dr.
technical
assistance
of the cesalin J.E. C.S.
Rodriguez Vestling
with
used in
of the Departof this
depart-
respectively. REFERENCES
1. 2. 3. 4. 5. 6.
Cuatrecasas, P., (1974) Ann. Rev. Biochem.,43, 169-214. Cuatrecasas, P., (1973) Biochemistry,12, 3558-3566. Collier, R.J., (1975) Bacterial. Rev.,39, 54-85. Peters, J.H., and Hausen, P., (1971) Eur. J. Biochem., 19, 502-508. R., (1975) Biochem. J.,152, 421-423. Roguet, R., and Bourrilon, Thompson, E.B., Tomkins, G.M., and Curran, J.P., (1966) Proc. Natl. Sci. USA,56, 296-303.
8’75
Acad.
Vol. 71, No. 3,1976
7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Lowry, O.H., Roberts, N.R., Leiner, K.Y., Wu, M.L., and Farr, A.L., (1954) J. Biol. Chem.,207, 1-17. Goa, J., (1953) Scand. J. Clin. Lab. Iov.,~, 218-222. Michell, R.J., and Hawthorne, J.N., (1965) Biochem. Biophys. Res. Commun., 21, 333-338. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., (1951) J. Biol. Chem.,193, 265-275. Marsh, B.B., (1959) Biochim. Biophys. Acta,32, 357-361. Smith, C.G., Lummis, W.L., and Grady, J.E., (1959) Cancer Research,19, 843-846. Chiang, C.K., Yamauchi, F., and Montgomery, R. Unpublished work. Butters, T.D., and Hughes, R.C., (1975) Biochem. J.,l50, 59-69. Dorling, P.R., and Le Page, R.N., (1973) Biochim. Biophys. Acta,318, 33-44. Solomonson, L.P., Liepkalns, V.A., and Spector, A.A., (1976) Biochemistry, 15, 892-897.
876
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MECHANISM OF ACTION OF CESALIN, J. Elting
AN ANTIT'UMOR PROTEIN*
and R. Montgomery
Department of Biochemistry University of Iowa Iowa City, Iowa 52242 Received
June
8.1976 SUMMARY
A protein, cesalin, isolated from Caesalpinia gilliesii is cytotoxic to KB cells in tissue culture. It has been shown to bind to the plasma membrane of this cell line and to inhibit Na+,K+-ATPase (ATP Phosphohydrolase EC 3.6.1.3). Similar studies with HTC cells show no cytotoxicity or inhibition of plasma membrane Na+,K+-ATPase. The Na+,K+-ATPase of human erythrocytes and rat brain and kidney tissues are not inhibited. 5'-Nucleotidase and Mg*-ATPase are not inhibited by cesalin in any cells tested. INTRODUCTION It
is well
membranes peptide
toxin
may alter hormones
plasma with
known
membrane
cell
secreted
after
penetration of elongation
of chick
lectin
a protein,
into
cesalin,
possesses GM-14013
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
and
the cells
GM-550
from
activity.
(2).
The receptors
inhibits
of which
protein
Varied concern
eftrans-
of uridine
into
with
concanavalin
of thymidine
into
(5). the seeds It
appears
from USPHS National
871
interacts
2 (3).
fibroblasts
incorporation
purified
antitumor
some
that
of the
cholerae
the cell, factor
surface shown
surface
ganglioside
transport
embryo
inhibits
transport
by grants
into
cell have
cyclase
to specific
stimulates
treatment
inhibiting
adenylate
binds
with
studies
by Vibrio
and stimulates
Phytohemagglutinin
which
Supported
on the exterior
have been observed,
pseudoacacia
We report
receptors
modification
(4) while
DNA without
gilliesii,
and,
interaction
processes.
A or Robinia
Copyright All rights
surface
of lectin
Numerous
diphtheriae
by enzymatic
lymphocytes
*
membranes
of some proteins
processes.
The enterotoxin
of Corynebacterium
synthesis
port
to specific
(1).
eucaryotic
the interaction
intracellular bind
on the membrane
fects
that
of Caesalpinia to act
in
Institutes
tissue
of Health.
Vol. 71, No. 3,1976
culture bind is
BIOCHEMICAL
by directly to plasma
altering
membranes
AND BIOPHYSICAL
transport
functions.
and to affect
RESEARCH COMMUNICATIONS
Cesalin
the Na+,K+-ATPase
has been shown of cells
for
to
which
it
cytotoxic. MATERIALS
ANDMETHODS
KB (human epidermoid carcinoma derived) cells are routinely subcultured by seeding at 4 x lo4 cells/cm2 in Falcon monolayer flasks in Eagle's minimum essential medium supplemented with 10% heat-inactivated fetal calf serum and 100 units/ml penicillin and 100 ug ml streptomycin. HTC (rat hepatoma derived) cells (6) are seeded at 2-2.5 x 10 4 cells/ml in spinner flasks with Swim's S-77 medium supplemented with cystine, glutamine, penicillin, streptomycin, 5% fetal calf serum and 5% calf serum. All media and supplements were purchased from Grand Island Biological Company. Ouabain was purchased from Sigma Chemical Co. FITC-conjugated IgG from goat anti-rabbit IgG serum was purchased from Miles Laboratories. Protein concentrations of solutions were determined by the method of Lowry (7) using crystalline bovine serum albumin as standard. Due to the presence of substances interfering with the Lowry method, total KB cell protein was determined by the microbiuret method of Goa (8). 5'-Nucleotidase was assayed by the method of Michell and Hawthorne (9) and released phosphate measured by the method of Lowry (10). Phosphate released during ATPase assays was determined by the method of Marsh (11) due to the instability of ATP in the system of Lowry. Growth inhibition was determined by adding cesalin at various concentrations in phosphate buffered saline (PBS) (10 mM Na phosphate, 0.14 M NaCl, 4 mM KCl, pH 7.5) to KB monolayer cultures according to the procedure of Smith, cultures. Growth of cesalin-treated cultures --et al. (12) or to HTC spinner and PBS-treated controls was monitored by hemocytometer counting of HTC cells or, for KB cells, determination of total cell protein after 4 days growth. Cesalin was prepared from the meal of Caesalpinia seeds (13). Na+,K+ ATPase was partially purified by preparation of a plasma membrane-enriched fraction from KB and HTC cells using the method of Butters and Hughes (14). Plasma membrane-enriched microsomes from rat brain and kidney were prepared according to the method described by Dorling and LePage (15) for rat liver plasma membranes. Na+,K+-ATPase was determined as ouabain-sensitive activity by incubation of the enzyme in buffer containing 25 mM HEPES, 3 mM MgC12, 0.13 M NaCl, 20 mM KCl, 3 n&l ATP (final concentrations) in the presence and absence of 10-3 M ouabain (16). The incubation was for 20 min. at 37' with enzyme concentrations at which the reaction rate was linear for at least 30 min. Reactions were started by the addition of ATP. RESULTS AND DlSCUSSION The growth shown
in Figure
(ED50)
is 2.0
centrations
response
of HTC and KB cells
1.
The concentration -2 - 5.0 x 10 ug/ml.
up to 11 pglml.
that Growth
Cesalin-treated
872
to various inhibits
growth
of HTC cells KB cells
levels
of cesalin
of KB cells
was unaffected appear
by phase
are by 50%
by concontrast
BIOCHEMICAL
Vol. 71, No. 3,1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1
1 CESALIN
of growth 1 Inhibition procedure see Materials
Figure
2 Effect of cesalin (lmg/ml) cells that have spontaneously of cells that exclude trypan
microscopic
examination
12-16
they
all
released. blue
of the
cells
Figure
2.
(0.25%
in Earle's
37"
begin
dye until still
C) during
to a new culture cesalin
first flask
treatment. of further
the
cesalin
continuous 30 minutes, death with
contact
by either Earle's Assay
balanced
This
exposure
cesalin.
tested,
could
or EDTA treatment salts
of the membrane
solution phosphatases
or complete showed
873
themselves
reattached
cesalin
be rescued
12-16
that
for from
cesalin
of in
hours left
after in
as little eventual
or multiple
culture
trypsin
mM in PBS, 7 min.,
as do the cells
(see above)
with
have
to detach
50%
shown in
7 hours
begin
not
is
after
that
have
24 hours
reattach
cells
with
they
to exclude
events
will
was lost
treated
After
ability
of the flask
treatment
just
of percentage
After
of these
thrs)
experimental HTC cells.
hrs.
C) or EDTA (0.5
to die
Cells
their
the surface
will
l-10
40
ADDN.
and by 24 hours in
course
property
and begin
time
30 CESALIN
for
addition.
37'
of cesalin
for
substratum
cesalin-treated
addition
trypsin
from 5 min.,
surface.
the shortest
normal
cesalin
The time
salts,
cesalin
with
the
after
are harvested
However,
the absence initial
hours
6 hours
by cesalin; KB cells -
show no reduction
dye.
balanced
the
from
cells
14-16
which
20 AFTER
on KB cells; w percentage released from substratum;blue.
to detach
exclude
Cells
of KB and HTC cells and Methods. O---O
to be morphologically
The treated
trypan
10 TIME
Figure
hours
%
lug/d
washings
medium. inhibits
ouabain-
as
I
BIOCHEMICAL
Vol. 71, No. 3,1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Inhibition
1
of Na+,K+-ATPase
Na+ K+-ATPase Altivity (umoles Pi/mg*hr)'
Cesalin Added (ug)
% Inhibition
0
4.98
+ 0.962
1
3.54
+ 0.83
29
10
3.09 + 0.69
38
100
3.53 + 1.0
29
' Na+,I?-ATPase
activity
by 1O-3 M ouabain. 6X by density
determined
as that
Enzyme source
gradient
is
ATPase
plasma
activity
membrane
inhibited
of KB cells
purified
centrifugation.
2 S.E.M.
sensitive
Na+,K+-ATPase
membranes. cesalin
not
The degree
is
was never
shown
Similarly, not
than further
is unaffected
not
1.
that
Na+,K+-ATPases
of Na+,K+ -ATPase
The total inhibited
by adding
by cesalin,
or Mg+I- -ATPase
5'-nucleotidase
of inhibition
in Table
greater
be increased
growth
but
ATPase
by various
activity
inhibited
by 10B3 M ouabain, cesalin.
is not
which
Na+,K+-ATPase
inhibited
at all
from human erythrocytes
levels
of
by cesalin inhibition
could
from HTC cells, under
and rat
in KB plasma
brain
these
whose
conditions.
and kidney
were
inhibited. Studieswith[l25Ij
of KB cells cesalin
acts
and not
cesalin to HTC cell
at the cell
surface
indicated
that
it
plasma
membranes.
comes from indirect
874
binds
to the plasma
membrane
Corroborative
evidence
fluorescent
antibody
that
Vol. 71, No. 3,1976
staining
BIOCHEMICAL
of cesalin-treated
washed
and incubated
again
washed
gated
goat
treated that toxic
cesalin
is known
be produced
by cesalin-treated
sizing
without
on the
shows
products
interacts
with
against
cesalin.
The cells
fluorescein membrane
fluorescence
membrane
of buffer-
antiserum.
after
were
was observed
staining
cesalin-specific
cell
cesalin,
isothiocyanate-conju-
or no non-specific
would
be expected
transport
Similar
of other
in preliminary changes
addition
of ouabain.
KB cells
is
that
60 min with
1 hour,
It
follows
by which
time
cyto-
in dysfunction
of
to be irreversible.
has been shown
by the
incubated
with
minimal
and ion-coupled
transport.
were
Intense
of Na+,K+-ATPase
ment of KB cells
lin
with
or controls
transport
nucleoside
antiserum
y-globulin.
was present
action
molecular
rabbit
Cells
and incubated
cells
controls
membrane
with
anti-rabbit
Inhibition ion
KB cells.
extensively
on cesalin-treated
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the cells' is nearly the plasma
in
metabolites. studies
While
uptake
membrane
This without
and
materials
of thymidine rupturing
to incorporate
treat-
glucose
of these
reduced,
unaffected.
Cesalin
to alter
the transport
significantly ability
to result
can
and uridine the cell
nucleosides
into
supports
the proposal
inhibiting
nucleic
macrothat
acid
cesa-
synthe-
enzymes.
ACKNOWLRDGMRNTS The authors cell
cultures
this
study.
wish and Mrs.
Mr.
Dennis
H. Jane Mead for
KB and HTC cells
ment of Microbiology, ment,
to thank
University
were
Drews
for
preparation
gifts
of Dr.
of Iowa,and
Dr.
technical
assistance
of the cesalin J.E. C.S.
Rodriguez Vestling
with
used in
of the Departof this
depart-
respectively. REFERENCES
1. 2. 3. 4. 5. 6.
Cuatrecasas, P., (1974) Ann. Rev. Biochem.,43, 169-214. Cuatrecasas, P., (1973) Biochemistry,12, 3558-3566. Collier, R.J., (1975) Bacterial. Rev.,39, 54-85. Peters, J.H., and Hausen, P., (1971) Eur. J. Biochem., 19, 502-508. R., (1975) Biochem. J.,152, 421-423. Roguet, R., and Bourrilon, Thompson, E.B., Tomkins, G.M., and Curran, J.P., (1966) Proc. Natl. Sci. USA,56, 296-303.
8’75
Acad.
Vol. 71, No. 3,1976
7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Lowry, O.H., Roberts, N.R., Leiner, K.Y., Wu, M.L., and Farr, A.L., (1954) J. Biol. Chem.,207, 1-17. Goa, J., (1953) Scand. J. Clin. Lab. Iov.,~, 218-222. Michell, R.J., and Hawthorne, J.N., (1965) Biochem. Biophys. Res. Commun., 21, 333-338. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., (1951) J. Biol. Chem.,193, 265-275. Marsh, B.B., (1959) Biochim. Biophys. Acta,32, 357-361. Smith, C.G., Lummis, W.L., and Grady, J.E., (1959) Cancer Research,19, 843-846. Chiang, C.K., Yamauchi, F., and Montgomery, R. Unpublished work. Butters, T.D., and Hughes, R.C., (1975) Biochem. J.,l50, 59-69. Dorling, P.R., and Le Page, R.N., (1973) Biochim. Biophys. Acta,318, 33-44. Solomonson, L.P., Liepkalns, V.A., and Spector, A.A., (1976) Biochemistry, 15, 892-897.
876
