Vol. 70, No. 1, 1976
of
BIOCHEMICAL
an
Direct
Spectrophotometric
Acyl-Enzyme
Intermediate
Eves
J.
Breaux
Chemistry
March
RESEARCH COMMUNICATIONS
Observation in
and
and
Northwestern Received
AND BIOPHYSICAL
Elastase
Myron
L.
Biochemistry
University,
Catalysis
Bender
Departments
Evanston,
Illinois
60201
8,1976
SUMMARYin the catalytic action of the pancreatic An acyl-enzyme intermediate serine protease, elastase, was spectrophotometrically observed. 4-Dimethylaminocinnamoyl-elastase was prepared by the reaction of 4-dimethylaminocinnamoylimidazole with a 20 fold excess of elastase for 0.5 hr in pH 5.2 buffer. The visible absorption spectrum of this acyl-enzyme is similar to that of the analogous aldehyde but is red-shifted when compared to that of the denatured acyl-enzyme or a model ester. Our results are the first direct evidence for the intermediacy of an acyl-enzyme in elastase catalysis. Introduction The involve of
an
this
as
recent
This
data
involved
in
example,
substrates
it
based
in
of
ES’,
has
indicated
catalysis
by
been
found the
leaving
contrast
to
derived
from
the
same
case the
Copyright 0 I976 by Academic Press, Inc. Ail rights of reproduction in any form reserved.
same
but
ability
of
from the
chymotrypsin acid
but 235
a
and
upon
may
protease,
series
of
different alcohol
different
the
However,
intermediate
and from
intermediacy
(3).
serine for
to
acyl-enzymes
acyl-enzyme
kcat acid
of
evidence
cinnamoyl-trypsin an
thought
The
chromophoric
pancreatic that
group
the
and
is
1 (1).
kinetic of
that
catalysis
equation
indirect
upon
(2)
from the
protease
observation
has
derived
a function is
serine
intermediate, is
kinetic be
in
cinnamoyl-chymotrypsin
substrates as
acyl-enzyme
spectrophotometric
such
For
pathway
acyl-enzyme
direct
not
mechanistic
elastase elastase alcohols
(4,5). ester increased
(4,5). trypsin
where alcohols
ester undergo
BIOCHEMICAL
Vol. 70, No. 1, 1976
enzyme-catalyzed ing
hydrolysis
deacylation
of
the
with
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the
same
kcat
acyl-enzyme
common
RCOR’
t EnzOH
+
R-
to
the
intermediate
0 I1
0 ,I
due
rate-determinformed,
ES’.
0
k cat
C-OEnz
-t R’CH
-3
” R-COH
t EnzOH
(1)
ES’ One
explanation
leaving
offered
groups
is
displacement ion
hedral acy
that
upon
hydroxide
(4)
the
explain
the
ET,
, to
by
give
the
equation
*
product
+
bEnz
in
elastase
an
attempt catalysis,
intermediates
Materials
to
in
and
determine we
the
catal
ytic
a
an
enzymatic
second
tetra-
without
the
intermedi-
t R’OH
8 RCOH
-)
t EnzOH
(2)
ETz which
have
acid
03 R-k-OH CbEnz
ET1 In
by
of
alcohol
a nucleophilic
ETr,
decomposition
with
2.
HOt EnzOH
OI R-&-GR’
involves
intermediate,
followed
kcat
of
step
tetrahedral
acyl-enzyme,
8 RCOR’
variation
rate-determining
equivalent
intermediate, of an
the
to
of
tried
to
pathway.
these
two
pathways
is
spectrophotometrically Our
results
involved detect
are
discussed
below.
Methods
4-Dimethylaminocinnamoylimidazole was synthesized in a manner similar to that used by Bernhard, Lau, and Noller (6). Imidazole and dimethylaminocinnamic acid (K and K Labs) were coupled, using either dicyclohexylcarbodiimide or isobutyl chloroformate, in dimethylformamidetetrahydrofuran (1: 4 by volume) to give 4 -dimethylaminocinnamoylimidazole, m. p. 164-167. The halflife of 4-dimethylaminocinnamoylimidazole in 5.2 acetate buffer was 4. 5 hrs. Elastase was obtained from a twice crystallized product of Sigma Chemical Co. 4-Dimethylaminocinnamoyl-elastase was prepared by the reaction 4-dimethylaminocinnamoylimidazole with a 20 fold excess of elastase 0. 5 hr at pH5.2 (0. 83% acetonitrile). The denatured acyl-enzyme was pared by adding either 30 mg of solid sodium dodecyl sulfate to 3 ml the native a cyl-enzyme solution or by adding 1 ml of a 4% solution sodium dodecyl sulfate to 3 ml of the native acyl-enzyme. The differ-m
236
4-
pH
of for preof of ce
BIOCHEMICAL
Vol. 70, No. 1, 1976
spectra cells agent
of
were recorded by adding enzyme ;a Cary 14 recording spectrophotometer to the sample cell only.
Results
and If
istic
a common
by the
3
kz
permits
conditions
E
ES’
is
the
is
The
accumulation
the
spectrophotometric acylation
than
chromophoric
For acylate
deacylation
allowing
is
are
reaction use
of of
substrate functional
at
direct
This
a large
excess
and
made
Pr
is
acyl-
the
both can
slow be
ES’,
possible but
by
deacylation
achieved
by
the
useof
9 R-COEnz
(4)
low
with
(e- g. , a substrate group) f__,a,
pH
(2).
which was
However,
spectrophotometric
elastase
the
denatured acylenzyme 3a 3b
cinnamoylimidazole,
chymotrypsin
is
R=cinnamoyl R=dimethylaminocinnamoyl
a-acetamido
example,
be
substrate,
intermediate,
detection,
non-specific
an
the ES’
group,
acyl-enzyme
acyl-enzyme 2a 2b la,2a,3a lb,Zb,3b
is
can
(3)
leaving
(kz > ’ KS).
acylation
mechan-
iEtP,
alcohol the
the
catalysis
complex,
ks
of
and
la lb
possess
and sample acylating
the
in
S
binding
t P,
the
involved
enzyme,
denaturant ->
a
reference adding
chymotrypsin
deacylation
PI
wherein islower
trypsin,and
where
acylation
acid.
much
the and
is
Michaelis-Menten
intermediate,
which
is
equation
ZE’S
product
both
intermediate
elastase,
non-covalent
EtS
enzyme
acyl-enzyme then
described is
to
RESEARCH COMMUNICATIONS
Discussion
pathway,
E-S
AND BIOPHYSICAL
enzyme
found
for
237
to
of was
acylation
a good
the slow, to
does
leaving
not group.
s toichiometrically
deacylation
observation
cinnamoylimidazole of
has
which
was
very
The
acyl-enzyme. necessitating compete
slow,
the favorably
Vol. 70, No. 1, 1976
Figure
1.
with
Absorption Curve lb, Curve 2b, Curve 3b,
buffer
region
as
the
sorption
-
characteristics of the
enzyme.
The
2,
is
with
the the
which acyl-enzyme
in
indications
the
of
of
the
the
a model
mixture
that
of
a
mixture
ting
2b;
4)
the
native
for
chromophoric
to
sin
(2,6).
that
nm.
in
found
the
Therefore,
the nm)
in
observation the
same
we 2
acylimidazole, are:
spectral
used
as
, which of
presence
2 a,
1) the
denatured
the of
Table
acylation of
the
forms
enzyme
an
had
ab-
visible
a large
denatured
acyl-enzyme
had
I; was
3) if
238
a
excess
acyl-enzyme the
visible was
then
acyl-enzyme
the and
a spectral derivatives
hydrolysis
a similar
denaturant
complete,
exhibited acyl-enzyme
an
catalyzes
acyl-enzyme
ester,
before
resembled
lar
absorbs
of 4 -dimethylaminocinnamoyl-elastase,
elastase 2)
that
reaction
agent,
(------).
1. that
acylimidazole; to
280
400
spectrum
Figure
serine-195
spectrum
enzyme
permitted ( -
absorption
shown
The
the
derivatives. (. . . . . . . ). ().
4-dimethylaminocinnamoylimidazole,
spectrum
to
However,
cinnamoyl-enzyme,
agent,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
spectra of 4 -di methylaminocinnamoyl 4 dimethylaminocinnmoylimidazole native 4-dimethylaminocinnamoyl-elastase denatured 4-dimethylaminocinnamoyl-elastase
hydrolysis.
acylating
of
BIOCHEMICAL
added
spectrum the
acyla-
red-shift, of
chymotryp-
simi.
Vol. 70, No. 1, 1976
BIOCHEMICAL
TABLE
Spectral
I:
Absorption Derivatives.
AND BIOPHYSICAL
Data
RESEARCH COMMUNICATIONS
of 4-Dimethylaminocinnamoyl
xmax
E
4-Dimethylaminocinnamoylimidazole*
425
25200
Native
401
27060
4-Dimethylaminocinnamaldehyde’
397
31575
Denatured
370
23500
369
25100
332
16300
Derivative
4..dimethylaminocinnamoyl-elastase’
Ethyl
4-dimethylaminocinnamoyl-elastaseb
4-cIimethylaminocinnamatee
4-Dimethylaminocinnamic
a. b. c. d.
acidd
pH 5. 2 acetate pH 5. 2 acetate pH5.2 acetate pH 5. 2 acetate
The
most
buffer, buffer, buffer, buffer,
0. 0. 0. 0.
interesting
is that
it is
red-shifted
a model
ester,
Table
I.
and spectral
red-shift
“out
sit.e,
or
the acyl-enzyme
into
the carbonyl.
equivalent phoric sons
models
have
been
for
chromophoric
derivative 2) an “out
of the plane”
of
of the
acyl-enzyme,
the
spectra
perturbed
The
net result
aminocinnamaldehyde,
of
to the denatured explanations
offered
(7)
plane”
in
to explain
or
pair
upon a similar
such
protonat
a way that
the acyl-enzyme
The
prediction
to aldehydes
I. 239
or
x-ray spectral
1) the carbonyl
orbitals
4-dimethylaminocinnamoylelastase Table
based
acyl-enzyme
is that
or ketone. similar
acyl-enzyme
acyl-chymotrypsins:
the lone
are
are
of 4-dimethylaminocinnamoyl-
is hydrogen-bonded
of an aldehyde acyl-enzymes
of
relative possible
of the acyl
active
property
Two
observed
group
acetonitrile. acetonitrile, 1% sodium dodecyl sulfate. ethanol, 1% sodium dodecyl sulfate. dimethyl sulfoxide.
spectral
elastase
data
83% 83% 83% 83%
is
ed in the
formed. of the
they
In
alkyl
cannot
an oxygen
delocalize
is the electronic that the chromo-
is substantiated
by a compariand 4-dimethyl-
Vol. 70, No. 1,1976
In addition can also
acylate
BIOCHEMICAL
to acylating
elastase,
chymotrypsin,
results
indicate
that
strates
is mechanistically
4-dimethylaminocinnamoylimidazole
trypsin,
and other
the elastase-catalyzed similar
catalyzed
hydrolyses
in that
common
to all
enzymes.
common
acyl-enzyme
by these
three
three
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
hydrolysis
to that
is involved
acyl-enzyme are
now attempting
in the
hydrolysis
(8).
Our
of non-specific
of chymotrypsin-
demonstrable We
proteases
sub-
or trypsinintermediates to determine
of specific
are if
a
substrates
enzymes.
Acknowledgement This of
research
was
supported
by grants
from
the National
Institutes
Health. REFERENCES
1. Bender, M. L. and Killheffer, J. V. (1973), CRC Crit. Rev. Biochem. , 1, 149-199. 2. Bender, M. L. Schonbaum, G. R., and Zerner, B. (1962), J. Amer. Chem. sot. , 84, 2540-2 3. Bender, M. L. and Kaiser, E. T. (1962), ibid., 84, 2556-2561. 4. Kaplan, H. , Symonds, V. B. , Dugas, H. , and Whitaker, D. R. (1970), Can J. Chem. 48, 649-658. Y. (1975), Biochim. Biophys. Acta, 410, 421-425. 5. Gold, R. and Shalitin, 6. Bernhard, S.A., Lau, S. J., and Noller, H. (1965), Biochem., 4, 1108-1118. 7. Bernhard, S. A. and Lau, S. J. (1971), Cold Spring Harbor Syrnp. on &ant. The Cold Spring Harbor Laboratory, Cold pp. 75-83. Biol. , Vol. XXXVI, Spring Harbor, New York. 8. Breaux, E. J. and Bender, M. L. , unpublished data.
240
of
BIOCHEMICAL
an
Direct
Spectrophotometric
Acyl-Enzyme
Intermediate
Eves
J.
Breaux
Chemistry
March
RESEARCH COMMUNICATIONS
Observation in
and
and
Northwestern Received
AND BIOPHYSICAL
Elastase
Myron
L.
Biochemistry
University,
Catalysis
Bender
Departments
Evanston,
Illinois
60201
8,1976
SUMMARYin the catalytic action of the pancreatic An acyl-enzyme intermediate serine protease, elastase, was spectrophotometrically observed. 4-Dimethylaminocinnamoyl-elastase was prepared by the reaction of 4-dimethylaminocinnamoylimidazole with a 20 fold excess of elastase for 0.5 hr in pH 5.2 buffer. The visible absorption spectrum of this acyl-enzyme is similar to that of the analogous aldehyde but is red-shifted when compared to that of the denatured acyl-enzyme or a model ester. Our results are the first direct evidence for the intermediacy of an acyl-enzyme in elastase catalysis. Introduction The involve of
an
this
as
recent
This
data
involved
in
example,
substrates
it
based
in
of
ES’,
has
indicated
catalysis
by
been
found the
leaving
contrast
to
derived
from
the
same
case the
Copyright 0 I976 by Academic Press, Inc. Ail rights of reproduction in any form reserved.
same
but
ability
of
from the
chymotrypsin acid
but 235
a
and
upon
may
protease,
series
of
different alcohol
different
the
However,
intermediate
and from
intermediacy
(3).
serine for
to
acyl-enzymes
acyl-enzyme
kcat acid
of
evidence
cinnamoyl-trypsin an
thought
The
chromophoric
pancreatic that
group
the
and
is
1 (1).
kinetic of
that
catalysis
equation
indirect
upon
(2)
from the
protease
observation
has
derived
a function is
serine
intermediate, is
kinetic be
in
cinnamoyl-chymotrypsin
substrates as
acyl-enzyme
spectrophotometric
such
For
pathway
acyl-enzyme
direct
not
mechanistic
elastase elastase alcohols
(4,5). ester increased
(4,5). trypsin
where alcohols
ester undergo
BIOCHEMICAL
Vol. 70, No. 1, 1976
enzyme-catalyzed ing
hydrolysis
deacylation
of
the
with
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the
same
kcat
acyl-enzyme
common
RCOR’
t EnzOH
+
R-
to
the
intermediate
0 I1
0 ,I
due
rate-determinformed,
ES’.
0
k cat
C-OEnz
-t R’CH
-3
” R-COH
t EnzOH
(1)
ES’ One
explanation
leaving
offered
groups
is
displacement ion
hedral acy
that
upon
hydroxide
(4)
the
explain
the
ET,
, to
by
give
the
equation
*
product
+
bEnz
in
elastase
an
attempt catalysis,
intermediates
Materials
to
in
and
determine we
the
catal
ytic
a
an
enzymatic
second
tetra-
without
the
intermedi-
t R’OH
8 RCOH
-)
t EnzOH
(2)
ETz which
have
acid
03 R-k-OH CbEnz
ET1 In
by
of
alcohol
a nucleophilic
ETr,
decomposition
with
2.
HOt EnzOH
OI R-&-GR’
involves
intermediate,
followed
kcat
of
step
tetrahedral
acyl-enzyme,
8 RCOR’
variation
rate-determining
equivalent
intermediate, of an
the
to
of
tried
to
pathway.
these
two
pathways
is
spectrophotometrically Our
results
involved detect
are
discussed
below.
Methods
4-Dimethylaminocinnamoylimidazole was synthesized in a manner similar to that used by Bernhard, Lau, and Noller (6). Imidazole and dimethylaminocinnamic acid (K and K Labs) were coupled, using either dicyclohexylcarbodiimide or isobutyl chloroformate, in dimethylformamidetetrahydrofuran (1: 4 by volume) to give 4 -dimethylaminocinnamoylimidazole, m. p. 164-167. The halflife of 4-dimethylaminocinnamoylimidazole in 5.2 acetate buffer was 4. 5 hrs. Elastase was obtained from a twice crystallized product of Sigma Chemical Co. 4-Dimethylaminocinnamoyl-elastase was prepared by the reaction 4-dimethylaminocinnamoylimidazole with a 20 fold excess of elastase 0. 5 hr at pH5.2 (0. 83% acetonitrile). The denatured acyl-enzyme was pared by adding either 30 mg of solid sodium dodecyl sulfate to 3 ml the native a cyl-enzyme solution or by adding 1 ml of a 4% solution sodium dodecyl sulfate to 3 ml of the native acyl-enzyme. The differ-m
236
4-
pH
of for preof of ce
BIOCHEMICAL
Vol. 70, No. 1, 1976
spectra cells agent
of
were recorded by adding enzyme ;a Cary 14 recording spectrophotometer to the sample cell only.
Results
and If
istic
a common
by the
3
kz
permits
conditions
E
ES’
is
the
is
The
accumulation
the
spectrophotometric acylation
than
chromophoric
For acylate
deacylation
allowing
is
are
reaction use
of of
substrate functional
at
direct
This
a large
excess
and
made
Pr
is
acyl-
the
both can
slow be
ES’,
possible but
by
deacylation
achieved
by
the
useof
9 R-COEnz
(4)
low
with
(e- g. , a substrate group) f__,a,
pH
(2).
which was
However,
spectrophotometric
elastase
the
denatured acylenzyme 3a 3b
cinnamoylimidazole,
chymotrypsin
is
R=cinnamoyl R=dimethylaminocinnamoyl
a-acetamido
example,
be
substrate,
intermediate,
detection,
non-specific
an
the ES’
group,
acyl-enzyme
acyl-enzyme 2a 2b la,2a,3a lb,Zb,3b
is
can
(3)
leaving
(kz > ’ KS).
acylation
mechan-
iEtP,
alcohol the
the
catalysis
complex,
ks
of
and
la lb
possess
and sample acylating
the
in
S
binding
t P,
the
involved
enzyme,
denaturant ->
a
reference adding
chymotrypsin
deacylation
PI
wherein islower
trypsin,and
where
acylation
acid.
much
the and
is
Michaelis-Menten
intermediate,
which
is
equation
ZE’S
product
both
intermediate
elastase,
non-covalent
EtS
enzyme
acyl-enzyme then
described is
to
RESEARCH COMMUNICATIONS
Discussion
pathway,
E-S
AND BIOPHYSICAL
enzyme
found
for
237
to
of was
acylation
a good
the slow, to
does
leaving
not group.
s toichiometrically
deacylation
observation
cinnamoylimidazole of
has
which
was
very
The
acyl-enzyme. necessitating compete
slow,
the favorably
Vol. 70, No. 1, 1976
Figure
1.
with
Absorption Curve lb, Curve 2b, Curve 3b,
buffer
region
as
the
sorption
-
characteristics of the
enzyme.
The
2,
is
with
the the
which acyl-enzyme
in
indications
the
of
of
the
the
a model
mixture
that
of
a
mixture
ting
2b;
4)
the
native
for
chromophoric
to
sin
(2,6).
that
nm.
in
found
the
Therefore,
the nm)
in
observation the
same
we 2
acylimidazole, are:
spectral
used
as
, which of
presence
2 a,
1) the
denatured
the of
Table
acylation of
the
forms
enzyme
an
had
ab-
visible
a large
denatured
acyl-enzyme
had
I; was
3) if
238
a
excess
acyl-enzyme the
visible was
then
acyl-enzyme
the and
a spectral derivatives
hydrolysis
a similar
denaturant
complete,
exhibited acyl-enzyme
an
catalyzes
acyl-enzyme
ester,
before
resembled
lar
absorbs
of 4 -dimethylaminocinnamoyl-elastase,
elastase 2)
that
reaction
agent,
(------).
1. that
acylimidazole; to
280
400
spectrum
Figure
serine-195
spectrum
enzyme
permitted ( -
absorption
shown
The
the
derivatives. (. . . . . . . ). ().
4-dimethylaminocinnamoylimidazole,
spectrum
to
However,
cinnamoyl-enzyme,
agent,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
spectra of 4 -di methylaminocinnamoyl 4 dimethylaminocinnmoylimidazole native 4-dimethylaminocinnamoyl-elastase denatured 4-dimethylaminocinnamoyl-elastase
hydrolysis.
acylating
of
BIOCHEMICAL
added
spectrum the
acyla-
red-shift, of
chymotryp-
simi.
Vol. 70, No. 1, 1976
BIOCHEMICAL
TABLE
Spectral
I:
Absorption Derivatives.
AND BIOPHYSICAL
Data
RESEARCH COMMUNICATIONS
of 4-Dimethylaminocinnamoyl
xmax
E
4-Dimethylaminocinnamoylimidazole*
425
25200
Native
401
27060
4-Dimethylaminocinnamaldehyde’
397
31575
Denatured
370
23500
369
25100
332
16300
Derivative
4..dimethylaminocinnamoyl-elastase’
Ethyl
4-dimethylaminocinnamoyl-elastaseb
4-cIimethylaminocinnamatee
4-Dimethylaminocinnamic
a. b. c. d.
acidd
pH 5. 2 acetate pH 5. 2 acetate pH5.2 acetate pH 5. 2 acetate
The
most
buffer, buffer, buffer, buffer,
0. 0. 0. 0.
interesting
is that
it is
red-shifted
a model
ester,
Table
I.
and spectral
red-shift
“out
sit.e,
or
the acyl-enzyme
into
the carbonyl.
equivalent phoric sons
models
have
been
for
chromophoric
derivative 2) an “out
of the plane”
of
of the
acyl-enzyme,
the
spectra
perturbed
The
net result
aminocinnamaldehyde,
of
to the denatured explanations
offered
(7)
plane”
in
to explain
or
pair
upon a similar
such
protonat
a way that
the acyl-enzyme
The
prediction
to aldehydes
I. 239
or
x-ray spectral
1) the carbonyl
orbitals
4-dimethylaminocinnamoylelastase Table
based
acyl-enzyme
is that
or ketone. similar
acyl-enzyme
acyl-chymotrypsins:
the lone
are
are
of 4-dimethylaminocinnamoyl-
is hydrogen-bonded
of an aldehyde acyl-enzymes
of
relative possible
of the acyl
active
property
Two
observed
group
acetonitrile. acetonitrile, 1% sodium dodecyl sulfate. ethanol, 1% sodium dodecyl sulfate. dimethyl sulfoxide.
spectral
elastase
data
83% 83% 83% 83%
is
ed in the
formed. of the
they
In
alkyl
cannot
an oxygen
delocalize
is the electronic that the chromo-
is substantiated
by a compariand 4-dimethyl-
Vol. 70, No. 1,1976
In addition can also
acylate
BIOCHEMICAL
to acylating
elastase,
chymotrypsin,
results
indicate
that
strates
is mechanistically
4-dimethylaminocinnamoylimidazole
trypsin,
and other
the elastase-catalyzed similar
catalyzed
hydrolyses
in that
common
to all
enzymes.
common
acyl-enzyme
by these
three
three
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
hydrolysis
to that
is involved
acyl-enzyme are
now attempting
in the
hydrolysis
(8).
Our
of non-specific
of chymotrypsin-
demonstrable We
proteases
sub-
or trypsinintermediates to determine
of specific
are if
a
substrates
enzymes.
Acknowledgement This of
research
was
supported
by grants
from
the National
Institutes
Health. REFERENCES
1. Bender, M. L. and Killheffer, J. V. (1973), CRC Crit. Rev. Biochem. , 1, 149-199. 2. Bender, M. L. Schonbaum, G. R., and Zerner, B. (1962), J. Amer. Chem. sot. , 84, 2540-2 3. Bender, M. L. and Kaiser, E. T. (1962), ibid., 84, 2556-2561. 4. Kaplan, H. , Symonds, V. B. , Dugas, H. , and Whitaker, D. R. (1970), Can J. Chem. 48, 649-658. Y. (1975), Biochim. Biophys. Acta, 410, 421-425. 5. Gold, R. and Shalitin, 6. Bernhard, S.A., Lau, S. J., and Noller, H. (1965), Biochem., 4, 1108-1118. 7. Bernhard, S. A. and Lau, S. J. (1971), Cold Spring Harbor Syrnp. on &ant. The Cold Spring Harbor Laboratory, Cold pp. 75-83. Biol. , Vol. XXXVI, Spring Harbor, New York. 8. Breaux, E. J. and Bender, M. L. , unpublished data.
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