Vol. 67, No. 1, 1975
BIOCHEMICAL
SITES
AND BIOPHYSICAL
OF CLEAVAGE
OF GLUCAGON
INSULIN-GLUCAGON
Frankie
RESEARCH COMMUNICATIONS
BY
PROTEASE
K. Baskin, William C. IX&worth, and Abbas E. Kitabchi
Departments of Medicine and Biochemistry University of Tennessee Center for the Health Sciences and Veterans’ Administration Hospital Memphis, Tennessee 38163 Received
August
22,1975 Summary
To study the mechanism of degradation of glucagon with purified insulin-glucagon The protease, glucagon was reacted with the enzyme at various times of incubation. proteolysis was followed by the production of flourescamine-reacting material as well as reaction with dansyl chloride, cleavage by acid hydrolysis, and identification by thin layer chromatography. For quantitative measurement of the degradation products, [14cl dansyl derivatives were produced, identified by autoradiography, and counted. In the degradation products in addition to histidine, the dansyl derivatives of For comtyrosine, phenylalanine, two leucines, alanine and lysine were identified. parison, glucagon was also reacted with chymotrypsin and the degradation products consisted of threonine, serine, two leucines and valine. Thus, insulin-glucagon protease degrades glucagon in a manner distinct from that of chymotrypsin.
Int reduction
An enzyme lated
from
skeletal
procedures
prolonged a more minutes)
can degrade muscle
including
The proteolytic crease
which
in ninhydrin
and direct
This
(18-24
enzyme
chromatography
of the enzyme
positive
incubation sensitive
(1).
affinity
nature
both insulin
materials hours)
has been previously
has been highly
purified
and shown to be a single
has been shown by its ability when incubated to produce
assay for proteolysis proteolysis
and glucagon
of insulin
with
a significant
with fluorescamine, and glucagon
insulin
iso-
by a series enzyme
(2).
to produce but this
increase the rapid
by the purified
(3).
of
an inrequired
Using
(within enzyme
2
Vol. 67, No. 1, 1975
BIOCHEMICAL
has been demonstrated two peptides
The degradation
growth
as measured
products
Glucagon
higher
V,,
tained
for study.
mole
The insulin-glucagon
protease
purified
as described
the final
step of affinity
pH 6.2 the enzyme The enzyme
column.
Glucagon
Indianapolis,
because
of the
could be ob-
and a quantitative
the amino
end groups
of the
Brie1 (5).
The amino
purchased
from
Pierce from
Schleicher
For the study of glucagon
against
chloride
O.OlM
phosphate,
over a small
purchased
was substituted handled,
Company,
from
and Schuell,
Inc.,
cellulose
the incubation 164
powder
and Company, with
dansyl
as described ammo
Illinois.
Kenne,
NaCl-0.02M
Schwartz/Mann
dansylated
Rockford,
on the
pH 7.5 for
for unlabeled
and stored
and individual
degradation,
enzyme,
by 0.2M
agarose
was the gift of the Eli Lilly
was diluted,
Chemical
of the purified
and
the insulin
was then passed
acid mixture
muscle
was performed
was dialyzed
of 100 PC/~ mole
rat skeletal
on insulin-agarose from
14C-dansyl
chloride
from
Due to instability
(lot #258-030-138-4)
activity
obtained
elution
preparation
Indiana.
The 14C-dansyl
(2).
chromatography After
3 hours,
were
with its
study was
of material
a qualitative
(4).
and Methods
was prepared
previously
day of the experiment.
a specific
deriv-
of this enzyme
amount
A
product.
Materials
acetate,
insulin
reactivity
to be studied
range was used to identify
for these
and collagen
the following
substrate
offers
including
in fluorescamine
this reason,
which
specific
albumin,
the reaction
and thus a greater
14C dansylation
in the lo-l4
degradation
For
as the initial
of this reaction
and proteins
hormone,
from
RESEARCH COMMUNICATIONS
to be highly
by changes
resulting
was chosen
appears
of peptides
have not been identified.
done.
method
ACTH,
not degraded
enzyme
number
proinsulin,
were
substrates
This
in that a large
and B chains, atives
(4).
AND BIOPHYSICAL
chloride. by
acids were
Polyamide
sheets
New Hampshire. mixture
consisted
of
Vol. 67, No. 1, 1975
the following:
BIOCHEMICAL
100 pg of enzyme
glucagon
in concentration
reaction
mixture
intervals
by the method
incubation
with fluorescamine
of glucagon
in 100 ~1 of distilled placed
H20.
in a 3 mm x 30 mm tube.
followed
by 25 111of 14C-dansyl
9.5 with
Na2C03.
drying,
dessicator.
50 J of 6.7N
tubes were reaction
sealed
mixture
polymide
under
(1:l
v/v)
On the back side of the plate, spotted.
The plates
and formic consisting
were
acid (10011.5 of benzene:
resolution
of glutamic
a-histidine,
a third
(3:l
acid, solvent
which
employed
des-
to identify
165
dissolved
to
in a
in a vacuum The The
these amino
syringe.
acids were
in a second
of water system
angle to the first
and C-lysine,
of 0.05M
in 1 ~1 of pyri-
consisting
UV light.
of ethyl
coated
a Hamilton ammo
in a solution
cu-lysine
system
was adjusted
for acid hydrolysis.
dansylated
under
consisting
residue,
and incubated
sheets with
was at a right
acid,
solvent
in a vacuum
was dried
were
by chromatography
and identified
system
and
on a 3 x 3 double
products
on the thin layer
aspartic
syringe
in a 105” oven for 18 hours.
and chromatographed
acid,
localized
v/v) were
and hydrolyzed
followed
acid (20: 1:2, v/v) and a fourth ethanol
with parafilm the mixture
then chromatographed
acetic
The spots were
time,
1 ml of an
was added to the dried
the non-radioactive
v/v)
at
and then redissolved
The pH of the mixture
The dansylated
and spotted
(6).
was then removed
then sealed
which
vacuum
plate.
removed
of the reaction
with a Hamilton
HCl was added to the residue
was then dried
thin layer
dine water
after
The
5 ml.
were
to that of Gray
Na2C03
chloride.
The tubes were
bath for 1 hour,
Aliquots
was lyophylized
The water
15 ~1 of 0.03M
37” water
tion.
was similar
25 4 was then removed
After
volume
to assay for completeness
and enzyme
sicator.
pH 7.5,
(4).
used for dansylation
mixture
buffer,
incubation,
at 37” for 60 min.
previously
RESEARCH COMMUNICATIONS
in 0. 01M phosphate
of 4.14 x 10m5M in total
described
The method
protein
was then incubated
for reaction
AND BIOPHYSICAL
direc-
For further arginine
acetate-methanol-acetic trisodium acids.
phosphate:
and
Vol. 67, No. 1, 1975
Figure
1:
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Chromatography of Dns-amino acids on polyamide thin layer plates. The direction of chromatography is indicated by the arrows. 1st direction: water/formic acid 100:3; 2nd direction: benzene/acetic acid 9:l. The numbers on the chromatogram represent the dansylated amino acids for the 60 minute incubation as follows: (1) Starting point (2) Dns-OH (3) cu-Dns-Histidine and C-Dns-C-Lysine (4) O-DnsTyrosine (5) di-Dns-Lysine (6) Dns-Alanine (7) Dns-NH2 (8) DnsPhenylalanine (9) di-Dns-Tyrosine (10) Dns-Leucine.
t Figure
2:
The method of chromatography was the same as in Figure 1. The numbers represent the following dansylated amino acids present in the initial incubation mixture. (1) Starting point (2) Dns-OH (3) CYDns-Histidine and C-Dns-Lysine (4) O-Dns-Tyrosine.
166
Vol. 67, No. 1, 1975
BIOCHEMICAL
The individual thin layer cut from
plate
shaking.
Liquiflourtoluene
X-ray
at room
scintillation
was then exposed
scintillation
plates
The residue solution
RESEARCH COMMUNICATIONS
dansyl
temperature
of the thin layer the vials.
film
The labeled
in individual
chloroform
from
of dental
of 1 day.
and placed
The segments
evaporated
packet
for a period
the plates
3 ml of redistilled
form
poly-soft
AND BIOPHYSICAL
amino vials
for
were
acids
to the were
then
and each eluted
with
1 hour with frequent
removed
and the chloro-
was then dissolved
(42: 1000) and counted
in 10 ml of
in a Searle
Isocap/300
with glucagon
resulted
spectrometer.
Results
The incubation in the appearance
Figure
3:
of the purified
and Discussion
insulin-glucagon
of five new N-terminal
amino
protease acids:
tyrosine,
leucine,
lysine,
The reference standard was spotted on the back of the double coated polyamide plates. The numbers represent the following Dansylated amino acids: (1) Starting point (2) and (3) Dns-OH (4) Dns-Arginine and cu-Dns-Histidine, C-Dns-Lysine, cu-Dns-Lysine (5) Dns-Glutamic acid (6) Dns-Serine (7) Dns-Glycine (8) Dns-Alanine (9) Dns-NH2 (10) Dns-Phe (11) di-Dns-Tyrosine (12) Dns-Isoleucine (13) Dns-Proline.
167
Vol. 67, No. 1, 1975
BIOCHEMICAL
AND BIOPHYSICAL
TABLE
FORMATION
OF [14CI-DANSYL
MIXTURE
COUNTS
QJ-Histidine
I
AMINO
OF INSULIN-GLUCAGON
ACIDS
FROM
PROTEASE
WITH
GLUCAGON
RATIO
T60
5,300
5,501
800
0-Tyrosine
INCUBATION
PER MINUTE
TO
C -Lysine
RESEARCH COMMUNICATIONS
Amino Acid o-Histidine 1.00
150
10,505
5,543
1.01
Leucine
0
10,450
1.90
di-Lysine
0
10,289
0.94
di-Tyrosine
0
10,337
0.94
Alanine
0
5,413
0.98
Phenylalanine
0
5,376
0.98
alanine,
and phenylalanine
at zero
time
(Figure
standard
pared
with the N-terminal
(Figure
tion of 2 tyrosines,
These material
results
products
3).
histidine
that 6 peptide
is produced
indicated,
1 lysine,
1 alanine,
bonds
in glucagon
of the enzyme increase
by the enzyme’s
over basal reaction
168
dansyl
mixture
amino
derivative
as shown in Table
com-
I, forma-
and 1 phenylalanine. are cleaved
of the production with glucagon.
incubation
dansylated
of the l4 C-labeled
group
agree with the studies
one, a six-fold
to the control
to the non-radioactive
Quantitation
2 leucines,
by incubation
in the present
1) as compared
2) and compared
acids
Thus it appears
(Figure
by this enzyme.
of fluorescamine-reactive In previous
value
studies
(4) and
in fluorescamine-reactive
with glucagon,
following
which
Vol. 67, No. 1, 1975
increase,
a plateau
of glucagon
BIOCHEMICAL
is reached.
are ser-lys
and either
thr-phe
These
(ll-12),
(5-6)
AND BIOPHYSICAL
results
tyr-leu
or asp-phe
suggest
(13-14),
(21-22)
RESEARCH COMMUNICATIONS
that the sites
arg-ala
and either
(18-19),
asp-tyr
of cleavage
trp-leu
(9-10)
(25-26)
or lys-tyr
(12-13). Using
the same procedure,
shown)
and as previously
were
identified
sites
distinct
These which parent
from
with
chymotrypsin
serine,
2 leucines,
Thus
this enzyme
acids.
(data not and valine clearly
attacks
by chymotrypsin.
are also different
glucagon
specificity
being
amino
those cleaved
sites of cleavage
marked
was reacted
shown (7), threonine,
as new N-terminal
can degrade
currently
glucagon
(7).
These
results,
of the enzyme
made to identify
from
other however,
for glucagon
the sites
proteolytic
enzymes
do not explain
and insulin.
of cleavage
the ap-
Attempts
are
of insulin.
Acknowledgements
This work Grant
was supported
AM 15509,
No. 4966-01.
General
in part Clinical
The authors
by USPHS Training Research
are grateful
Center
Grant Grant
AM 05497,
RR 00211,
to M. Heinemann
and M.A.
Research
and VA Project Babal for their
assistance. References 1.
Duckworth,
W. C. and Kitabchi,
2.
Duckworth, Acad. Sci.
W. C., Heinemann, USA 69, 3698-3702
3.
Burghen, 633-642
4.
Duckworth, W. C., Heinemann, M.A. Biophys. Acta 377, 421-430 (1975).
5.
Briel,
6.
Gray, W.R. Methods in Enzymology 11, pp. 139-151, (ed. ), Academic Press, Inc., New York (1967).
7.
Bromer, Pergamon
G. A., (1972).
Kitabchi,
G. and Neuhoff,
A. E.
Diabetes
M.A. and Kitabchi, (1972).
A. E. and Brush,
V.
23, 536-543
Physiol.
J. S.
A. E.
W. W. Glucagon. pp. 27-30, Press, New York (1972). 169
Proc.
Endocrinology
and Kitabchi,
Chem.
(1974).
A.E.
353, 540-553
P. J. Lefebvre
Nat.
91,
Biochim.
(1972). Sidney
P. Colowick
and R. H. Unger
(eds. ),
BIOCHEMICAL
SITES
AND BIOPHYSICAL
OF CLEAVAGE
OF GLUCAGON
INSULIN-GLUCAGON
Frankie
RESEARCH COMMUNICATIONS
BY
PROTEASE
K. Baskin, William C. IX&worth, and Abbas E. Kitabchi
Departments of Medicine and Biochemistry University of Tennessee Center for the Health Sciences and Veterans’ Administration Hospital Memphis, Tennessee 38163 Received
August
22,1975 Summary
To study the mechanism of degradation of glucagon with purified insulin-glucagon The protease, glucagon was reacted with the enzyme at various times of incubation. proteolysis was followed by the production of flourescamine-reacting material as well as reaction with dansyl chloride, cleavage by acid hydrolysis, and identification by thin layer chromatography. For quantitative measurement of the degradation products, [14cl dansyl derivatives were produced, identified by autoradiography, and counted. In the degradation products in addition to histidine, the dansyl derivatives of For comtyrosine, phenylalanine, two leucines, alanine and lysine were identified. parison, glucagon was also reacted with chymotrypsin and the degradation products consisted of threonine, serine, two leucines and valine. Thus, insulin-glucagon protease degrades glucagon in a manner distinct from that of chymotrypsin.
Int reduction
An enzyme lated
from
skeletal
procedures
prolonged a more minutes)
can degrade muscle
including
The proteolytic crease
which
in ninhydrin
and direct
This
(18-24
enzyme
chromatography
of the enzyme
positive
incubation sensitive
(1).
affinity
nature
both insulin
materials hours)
has been previously
has been highly
purified
and shown to be a single
has been shown by its ability when incubated to produce
assay for proteolysis proteolysis
and glucagon
of insulin
with
a significant
with fluorescamine, and glucagon
insulin
iso-
by a series enzyme
(2).
to produce but this
increase the rapid
by the purified
(3).
of
an inrequired
Using
(within enzyme
2
Vol. 67, No. 1, 1975
BIOCHEMICAL
has been demonstrated two peptides
The degradation
growth
as measured
products
Glucagon
higher
V,,
tained
for study.
mole
The insulin-glucagon
protease
purified
as described
the final
step of affinity
pH 6.2 the enzyme The enzyme
column.
Glucagon
Indianapolis,
because
of the
could be ob-
and a quantitative
the amino
end groups
of the
Brie1 (5).
The amino
purchased
from
Pierce from
Schleicher
For the study of glucagon
against
chloride
O.OlM
phosphate,
over a small
purchased
was substituted handled,
Company,
from
and Schuell,
Inc.,
cellulose
the incubation 164
powder
and Company, with
dansyl
as described ammo
Illinois.
Kenne,
NaCl-0.02M
Schwartz/Mann
dansylated
Rockford,
on the
pH 7.5 for
for unlabeled
and stored
and individual
degradation,
enzyme,
by 0.2M
agarose
was the gift of the Eli Lilly
was diluted,
Chemical
of the purified
and
the insulin
was then passed
acid mixture
muscle
was performed
was dialyzed
of 100 PC/~ mole
rat skeletal
on insulin-agarose from
14C-dansyl
chloride
from
Due to instability
(lot #258-030-138-4)
activity
obtained
elution
preparation
Indiana.
The 14C-dansyl
(2).
chromatography After
3 hours,
were
with its
study was
of material
a qualitative
(4).
and Methods
was prepared
previously
day of the experiment.
a specific
deriv-
of this enzyme
amount
A
product.
Materials
acetate,
insulin
reactivity
to be studied
range was used to identify
for these
and collagen
the following
substrate
offers
including
in fluorescamine
this reason,
which
specific
albumin,
the reaction
and thus a greater
14C dansylation
in the lo-l4
degradation
For
as the initial
of this reaction
and proteins
hormone,
from
RESEARCH COMMUNICATIONS
to be highly
by changes
resulting
was chosen
appears
of peptides
have not been identified.
done.
method
ACTH,
not degraded
enzyme
number
proinsulin,
were
substrates
This
in that a large
and B chains, atives
(4).
AND BIOPHYSICAL
chloride. by
acids were
Polyamide
sheets
New Hampshire. mixture
consisted
of
Vol. 67, No. 1, 1975
the following:
BIOCHEMICAL
100 pg of enzyme
glucagon
in concentration
reaction
mixture
intervals
by the method
incubation
with fluorescamine
of glucagon
in 100 ~1 of distilled placed
H20.
in a 3 mm x 30 mm tube.
followed
by 25 111of 14C-dansyl
9.5 with
Na2C03.
drying,
dessicator.
50 J of 6.7N
tubes were reaction
sealed
mixture
polymide
under
(1:l
v/v)
On the back side of the plate, spotted.
The plates
and formic consisting
were
acid (10011.5 of benzene:
resolution
of glutamic
a-histidine,
a third
(3:l
acid, solvent
which
employed
des-
to identify
165
dissolved
to
in a
in a vacuum The The
these amino
syringe.
acids were
in a second
of water system
angle to the first
and C-lysine,
of 0.05M
in 1 ~1 of pyri-
consisting
UV light.
of ethyl
coated
a Hamilton ammo
in a solution
cu-lysine
system
was adjusted
for acid hydrolysis.
dansylated
under
consisting
residue,
and incubated
sheets with
was at a right
acid,
solvent
in a vacuum
was dried
were
by chromatography
and identified
system
and
on a 3 x 3 double
products
on the thin layer
aspartic
syringe
in a 105” oven for 18 hours.
and chromatographed
acid,
localized
v/v) were
and hydrolyzed
followed
acid (20: 1:2, v/v) and a fourth ethanol
with parafilm the mixture
then chromatographed
acetic
The spots were
time,
1 ml of an
was added to the dried
the non-radioactive
v/v)
at
and then redissolved
The pH of the mixture
The dansylated
and spotted
(6).
was then removed
then sealed
which
vacuum
plate.
removed
of the reaction
with a Hamilton
HCl was added to the residue
was then dried
thin layer
dine water
after
The
5 ml.
were
to that of Gray
Na2C03
chloride.
The tubes were
bath for 1 hour,
Aliquots
was lyophylized
The water
15 ~1 of 0.03M
37” water
tion.
was similar
25 4 was then removed
After
volume
to assay for completeness
and enzyme
sicator.
pH 7.5,
(4).
used for dansylation
mixture
buffer,
incubation,
at 37” for 60 min.
previously
RESEARCH COMMUNICATIONS
in 0. 01M phosphate
of 4.14 x 10m5M in total
described
The method
protein
was then incubated
for reaction
AND BIOPHYSICAL
direc-
For further arginine
acetate-methanol-acetic trisodium acids.
phosphate:
and
Vol. 67, No. 1, 1975
Figure
1:
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Chromatography of Dns-amino acids on polyamide thin layer plates. The direction of chromatography is indicated by the arrows. 1st direction: water/formic acid 100:3; 2nd direction: benzene/acetic acid 9:l. The numbers on the chromatogram represent the dansylated amino acids for the 60 minute incubation as follows: (1) Starting point (2) Dns-OH (3) cu-Dns-Histidine and C-Dns-C-Lysine (4) O-DnsTyrosine (5) di-Dns-Lysine (6) Dns-Alanine (7) Dns-NH2 (8) DnsPhenylalanine (9) di-Dns-Tyrosine (10) Dns-Leucine.
t Figure
2:
The method of chromatography was the same as in Figure 1. The numbers represent the following dansylated amino acids present in the initial incubation mixture. (1) Starting point (2) Dns-OH (3) CYDns-Histidine and C-Dns-Lysine (4) O-Dns-Tyrosine.
166
Vol. 67, No. 1, 1975
BIOCHEMICAL
The individual thin layer cut from
plate
shaking.
Liquiflourtoluene
X-ray
at room
scintillation
was then exposed
scintillation
plates
The residue solution
RESEARCH COMMUNICATIONS
dansyl
temperature
of the thin layer the vials.
film
The labeled
in individual
chloroform
from
of dental
of 1 day.
and placed
The segments
evaporated
packet
for a period
the plates
3 ml of redistilled
form
poly-soft
AND BIOPHYSICAL
amino vials
for
were
acids
to the were
then
and each eluted
with
1 hour with frequent
removed
and the chloro-
was then dissolved
(42: 1000) and counted
in 10 ml of
in a Searle
Isocap/300
with glucagon
resulted
spectrometer.
Results
The incubation in the appearance
Figure
3:
of the purified
and Discussion
insulin-glucagon
of five new N-terminal
amino
protease acids:
tyrosine,
leucine,
lysine,
The reference standard was spotted on the back of the double coated polyamide plates. The numbers represent the following Dansylated amino acids: (1) Starting point (2) and (3) Dns-OH (4) Dns-Arginine and cu-Dns-Histidine, C-Dns-Lysine, cu-Dns-Lysine (5) Dns-Glutamic acid (6) Dns-Serine (7) Dns-Glycine (8) Dns-Alanine (9) Dns-NH2 (10) Dns-Phe (11) di-Dns-Tyrosine (12) Dns-Isoleucine (13) Dns-Proline.
167
Vol. 67, No. 1, 1975
BIOCHEMICAL
AND BIOPHYSICAL
TABLE
FORMATION
OF [14CI-DANSYL
MIXTURE
COUNTS
QJ-Histidine
I
AMINO
OF INSULIN-GLUCAGON
ACIDS
FROM
PROTEASE
WITH
GLUCAGON
RATIO
T60
5,300
5,501
800
0-Tyrosine
INCUBATION
PER MINUTE
TO
C -Lysine
RESEARCH COMMUNICATIONS
Amino Acid o-Histidine 1.00
150
10,505
5,543
1.01
Leucine
0
10,450
1.90
di-Lysine
0
10,289
0.94
di-Tyrosine
0
10,337
0.94
Alanine
0
5,413
0.98
Phenylalanine
0
5,376
0.98
alanine,
and phenylalanine
at zero
time
(Figure
standard
pared
with the N-terminal
(Figure
tion of 2 tyrosines,
These material
results
products
3).
histidine
that 6 peptide
is produced
indicated,
1 lysine,
1 alanine,
bonds
in glucagon
of the enzyme increase
by the enzyme’s
over basal reaction
168
dansyl
mixture
amino
derivative
as shown in Table
com-
I, forma-
and 1 phenylalanine. are cleaved
of the production with glucagon.
incubation
dansylated
of the l4 C-labeled
group
agree with the studies
one, a six-fold
to the control
to the non-radioactive
Quantitation
2 leucines,
by incubation
in the present
1) as compared
2) and compared
acids
Thus it appears
(Figure
by this enzyme.
of fluorescamine-reactive In previous
value
studies
(4) and
in fluorescamine-reactive
with glucagon,
following
which
Vol. 67, No. 1, 1975
increase,
a plateau
of glucagon
BIOCHEMICAL
is reached.
are ser-lys
and either
thr-phe
These
(ll-12),
(5-6)
AND BIOPHYSICAL
results
tyr-leu
or asp-phe
suggest
(13-14),
(21-22)
RESEARCH COMMUNICATIONS
that the sites
arg-ala
and either
(18-19),
asp-tyr
of cleavage
trp-leu
(9-10)
(25-26)
or lys-tyr
(12-13). Using
the same procedure,
shown)
and as previously
were
identified
sites
distinct
These which parent
from
with
chymotrypsin
serine,
2 leucines,
Thus
this enzyme
acids.
(data not and valine clearly
attacks
by chymotrypsin.
are also different
glucagon
specificity
being
amino
those cleaved
sites of cleavage
marked
was reacted
shown (7), threonine,
as new N-terminal
can degrade
currently
glucagon
(7).
These
results,
of the enzyme
made to identify
from
other however,
for glucagon
the sites
proteolytic
enzymes
do not explain
and insulin.
of cleavage
the ap-
Attempts
are
of insulin.
Acknowledgements
This work Grant
was supported
AM 15509,
No. 4966-01.
General
in part Clinical
The authors
by USPHS Training Research
are grateful
Center
Grant Grant
AM 05497,
RR 00211,
to M. Heinemann
and M.A.
Research
and VA Project Babal for their
assistance. References 1.
Duckworth,
W. C. and Kitabchi,
2.
Duckworth, Acad. Sci.
W. C., Heinemann, USA 69, 3698-3702
3.
Burghen, 633-642
4.
Duckworth, W. C., Heinemann, M.A. Biophys. Acta 377, 421-430 (1975).
5.
Briel,
6.
Gray, W.R. Methods in Enzymology 11, pp. 139-151, (ed. ), Academic Press, Inc., New York (1967).
7.
Bromer, Pergamon
G. A., (1972).
Kitabchi,
G. and Neuhoff,
A. E.
Diabetes
M.A. and Kitabchi, (1972).
A. E. and Brush,
V.
23, 536-543
Physiol.
J. S.
A. E.
W. W. Glucagon. pp. 27-30, Press, New York (1972). 169
Proc.
Endocrinology
and Kitabchi,
Chem.
(1974).
A.E.
353, 540-553
P. J. Lefebvre
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91,
Biochim.
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P. Colowick
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