Vol. 64, No. 1,1975
BIOCHEMICAL
COLLAGENASE Euqene
A.
PRODUCTION
Bauer,
George and
Division of Washington
Received
March
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
BY HUMAN P.
Stricklin,
Arthur
Z.
Dermatoloqy, Universjty St. Louis,
SKIN
FIBROBLASTS
J.
John
Jeffrey
Eisen
Department School of Missouri 63110
of Medicine Medicine
18,1975 SUMMARY
Normal human skin fibroblasts, when cultured in serum free medium, produce collapenase in an inactive form. The enzyme in the crude medium can be activated by a brief preincubation with trypsin or by autoactivation. Once activated, the fibroblast collagenase is identical in its mechanism of action to human skin collagenase obtained from organ cultures. In addition, an inhibitor of collagenase is also present in the medium of fibroblast cultures. The inhibitor appears to be produced by the cells and its molecular weight is slightly higher than that of the enzyme. The presence of this inhibitor may account for previous inability to detect collagenase in human skin fibroblast cultures. It is also possible that some of the inactive enzyme exists in the medium in the form of a proenzyme. INTRODUCTION The cells of
of
regulate
the
connective
tance at
definition
to
for
the
which
of in
initiation
Human source
point
of
skin
in
changes,
control
of
is
collaqenase
Abbreviations: 3.005 11 CaCl?;
has term
longer
essential
if
biosynthesis
Copyright o I975 by Academic Press. Inc. AN rights of reproduction in any form reserved.
organ
an
systems it
human ultimate
the
animal
goal
enzyme (1,Z)
is
and
of in
in
the
crucial that
specific
study
impor-
it
stands
enzyme
required
depradation.
short
Tris-CaCl? SBTI, soybean
an
is
metabolism;
culture
for
which
is
Collagenase
collagen
in
by
collagenase
mammalian
organ
he maintained
defined
of
collagen
of
collagenase
can
mechanisms
dynamics.
function
a unique
exact
production
tissue the
the
buffer, trypsin
served.previously experiments periods
the
as
of
various are
(3).
to
time
232
important
However and
parameters
be
a system,
subjected
involved
be adequately
0.05 M Tris-HCl inhibitor.
an
(pH
examined
7.5)
with
to in
well
the in
both
BIOCHEMICAL
Vol. 64, No. 1,1975
Cell
normal
and
diseased
states.
system
for
studying
a variety
studies in
in
this
we were
in
the to
This
was
whole Werb
culture
medium
and
collagenase
a neutral
cells
with
to
in
culture
fibroblasts
report
human
be
an
and
ideal
initial succeeded
(5,6)
using
immuno-
describes
medium
and
short
term
an the
fibroblasts.
when
fibroblasts that
activated
organ
cultures
it
human
due
to
from
skin the
the
inactive
form.
detection
of
The is
enzyme
identical of
human
skin
to
cells
both
by
stimulating
the
that
the
conversion
increased
activity.a
cultures,
that
even
extremely that
collagenase
the
in
exists
in
to
collagenase
the
recently,
collagenase
unlikely
or
the
demonstrate
fibroblast
fact
(7,B).
More
of
for
was
from
cells.
was
responsible in
able
those
synovial
medium
serum
were
production
culture
collagenase
withdrawing
of
collagenase,
culture skin
increased
be
of fibroblast
the human
medium
secreted in
that of
was
could
medium
the
fibroblasts,
rabbit
are
presence in
briefly
collagenase
serum,
skin
by
enzyme
detect
the
fact
concluding
active
the
the
obtained by
enzyme
into
for
culture
particles,
of
the
This
the
(ID)
to
of
secreted
from
synovial in
latex
absence
amounts
the
fibroblasts
inhibitor
rabbit
protease
failure
normal
a potent (9),
of
a precursor
the
(4)
activity
to
Burleigh
Reynolds
and
in
to
systems
skin
enzymatic
attributed
activity
and
The
proven
enzyme
evidence
detect
serum,
Indeed,
of
immunologic
unable
medium.
Nerb
human
RESEARCH COMMUNICATIONS
techniques.
Despite
rich
in
have
other
employing
collagenase
histochemical
cultures
of
laboratory
localizing
AND BIOPHYSICAL
an
inact
cu
small enzyme
tures ve
is
of form
in
isolated
(3).
METHODS Human skin -punch chased from were passed
fibroblasts: Primar,y cell biopsy obtained from healthy the American Type Culture into glass roller bottles
aLatex stimulation by human cells
has (lk1.S.
Sly
lines were volunteers Collection (15B5 cm2,
also been employed to and Z. Nerb, personal
233
initiated from a 3 mm (11) or were pur(Rockville, Md.). Cells Rellco) and grown in
induce collaoenase communicat;on).
production
BIOCHEMICAL
Vol. 64, No. 1,1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Dulbecco's Modified Eagle'sMedium - HG + glutamine (GIHCO) with 0.03 M HEPES buffer (pH 7.6), 20% fetal calf serum and 200 units of penicillin and 2Wl ug of streptomycin/ml at 36°C. At confluence (5-10 x 107 cells) serum-containing medium was removed, cells were washed 4 times with 100 ml of Hank's Balanced Salt Solution (GIBCO) and cultures were maintained in 100 ml of the serum-free medium for 48-96 hr. Preparation of fibroblast collagenase. Crude, serum-free culture medium, concentrated about ZO-fold by a vacuum dialysis at 4", was dialyzed against Tris-CaC12 buffer and was assayed for collagenase activity either alone or after preincubation with trypsin to detect latent activity. The optimum conditions of preincubation were determined usins 0.1-20 ~g of trypsin (Sigma Chemical) for time periods of 0.5-20 min at both 25" and 37". In each case, preincubation was followed by the addition of at least a 5-fold molar excess of SBTI (Sigma Chemical) and the entire mixture was assayed on radioactive collagen fibrils as described below. Gel filtration was carried out on a column (1 x 40 cm) of Sephadex G-150 equilibrated with Tris-CaC12 buffer with 0.1 M NaCl at 4". Column calibration for determining molecular weight was done according to Andrews (12,13). Assays. Collagenase activity was measured at 37" by using native, reconstituted 14C-glycine labeled collagen fibrils as a substrate (14) or at 27" by determining the specific viscosity of collagen in solution as a function of time in Ostwald viscometers having flow times for water of 26-35 set (3). Thermally denatured collagen-enzyme reaction mixtures were subjected to electrophoresis in polyacrylamide gels (15). Trypsin activity was determined by measuring the rate of hydrolysis of Tos-ArgOMe at 25" (16). Protein was determined by the method of Lowry et al. (17). Purified alphal-antitr,ypsin (18) was the gift of Dr. Jo Seltzer. AND -_____ DISCUSSION
RESULTS Crude
culture
undetectable
levels
However, The
brief
optimum
medium
presented
under
these ZZO-fold.
preincubation
times some
collagenase the
collagen
autoactivation genase
was
in
for
some
of
the
was in
its
the
ensuing
12
Once
occurring. mechanism
hr,
of
234
which 37",
of
culture
vg)
was
trypsin
at
increased or
lancer
that
activated, to
displayed
no
completely
indicating
action
(43
activity.
activity.
medium, at
of
trypsin
enzyme
incubation
2 vg was
of
enzyme
medium
with
activity
period.
preparation
culture 5 min
crude
a 6-hr
such
almost
assay
increased
one
concentrations
unactivated
in
of
collagenase
destroyed
a 6-hr
markedly
Concentrated
preincubation
contained
during
trypsin
I.
Table
after
fibroblasts
activation
Higher
process identical
the
conditions
substrate
skin
collagenase with
for
cultures, activity
human
active
by
approximately
In
from
preincubation
activated
and
of
conditions
are
maximally 37"
medium
fibroblast the
enzyme
degraded an
apparent colla-
produced
by
BIOCHEMICAL
Vol. 64, No. 1,1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Effect
of
Preincubation Temperature
Preincubation
I
Conditions
Trypsin (Kl)
(5
on
Collagenase
Activity
Preincubation (10
min)
Time min)
Collagenase 25"
cl 0.5 1 2
(20min)
Activitya
37 14 16 NDb
2:
20 32 34 1338
255 898 1416 1708 1653
1301 1286 1263
1; 20
858 583 1301
0
11
20
36
0.51 2 5 10 20
18 243: 1481 1630 1683
ND 229:
1; 1864 1647 1404 1229
37"
18;: 2078
Crude culture medium (43 ug) was preincubated with varying concentrations of trypsin for 5, 10 or 20 min at either 25" or 37" after which the trypsin was inhibited by a 5-fold molar excess of SBTI for 10 min. This reaction mixture was then incubated with 2DD ug native 14C-glycine labeled collagen fibrils (3932 cpm/substrate) for 45 min at 37". A trypsin blank (20 ug) released 10.1% of the total counts. acollagenase activity, cpm above trypsin blank bND, assay not done
human
skin
explants
reaction
of
results
serum
product
free
of The
time there
Fig.
and
that
normal
and was
culture
these kinetics
2.
a critical
assessed
by
examining
collagen
in
solution
the at
human
skin
fibroblasts
and
that
the
medium
products 27"
of
(Fig.
1).
secrete
enzyme
is
the These
collagenase
a normal
gene
cells. of
trypsin-dependent
Activation
temperature
as
enzyme
indicate
into
in
the
(3)
was of
collagenase
dependent
on
preincubation.
concentration
trypsin For
of
235
trypsin
activation
are
concentration any
given below
as
preincubation which
virtually
shown well
as time no
Vol. 64, No. 1, 1975
Fig. 1. reaction reaction note the
Acrylamide mixture mixture; appearance
activation
there
could
be
(Fig.
2B)
These
kinetics
enzyme
was
or (20).
In
effort
an
culture
to
addition,
with
the
preincubation
consistent
medium
the was
before
any
trypsin
with
to
gel
to
of
filtration
which
concentration
conversion
process,
concen-
began,
temperature
activation
236
given
trypsin
degradation
sub.jected
collagenase-collagen left is the 0 time in specific viscosity,
activation
higher
either
a time-dependent
elucidate
at
period
preincubation
be with
In a lag
increasing could
medium
?A).
typically by
by
(19,ZO)
fibroblast
electrophoresis of fibroblast thermal denaturation. On the the right, after a 41% reduction of newly formed .A and c,B bands.
(Fig.
shortened or
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
gel
after on
occurred
tration,
the
BIOCHEMICAL
37"
of an
(Table
proenzyme
inhibitor
inactive on
I). to in
crude Sephadex
G-150
BIOCHEMICAL
Vol. 64, No. 1,1975
0
0.2 0.4 TRYPSIN ADDED
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1.00 (LJQ)
4 PREINCUBATION
TIME
(Mln )
Fig. 2. Kinetics of trypsin-dependent activation of fibroblast collagenase. (A) Preincubation was carried out at 25" for 5 min using varying amounts of trypsin and 25 fig (CRL 1146) or 17 ug (WU 1213) of crude culture medium. After the addition of 10 I.rg SBTI, the entire mixture was assayed with 200 Mug 14C-labeled collagen fibrils (3506 cpm/substrate) for 90 min at 37". (B) 17 ug crude medium (NJ 1213) was preincubated with varying amounts of trypsin for varying time at 25" after which collagenase activity was assessed as in "A".
12
16 20 FRACTION
24 28 NUMBER
32
Fig. 3. Gel filtration of fibroblast culture medium on Sephadex C-150. A sample containing 0.44 mg/ml protein was applied to a column (1 x 40 cm) at 4" and effluent fractions of 1.3 ml were collected at a rate of 3.9 ml/hr. a-0, absorbance at 280 nm; O---U, collagenase activity. (Insert) Calibration of Sephadex column for molecular weight determination. BSA, bovine serum albumin; OA, ovalbumin; Chy, chymotrypsinogen. 1,2 and 3 refer to 3 different filtration procedures to determine the approximate molecular weight of fibroblast collagenase.
(Fig. tivity
3).
Fractions
and for
was detected
were
inhibitory at a position
assayed
for
activity.
trypsin-dependent A single
corresponding
237
collagenase
ac-
peak of enzyme activity
to a molecular
weight
of approx-
Vol.
64,No.
1, 1975
imately
40,000
column
retained
Of
daltons
the
emerged
the
(Fig.
the 3,
collagenase
trypsin inhibitor
of
completely
failed
a slightly
while
Conversely,
approximately
The
inhibit
Inhibitor
Exe.
II
III
IV
Enzvme
0 3.0 7.5
Collagenase
G-150 Fractions 15-17
0 3.0 6.0
Collagenase
0 3.0 3.0 6.0
Trypsin
G-150 Fractions 15-17
0
Alphal-at
and
0 A1phal-at
in
inhibitor
activatable. of
collagenase
molecular
weight
abolished no
over
of
excess (Table
by
II,
weight, Exp.
of
units
Collagenase
Trypsin
cpm above x 103
IV).
Enzyme
the
Activity
(AK!)
3.0 3.9
activity, activity,
90%
inhibition
blank
238
Inhibition I 0, \ \ 10I
8.7 8.7 8.7
1333a 168 78
16.0 16.0 16.0
547a 166 60
3.0 6.0 3.0 3.0
18625b 17500 16319 17290
9.9 9.9 9.9 3.9 9.9
1529a 1647 1601 1479 1601
3.0 6.0 3.0
lOOO0 300
16omb
Aliquots of previously activated fibroblast collagenase (CRL 1146) or trypsin were incubated with increasing quantities of either mediumderived inhibitor (Sephadex G-150 fractions 15-17) or alphal-antitrypsin (alphal-at). These mixtures were then assayed for collagenase activity as described in Fig. 2 or for trypsin activity using Tos-Arg-OMe. acollaqenase bTrypsin
than
a serum
a 2-fold
Effects Alphal-antitrypsin
Protein
3.0 6.9 9.7 19.3 V
an
trypsin
the
II
-
G-150 Fractions 15-17
was
collagenase
Enzyme-Inhibitory Inhibitor
Proter
and
on
alphal-antitrypsin,
fibroblast
hg)
____I
of
COMMUNICATIONS
detected
virtually
size,
Table Comparison Medium-derived
enzyme
inhibitor
displaying
same
RESEARCH
higher
purified the
to
of
15-17).
activity,
II).
(Table
detection at
fractions
The
autoactivate
the
column
BIOPHYSICAL
insert).
to
was
from
AND
3,
ability
interest
enzyme
of
(Fig.
its
particular
which
BIOCHEMICAL
0 87.4 94.1
69O7 8917
0 5.5 12.4 7.2
0 0 0 3.3 0
0 37.4 98.1
BIOCHEMICAL
Vol. 64, No. 1,1975
Additionally, of
the
material
reacting
results
suggest
blasts
rather
that of
than
major
the
collagenase enzyme
with
also
to
detect
(6).
The
presence
of
gen
may
enzyme also
from be
the
present,
whole
the
residual
serum
in
present
an in
is in
of
the
collaqenase
inhibitor
in
as
material
suggested
filtration for
other
the
culture and
The
medium.
gel
These
diffusion. from
fibroblast
activity
by
gel
devoid
fibro-
medium.
form
trypsin-activatable
in
were
derived
the
inactive the
activity serum
inhibitor
therefore, exists
inability
the
that
features,
is
inhibitory
anti-bovine
from
the
of
containing
strongly
The
the
fractions
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
system
that
a potent
inhibitor
may
human
fibroblast
after indicates vertebrate
are inhibitor
explain cultures
partial
separation that
a zymo-
collagenases
(21-25). ACKNOHLEDGMENTS This was supported by Dr. Bauer is the recipient AM 013077 from the National to Mr. Hilliam Fiehler for
USPHS Grants of Research Institutes his excellent
AM 12123, AM 05611 and Career Development Award of Health. Our appreciation technical assistance.
HD 05291. 1 K04 ooes .
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Eisen, A.Z., Bauer, E.A. and Jeffrey, J.J. (1970) 3. Invest. Derm. 55, 359-373. Harris, E.D. and Krane, S.M. (1974) New Eng. J. Med. 291, 557563, 605-609, 652-661. Eisen, A.Z., Jeffrey, J.J. and Gross, J. (1968) Biochim. Biophys. Acta 151, 637-645. Pollack, R., Ed. (1973) Readings in Mammalian Cell Culture, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Reddick, M.E., Bauer, E.A. and Eisen, A.Z. (1973) Clin. Res. 21, 481A. Reddick, M.E., Bauer, E.A. and Eisen, A.Z. (1974) J. Invest. Derm. 2, 361-366. Eisen, A.Z., Bloch, K.J. and Sakai, T. (1970) J. Lab. Clin. Med. 75, 258-263. Esen, A.Z., Rauer, E.A. and Jeffrey, J.J. (1971) Proc. Nat. Acad. Sci. 66, 248-251. Werb, Z. and Burleigh, M.C. (1974) Biochem. 3. 137, 373-385. Herb, Z. and Reynolds, J.J. (1974) J. Exp. Med. 140, 1481-1497. Boyle, J.A. and Seegmiller, J.E. (1971) in Meth. Enzymol., W.B. Jakoby, Ed., Vol. 22, pp. 161-165, Academic Press, New York. Andrews, P. (1964) Biochem. J. 91, 222-233. Andrews, P. (1965) Biochem. J. 96, 595-606. Nagai, Y., Lapiere, C.M. and Gross, J. (1966) Biochemistry 5, 3123-3130.
239
Vol. 64, No. 1, 1975
15.
18. 19. 20. 21. 22. 23. 24. 25.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Nagai, Y., Gross, J. and Piez, K.A. (1964) Ann. N.Y. Acad. Sci. g, 494-500. l+-~i;;l,~B.C. (1959) Can. J. Biochem. Physiol. 37, 1393-1399. Rosebrough, N., Farr, A. and Randall, R. (1951) J. Eiol. Chem.'&I 265-275. Pannell, R., Johnson, Cl. and Travis, J. (1974) Biochemistry 13, 5439-5445. Kay, J. and Kassell, B. (1971) J. Biol. Chem. 246, 6661-6665. Kunitz. M. and Northroo. J.H. (1948) CrvstalliEEnzymes, Ed. 2, Columbia Un iversity Press, New York.' " Vaes, G. ( 1972) Biochem. J. 126, 275-289. Vaes, G. ( 1972) FEBS Lett. 28, 198-200. 10 Harper, E., Bloch, K.J. and Gross, J. (197 1) Biochemistry --) 3035-3041. Kruze, D. and Wojtecka, E. (1972) Biochim . Biophys. Acta 285, 436-446. (1973) Nature Oronsky, A.L., Perper, R.J. and Schroder, H.C. g&, 417-419.
240
BIOCHEMICAL
COLLAGENASE Euqene
A.
PRODUCTION
Bauer,
George and
Division of Washington
Received
March
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
BY HUMAN P.
Stricklin,
Arthur
Z.
Dermatoloqy, Universjty St. Louis,
SKIN
FIBROBLASTS
J.
John
Jeffrey
Eisen
Department School of Missouri 63110
of Medicine Medicine
18,1975 SUMMARY
Normal human skin fibroblasts, when cultured in serum free medium, produce collapenase in an inactive form. The enzyme in the crude medium can be activated by a brief preincubation with trypsin or by autoactivation. Once activated, the fibroblast collagenase is identical in its mechanism of action to human skin collagenase obtained from organ cultures. In addition, an inhibitor of collagenase is also present in the medium of fibroblast cultures. The inhibitor appears to be produced by the cells and its molecular weight is slightly higher than that of the enzyme. The presence of this inhibitor may account for previous inability to detect collagenase in human skin fibroblast cultures. It is also possible that some of the inactive enzyme exists in the medium in the form of a proenzyme. INTRODUCTION The cells of
of
regulate
the
connective
tance at
definition
to
for
the
which
of in
initiation
Human source
point
of
skin
in
changes,
control
of
is
collaqenase
Abbreviations: 3.005 11 CaCl?;
has term
longer
essential
if
biosynthesis
Copyright o I975 by Academic Press. Inc. AN rights of reproduction in any form reserved.
organ
an
systems it
human ultimate
the
animal
goal
enzyme (1,Z)
is
and
of in
in
the
crucial that
specific
study
impor-
it
stands
enzyme
required
depradation.
short
Tris-CaCl? SBTI, soybean
an
is
metabolism;
culture
for
which
is
Collagenase
collagen
in
by
collagenase
mammalian
organ
he maintained
defined
of
collagen
of
collagenase
can
mechanisms
dynamics.
function
a unique
exact
production
tissue the
the
buffer, trypsin
served.previously experiments periods
the
as
of
various are
(3).
to
time
232
important
However and
parameters
be
a system,
subjected
involved
be adequately
0.05 M Tris-HCl inhibitor.
an
(pH
examined
7.5)
with
to in
well
the in
both
BIOCHEMICAL
Vol. 64, No. 1,1975
Cell
normal
and
diseased
states.
system
for
studying
a variety
studies in
in
this
we were
in
the to
This
was
whole Werb
culture
medium
and
collagenase
a neutral
cells
with
to
in
culture
fibroblasts
report
human
be
an
and
ideal
initial succeeded
(5,6)
using
immuno-
describes
medium
and
short
term
an the
fibroblasts.
when
fibroblasts that
activated
organ
cultures
it
human
due
to
from
skin the
the
inactive
form.
detection
of
The is
enzyme
identical of
human
skin
to
cells
both
by
stimulating
the
that
the
conversion
increased
activity.a
cultures,
that
even
extremely that
collagenase
the
in
exists
in
to
collagenase
the
recently,
collagenase
unlikely
or
the
demonstrate
fibroblast
fact
(7,B).
More
of
for
was
from
cells.
was
responsible in
able
those
synovial
medium
serum
were
production
culture
collagenase
withdrawing
of
collagenase,
culture skin
increased
be
of fibroblast
the human
medium
secreted in
that of
was
could
medium
the
fibroblasts,
rabbit
are
presence in
briefly
collagenase
serum,
skin
by
enzyme
detect
the
fact
concluding
active
the
the
obtained by
enzyme
into
for
culture
particles,
of
the
This
the
(ID)
to
of
secreted
from
synovial in
latex
absence
amounts
the
fibroblasts
inhibitor
rabbit
protease
failure
normal
a potent (9),
of
a precursor
the
(4)
activity
to
Burleigh
Reynolds
and
in
to
systems
skin
enzymatic
attributed
activity
and
The
proven
enzyme
evidence
detect
serum,
Indeed,
of
immunologic
unable
medium.
Nerb
human
RESEARCH COMMUNICATIONS
techniques.
Despite
rich
in
have
other
employing
collagenase
histochemical
cultures
of
laboratory
localizing
AND BIOPHYSICAL
an
inact
cu
small enzyme
tures ve
is
of form
in
isolated
(3).
METHODS Human skin -punch chased from were passed
fibroblasts: Primar,y cell biopsy obtained from healthy the American Type Culture into glass roller bottles
aLatex stimulation by human cells
has (lk1.S.
Sly
lines were volunteers Collection (15B5 cm2,
also been employed to and Z. Nerb, personal
233
initiated from a 3 mm (11) or were pur(Rockville, Md.). Cells Rellco) and grown in
induce collaoenase communicat;on).
production
BIOCHEMICAL
Vol. 64, No. 1,1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Dulbecco's Modified Eagle'sMedium - HG + glutamine (GIHCO) with 0.03 M HEPES buffer (pH 7.6), 20% fetal calf serum and 200 units of penicillin and 2Wl ug of streptomycin/ml at 36°C. At confluence (5-10 x 107 cells) serum-containing medium was removed, cells were washed 4 times with 100 ml of Hank's Balanced Salt Solution (GIBCO) and cultures were maintained in 100 ml of the serum-free medium for 48-96 hr. Preparation of fibroblast collagenase. Crude, serum-free culture medium, concentrated about ZO-fold by a vacuum dialysis at 4", was dialyzed against Tris-CaC12 buffer and was assayed for collagenase activity either alone or after preincubation with trypsin to detect latent activity. The optimum conditions of preincubation were determined usins 0.1-20 ~g of trypsin (Sigma Chemical) for time periods of 0.5-20 min at both 25" and 37". In each case, preincubation was followed by the addition of at least a 5-fold molar excess of SBTI (Sigma Chemical) and the entire mixture was assayed on radioactive collagen fibrils as described below. Gel filtration was carried out on a column (1 x 40 cm) of Sephadex G-150 equilibrated with Tris-CaC12 buffer with 0.1 M NaCl at 4". Column calibration for determining molecular weight was done according to Andrews (12,13). Assays. Collagenase activity was measured at 37" by using native, reconstituted 14C-glycine labeled collagen fibrils as a substrate (14) or at 27" by determining the specific viscosity of collagen in solution as a function of time in Ostwald viscometers having flow times for water of 26-35 set (3). Thermally denatured collagen-enzyme reaction mixtures were subjected to electrophoresis in polyacrylamide gels (15). Trypsin activity was determined by measuring the rate of hydrolysis of Tos-ArgOMe at 25" (16). Protein was determined by the method of Lowry et al. (17). Purified alphal-antitr,ypsin (18) was the gift of Dr. Jo Seltzer. AND -_____ DISCUSSION
RESULTS Crude
culture
undetectable
levels
However, The
brief
optimum
medium
presented
under
these ZZO-fold.
preincubation
times some
collagenase the
collagen
autoactivation genase
was
in
for
some
of
the
was in
its
the
ensuing
12
Once
occurring. mechanism
hr,
of
234
which 37",
of
culture
vg)
was
trypsin
at
increased or
lancer
that
activated, to
displayed
no
completely
indicating
action
(43
activity.
activity.
medium, at
of
trypsin
enzyme
incubation
2 vg was
of
enzyme
medium
with
activity
period.
preparation
culture 5 min
crude
a 6-hr
such
almost
assay
increased
one
concentrations
unactivated
in
of
collagenase
destroyed
a 6-hr
markedly
Concentrated
preincubation
contained
during
trypsin
I.
Table
after
fibroblasts
activation
Higher
process identical
the
conditions
substrate
skin
collagenase with
for
cultures, activity
human
active
by
approximately
In
from
preincubation
activated
and
of
conditions
are
maximally 37"
medium
fibroblast the
enzyme
degraded an
apparent colla-
produced
by
BIOCHEMICAL
Vol. 64, No. 1,1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Effect
of
Preincubation Temperature
Preincubation
I
Conditions
Trypsin (Kl)
(5
on
Collagenase
Activity
Preincubation (10
min)
Time min)
Collagenase 25"
cl 0.5 1 2
(20min)
Activitya
37 14 16 NDb
2:
20 32 34 1338
255 898 1416 1708 1653
1301 1286 1263
1; 20
858 583 1301
0
11
20
36
0.51 2 5 10 20
18 243: 1481 1630 1683
ND 229:
1; 1864 1647 1404 1229
37"
18;: 2078
Crude culture medium (43 ug) was preincubated with varying concentrations of trypsin for 5, 10 or 20 min at either 25" or 37" after which the trypsin was inhibited by a 5-fold molar excess of SBTI for 10 min. This reaction mixture was then incubated with 2DD ug native 14C-glycine labeled collagen fibrils (3932 cpm/substrate) for 45 min at 37". A trypsin blank (20 ug) released 10.1% of the total counts. acollagenase activity, cpm above trypsin blank bND, assay not done
human
skin
explants
reaction
of
results
serum
product
free
of The
time there
Fig.
and
that
normal
and was
culture
these kinetics
2.
a critical
assessed
by
examining
collagen
in
solution
the at
human
skin
fibroblasts
and
that
the
medium
products 27"
of
(Fig.
1).
secrete
enzyme
is
the These
collagenase
a normal
gene
cells. of
trypsin-dependent
Activation
temperature
as
enzyme
indicate
into
in
the
(3)
was of
collagenase
dependent
on
preincubation.
concentration
trypsin For
of
235
trypsin
activation
are
concentration any
given below
as
preincubation which
virtually
shown well
as time no
Vol. 64, No. 1, 1975
Fig. 1. reaction reaction note the
Acrylamide mixture mixture; appearance
activation
there
could
be
(Fig.
2B)
These
kinetics
enzyme
was
or (20).
In
effort
an
culture
to
addition,
with
the
preincubation
consistent
medium
the was
before
any
trypsin
with
to
gel
to
of
filtration
which
concentration
conversion
process,
concen-
began,
temperature
activation
236
given
trypsin
degradation
sub.jected
collagenase-collagen left is the 0 time in specific viscosity,
activation
higher
either
a time-dependent
elucidate
at
period
preincubation
be with
In a lag
increasing could
medium
?A).
typically by
by
(19,ZO)
fibroblast
electrophoresis of fibroblast thermal denaturation. On the the right, after a 41% reduction of newly formed .A and c,B bands.
(Fig.
shortened or
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
gel
after on
occurred
tration,
the
BIOCHEMICAL
37"
of an
(Table
proenzyme
inhibitor
inactive on
I). to in
crude Sephadex
G-150
BIOCHEMICAL
Vol. 64, No. 1,1975
0
0.2 0.4 TRYPSIN ADDED
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1.00 (LJQ)
4 PREINCUBATION
TIME
(Mln )
Fig. 2. Kinetics of trypsin-dependent activation of fibroblast collagenase. (A) Preincubation was carried out at 25" for 5 min using varying amounts of trypsin and 25 fig (CRL 1146) or 17 ug (WU 1213) of crude culture medium. After the addition of 10 I.rg SBTI, the entire mixture was assayed with 200 Mug 14C-labeled collagen fibrils (3506 cpm/substrate) for 90 min at 37". (B) 17 ug crude medium (NJ 1213) was preincubated with varying amounts of trypsin for varying time at 25" after which collagenase activity was assessed as in "A".
12
16 20 FRACTION
24 28 NUMBER
32
Fig. 3. Gel filtration of fibroblast culture medium on Sephadex C-150. A sample containing 0.44 mg/ml protein was applied to a column (1 x 40 cm) at 4" and effluent fractions of 1.3 ml were collected at a rate of 3.9 ml/hr. a-0, absorbance at 280 nm; O---U, collagenase activity. (Insert) Calibration of Sephadex column for molecular weight determination. BSA, bovine serum albumin; OA, ovalbumin; Chy, chymotrypsinogen. 1,2 and 3 refer to 3 different filtration procedures to determine the approximate molecular weight of fibroblast collagenase.
(Fig. tivity
3).
Fractions
and for
was detected
were
inhibitory at a position
assayed
for
activity.
trypsin-dependent A single
corresponding
237
collagenase
ac-
peak of enzyme activity
to a molecular
weight
of approx-
Vol.
64,No.
1, 1975
imately
40,000
column
retained
Of
daltons
the
emerged
the
(Fig.
the 3,
collagenase
trypsin inhibitor
of
completely
failed
a slightly
while
Conversely,
approximately
The
inhibit
Inhibitor
Exe.
II
III
IV
Enzvme
0 3.0 7.5
Collagenase
G-150 Fractions 15-17
0 3.0 6.0
Collagenase
0 3.0 3.0 6.0
Trypsin
G-150 Fractions 15-17
0
Alphal-at
and
0 A1phal-at
in
inhibitor
activatable. of
collagenase
molecular
weight
abolished no
over
of
excess (Table
by
II,
weight, Exp.
of
units
Collagenase
Trypsin
cpm above x 103
IV).
Enzyme
the
Activity
(AK!)
3.0 3.9
activity, activity,
90%
inhibition
blank
238
Inhibition I 0, \ \ 10I
8.7 8.7 8.7
1333a 168 78
16.0 16.0 16.0
547a 166 60
3.0 6.0 3.0 3.0
18625b 17500 16319 17290
9.9 9.9 9.9 3.9 9.9
1529a 1647 1601 1479 1601
3.0 6.0 3.0
lOOO0 300
16omb
Aliquots of previously activated fibroblast collagenase (CRL 1146) or trypsin were incubated with increasing quantities of either mediumderived inhibitor (Sephadex G-150 fractions 15-17) or alphal-antitrypsin (alphal-at). These mixtures were then assayed for collagenase activity as described in Fig. 2 or for trypsin activity using Tos-Arg-OMe. acollaqenase bTrypsin
than
a serum
a 2-fold
Effects Alphal-antitrypsin
Protein
3.0 6.9 9.7 19.3 V
an
trypsin
the
II
-
G-150 Fractions 15-17
was
collagenase
Enzyme-Inhibitory Inhibitor
Proter
and
on
alphal-antitrypsin,
fibroblast
hg)
____I
of
COMMUNICATIONS
detected
virtually
size,
Table Comparison Medium-derived
enzyme
inhibitor
displaying
same
RESEARCH
higher
purified the
to
of
15-17).
activity,
II).
(Table
detection at
fractions
The
autoactivate
the
column
BIOPHYSICAL
insert).
to
was
from
AND
3,
ability
interest
enzyme
of
(Fig.
its
particular
which
BIOCHEMICAL
0 87.4 94.1
69O7 8917
0 5.5 12.4 7.2
0 0 0 3.3 0
0 37.4 98.1
BIOCHEMICAL
Vol. 64, No. 1,1975
Additionally, of
the
material
reacting
results
suggest
blasts
rather
that of
than
major
the
collagenase enzyme
with
also
to
detect
(6).
The
presence
of
gen
may
enzyme also
from be
the
present,
whole
the
residual
serum
in
present
an in
is in
of
the
collaqenase
inhibitor
in
as
material
suggested
filtration for
other
the
culture and
The
medium.
gel
These
diffusion. from
fibroblast
activity
by
gel
devoid
fibro-
medium.
form
trypsin-activatable
in
were
derived
the
inactive the
activity serum
inhibitor
therefore, exists
inability
the
that
features,
is
inhibitory
anti-bovine
from
the
of
containing
strongly
The
the
fractions
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
system
that
a potent
inhibitor
may
human
fibroblast
after indicates vertebrate
are inhibitor
explain cultures
partial
separation that
a zymo-
collagenases
(21-25). ACKNOHLEDGMENTS This was supported by Dr. Bauer is the recipient AM 013077 from the National to Mr. Hilliam Fiehler for
USPHS Grants of Research Institutes his excellent
AM 12123, AM 05611 and Career Development Award of Health. Our appreciation technical assistance.
HD 05291. 1 K04 ooes .
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Eisen, A.Z., Bauer, E.A. and Jeffrey, J.J. (1970) 3. Invest. Derm. 55, 359-373. Harris, E.D. and Krane, S.M. (1974) New Eng. J. Med. 291, 557563, 605-609, 652-661. Eisen, A.Z., Jeffrey, J.J. and Gross, J. (1968) Biochim. Biophys. Acta 151, 637-645. Pollack, R., Ed. (1973) Readings in Mammalian Cell Culture, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Reddick, M.E., Bauer, E.A. and Eisen, A.Z. (1973) Clin. Res. 21, 481A. Reddick, M.E., Bauer, E.A. and Eisen, A.Z. (1974) J. Invest. Derm. 2, 361-366. Eisen, A.Z., Bloch, K.J. and Sakai, T. (1970) J. Lab. Clin. Med. 75, 258-263. Esen, A.Z., Rauer, E.A. and Jeffrey, J.J. (1971) Proc. Nat. Acad. Sci. 66, 248-251. Werb, Z. and Burleigh, M.C. (1974) Biochem. 3. 137, 373-385. Herb, Z. and Reynolds, J.J. (1974) J. Exp. Med. 140, 1481-1497. Boyle, J.A. and Seegmiller, J.E. (1971) in Meth. Enzymol., W.B. Jakoby, Ed., Vol. 22, pp. 161-165, Academic Press, New York. Andrews, P. (1964) Biochem. J. 91, 222-233. Andrews, P. (1965) Biochem. J. 96, 595-606. Nagai, Y., Lapiere, C.M. and Gross, J. (1966) Biochemistry 5, 3123-3130.
239
Vol. 64, No. 1, 1975
15.
18. 19. 20. 21. 22. 23. 24. 25.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Nagai, Y., Gross, J. and Piez, K.A. (1964) Ann. N.Y. Acad. Sci. g, 494-500. l+-~i;;l,~B.C. (1959) Can. J. Biochem. Physiol. 37, 1393-1399. Rosebrough, N., Farr, A. and Randall, R. (1951) J. Eiol. Chem.'&I 265-275. Pannell, R., Johnson, Cl. and Travis, J. (1974) Biochemistry 13, 5439-5445. Kay, J. and Kassell, B. (1971) J. Biol. Chem. 246, 6661-6665. Kunitz. M. and Northroo. J.H. (1948) CrvstalliEEnzymes, Ed. 2, Columbia Un iversity Press, New York.' " Vaes, G. ( 1972) Biochem. J. 126, 275-289. Vaes, G. ( 1972) FEBS Lett. 28, 198-200. 10 Harper, E., Bloch, K.J. and Gross, J. (197 1) Biochemistry --) 3035-3041. Kruze, D. and Wojtecka, E. (1972) Biochim . Biophys. Acta 285, 436-446. (1973) Nature Oronsky, A.L., Perper, R.J. and Schroder, H.C. g&, 417-419.
240
