Vol.
170,
No.
July
31, 1990
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
769-774
Pages
CO-PURIFICATION
OF PROTEASES WITH
BASIC FIBROBLAST
GROWTHFACTOR (FGF)
1 2 L. Ho, Michael R. Carpenter, 2 B. Smillie and Angelo G. Gambarini
Paul0 Lawrence
1
I
Departamento Universidade de Medical Function,
Received
de Sbo
Bioquimica, Paulo, CP. Brasil
Instituto de Quimica, 20780, 01498, S&o Paulo,
Research Council Group in Protein Department of Biochemistry, University Edmonton, Canada T6G 2H7 June
14,
Structure and of Alberta,
1990
SUMMARY : Acidic and basic fibroblast growth factors (FGFs) are proteins of 16-18 kDa. Other forms of 25-30 kDa related to this growth factor family have recently been described. All these components bind tightly to heparin-Sepharose, a property that allows the purification of several FGF-related proteins. During the purification of acidic and basic FGFs from bovine pituitary glands, we detected the presence of 28-30 kDa components that are immunoreactive against antibasic FGF antisera. However, microsequencing analysis revealed that the 28-30 kDa components are lysosomal proteases that co-elute with basic FGF from heparinSepharose columns. The involvement of these proteases in the etiology of microheterogenous forms of FGFs and/or release of FGFs from the extracellular matrix is discussed. 0 1990 Academic Press,Inc.
Fibroblast thought
to
be comprised
18 kDa proteins cells
through
Subsequently, were
identified,
which
growth of
factors
acidic
from
affinity
molecular
characterized
were
and basic
can be purified
heparin-Sepharose higher
(FGFs)
weight
initially
proteins normal
of
and tumor
chromatography forms
and shown to
of
16-
25-30
(1). kDa
be FGF-related
ABBREVIATIONS: KGF: Keratinocyte Growth Factor, FGF: Fibroblast Growth Factor, bFGF: basic Fibroblast Growth Factor, SDS-PAGE: Sodium Dodecyl Sulphate Polyacrylamide Gel HPLC: High Electrophoresis, PVDF: Polyvinilydenedifluoride, Performance Liquid Chromatography, PTH-aas: Phenylthiohydantoin-amino acids, ECM: Extracellular Matrix.
769
0006-291X/90 $1.50 Copyrighf 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
170,
No.
2, 1990
BIOCHEMICAL
polypeptides. of
This
oncogenes All
(7).
is
int-2 these
processing or
(ll),
with
bFGF
and
no thiol-
due
with
the
FGFs
(6)
to
CUG start of
now
the
products and
proteolytic (5,9)
codon
pituitary kDa
(10). acidic
proteins columns
but
that are
and
that
co-
and
were
antisera.
shown
FGF-6
homology.
glycosylation
bFGF have
COMMUNICATIONS
30-50%
heparin-Sepharose
results
homology
found
28-30
against
microsequencing
FGF-5
protein
detected
from
immunoreactive
share
also
purification
we
and
share
through
the
(2)
(4,5),
in
translation
FGFs
eluted
are
RESEARCH
KGF
components
differences
During
of
h&/KS-3
forms (8),
BIOPHYSICAL
case
FGF-like
alternative
basic
the
(3),
Microheterogenous
AND
However, these
components
related
to
serine-
proteases.
METHODS PURIFICATION OF bFGF AND THE 28-30 kDa the 28-30 kDa components were purified glands as previously described (11).
COMPONENTS from bovine
- bFGF and pituitary
SDS-PAGE AND WESTERN-BLOTTING - SDS-PAGE was carried out according to Laemmli (14) or Moos et al using (13), polyacrylamide gels of different concentrations. Westernblotting was performed according to Burnette (15). The antibFGF antisera were generous gifts from Dr. A. Baird (The Salk Institute, La Jolla, CA, USA) and were raised against synthetic peptides homologous to the sequences 30-50 or 6987 of bFGF (1-146). Details are described elsewhere (11). N-TERMINAL SEQUENCING - Proteins were separated by SDS-PAGE and electroblotted onto a PVDF membrane (Immobilon, Millipore). The bands of interest were visualized by Coomassie Blue staining, cut and sequenced in an Applied Biosystem Model 470A gas-phase sequenator with on-line HPLC detection of PTH-aas (12,13).
RESULTS IMMUNOREACTIVE our
previous
HIGH results
antisera
anti-bFGF
kDa
were
that
columns
MOLECULAR
(Figure
(ll), revealed
eluted 1).
along After
WEIGHT
the
FORMS
Western-blotting
immunoreactive with long 770
bFGF
from
exposure
-
According assays
to using
components
of
30
heparin-Sepharose times,
28
kDa
Vol.
170,
No.
2, 1990
BIOCHEMICAL
AND
a
BIOPHYSICAL
b
RESEARCH
COMMUNICATIONS
c
Fiqure 1 - Western-blotting of pituitary fractions eluted from heparin-Sepharose columns with 1.9M NaCl as previously described (11). (a) Nitrocellulose membrane stained with colloidal gold showing the 28 kDa and 30 kDa components. The arrow indicates two 17-18 kDa forms of bFGF identified by microsequencing; ( b 1 az;z~;;; i~~~ba:~~5~~~~~~~~~~lyA wi;i anti-bFGF (69-87) described (11). The reaction were visualized after exposure to X-ray films: (c) same as in (b) using anti-bFGF (30-50) antiserum.
could
components
clarify
also
whether
forms
or
out,
using
the
either al
the
bands
(Figure
depending
acrylamide shown).
all
bands
same
called sequences
were
order
to
FGF-related was
carried
by Matsuidara
(12)
OF 28 AND 30 kDa COMPONENTS - The la)
can
on
the
the
and the
presence
28
available
the
in
system
length
of
convenience,
the
of
same
NBRF-PIR
771
two (13,
or
all When Protein
28 kDa
or
three
14),
the
gel
the
method
N-terminal
microheterogenous
component.
kDa
into
irrespective
of
For
N-terminus.
be resolved SDS-PAGE
showed
analysed
the
In
analysis
method described
Nevertheless,
suggesting
shown).
kDa components sequencing
concentration
not
(not
(13).
SEQUENCE ANALYSIS component
28-30
N-terminal
not,
by Moos et
be detected
the
(results used,
sequence,
forms
with
the
these
forms
were
compared
with
the
Sequence
Library
Vol.
170,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Table I N-terminal sequence homologies between 28 kDa component and thiol-proteases (A) and 30 kDa component and serine-proteases The identical amino acids are (8) underlined in each case; (*) represents the cysteinyl active site of thiol-proteases
(A)
1
"28 KDa component" Cathepsin L (Chicken) Cathepsin L (Human) Cathepsin L (Bovine) Cathepsin H (Rat) Papain
5
10
1
"30 KDa component" Elastase (Human leukocyte) Cathepsin G (Human) Factor X (Bovine) Chymotrypsin (Bovine)
the
I),
homology
with
N-terminal
cathepsin
L,
a protease
heterogenous SDS-PAGE bands for
and
presented this
component.
The
homologous
to
lysosomes
(17).
in
the
with
described
by
same
Homology (%I 73 73 66 60 53
20
Homology (%I
Table
These results as that
are
Western-blottings
of a
suggest
the
All
these I)
and
the
30
kDa
I
is
highly
serine-proteases,
that
recognized
(Figure
using
protease
proteases,
also
(Table
called
elastase,
are
(13).
sequence
sequences
like
1) bands,
al
et
in
of
(16).
three
Moos
degree
proteases,
(Figure
into
shown
N-terminal
57 47 42 38
high
thiol-
collectively
sequence
a
origin
N-terminal
were
bFGF
of
components
resolved
identified
proteins, epitopes
kDa
leukocyte
particularly
15
showed
lysosomal
be
reason,
10
sequence
of 30
the
5
sequences
can
system
25
IVGGRKARPQELPFLASIQNQ IVGGRRARPHAW~NVSLQLR =~HS~YN@L~I3 -IVGGRDCAEGECPWQALLtiE --IVNGEEAVPGgWFWQ%LQEK ---
consensus
The
20
APDSIDYRKKGYVT-P APRSVDWREKGYVT-PVKDQGICGSCWA ZR&~%&KGY~T-~VKNQGQCGSCWA DDZ~~W&KGGVT-&KYQGACGSCWA -----YPSSMDWRKKGNWSPVKNQGACGSCWT IPEi!VDWRQKGAV!C-i%KNQGSCGSCWA ------*****
(B)
(Table
15
the
the
in
28 and 30 kDa share
may by
found
antisera
similar used
1).
DISCUSSION Here we report and serine-
proteases
that
proteins
by N-terminal 772
identified microsequencing
as thiolcan be
Vol.
170,
No.
BIOCHEMICAL
2, 1990
purified
along
are
These
by
to
internal
results
(not
containing
28-30
kDa It
molecular
weight
components has
of
may play FGFs
suggest
a role
from
process glands.
It
suggested
that
was
liberated
through
matrix the
Cathepsin
L
that
degrade
collagens,
(17,20).
These
enzymes
are
that
the
cells
hydrolysis under
(21,22).
proteases of
normal
pathological
of
they
the
stored
could
be
enzymes lysosomal
and proteoglycans
produced
and
is
described
ECM degrading
elastins
ECM components
action
and
are
are
it
secreted
elastase
described
the
extraction
which
Therefore,
we have
and/or
some
actively
FGFs
we have
are
leukocyte
proteases
transformed
basic
Therefore,
from
of
factor N-terminal
by
the
FGFs
(ECM),
action and
and
during
pituitary
extracellular
-
this
the
proteases
bovine
the
(19)
in
the
high
growth
(8,18,19).
that
serine these
procedures
proteases
to
whether
acidic
purification
tissue-specific
reasonable
in
the
thiol-and
that
by
preparations
exhibit
shown
presented
during
known
resin
Preliminary
bFGF
display
also
the
peptides
bFGF.
that
not
been
microheterogeneity generated
is
to against
of
components
activities.
It
strongly
raised
indicate
COMMUNICATIONS
heparin-Sepharose
sequences
protease
activity.
bind
antisera
shown)
the
RESEARCH
using
proteases
recognized
corresponding
BIOPHYSICAL
bFGF
with
chromatography. and
AND
and secreted
its
is
may play release
conceivable a role
of
by
stored
in
the FGFs
conditions.
ACKNOWLEDGMENTS - P.L.H. and A.G.G. would like to thank Drs. H.A. Armelin and M.C.S. Armelin for helpful discussions P.L.H. is on leave of and comments on the manuscript. absence from Institute Butantan, Sao Paulo, Brasil. This work was supported by the following brazilian research agencies : FINEP, FAPESP and CNPq. REFERENCES 1.
Lobb,R.R., Biochem.
154,
Harper, 1-14.
J.W.
and
773
Fett,
J.W.
(1986)
Anal.
Vol.
170,
No.
2, 1990
BIOCHEMICAL
AND
2.
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Rubin, J-S., Osada, H., Finch, P.W., Taylor, W.G., Rudikoff, S. and Aaronson, S.A. (1989) Proc. Natl. Acad. Sci. USA 86, 802-806. 3. Dickson, C. and Peters, G. (1987) Nature 326, 833. 4. Taira, M., Yoshida, T., Miyagawa, K., Sakamoto, H., Terada, M. and Sugimura,T. (1987) Proc. Natl. Acad. SCi. USA 84, 2980-2984. 5. Delli-Bovi, P., Curatola, A.M., Kern, F.G., Greco, A., M. and Basilica, C. (1987) Cell 50, 729-737. Ittman, 6. Zhan, X., Bates, B., Hu, X. and Goldfarb, M. (1988) Mall. Cell. Biol. 8, 3487-3495. Raybaud, F., Mattei, M., 7. Marics, I., Adelaide, J., Coullier, F., Planche, J., Lapeyriere, 0. and Birbaum, D. (1989) Oncogene 4, 335-340. Smith, S., Sullivan, R., Shing, Y., 8. Klagsbrun, M., Smith, J.A. and Sasse, J. (1987) Proc. Davidson, S., USA 84, 1839-1843. Natl. Acad. Sci. 9. Delli-Bovi, P., Curatola, A.M., Newman, K-M., SatO, Y., Moscatelli, D., Hewick, R.M., Rifkin, D.R. and Basilica, C. (1988) Mall. Cell. Biol. 8, 2933-2941. lO.Prats, H., Kaghad, M., Prats, A.C., Klagsbrun, M., Lelias, J.M., Liauzun, P., Chalon, P., Tauber, J.P., Smith, J.A. and Caput, D. (1989) Proc. Natl. Amalric, F., Acad. Sci. USA 86, 1836-1840. ll.Ho, P.L., Jakes, R., Northrop, F.D. and Gambarini, A.G. (1988) Biochem. Int. 17, 973-980. 12.Matsuidara, P. (1987) J. Biol. Chem. 262, 10035-10038. 13.Moos Jr, M., Nguyen, N.Y. and Lui, T.Y. (1988) J. Biol. Chem. 263, 6005-6008. 14.Laemmli, U.K. (1970) Nature 227, 680-685. 15.Burnette, W.N. (1981) Anal. Biochem. 112, 195-203. 16.Dufour, E., Obled, A., Valin, C., Bechet, D., RibaudeauDumas,B. and Huet,J.C. (1987) Biochemistry 26, 5689-5695. l'ir.Sinha, S., Watorek, W., Karr, S., Giles, J., Bode, W. and Travis,J.(1987) Proc. Natl. Acad. Sci. USA 84, 2228-2232. 18.Gospodarowicz, D., Neufeld, G. and Schweigerer, L. (1986) Cell Differ. 19, 1-17. lg.Burgess, W.D. and Maciag, T. (1989) Annu. Rev. Biochem. 58, 574-606. 20.Erickson, A.H. (1989) J. Cell. Biochem. 40, 31-41. al.Troen, B-R., Ascherman, D., Atlas, D. and Gottesman,M.M. (1988) J. Biol. Chem. 263, 254-261. 22.Tryggvason, K., Hoyhtya, M. and Salo, T. (1987) Biochem. Biophys. Acta 907, 191-217.
774
170,
No.
July
31, 1990
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
769-774
Pages
CO-PURIFICATION
OF PROTEASES WITH
BASIC FIBROBLAST
GROWTHFACTOR (FGF)
1 2 L. Ho, Michael R. Carpenter, 2 B. Smillie and Angelo G. Gambarini
Paul0 Lawrence
1
I
Departamento Universidade de Medical Function,
Received
de Sbo
Bioquimica, Paulo, CP. Brasil
Instituto de Quimica, 20780, 01498, S&o Paulo,
Research Council Group in Protein Department of Biochemistry, University Edmonton, Canada T6G 2H7 June
14,
Structure and of Alberta,
1990
SUMMARY : Acidic and basic fibroblast growth factors (FGFs) are proteins of 16-18 kDa. Other forms of 25-30 kDa related to this growth factor family have recently been described. All these components bind tightly to heparin-Sepharose, a property that allows the purification of several FGF-related proteins. During the purification of acidic and basic FGFs from bovine pituitary glands, we detected the presence of 28-30 kDa components that are immunoreactive against antibasic FGF antisera. However, microsequencing analysis revealed that the 28-30 kDa components are lysosomal proteases that co-elute with basic FGF from heparinSepharose columns. The involvement of these proteases in the etiology of microheterogenous forms of FGFs and/or release of FGFs from the extracellular matrix is discussed. 0 1990 Academic Press,Inc.
Fibroblast thought
to
be comprised
18 kDa proteins cells
through
Subsequently, were
identified,
which
growth of
factors
acidic
from
affinity
molecular
characterized
were
and basic
can be purified
heparin-Sepharose higher
(FGFs)
weight
initially
proteins normal
of
and tumor
chromatography forms
and shown to
of
16-
25-30
(1). kDa
be FGF-related
ABBREVIATIONS: KGF: Keratinocyte Growth Factor, FGF: Fibroblast Growth Factor, bFGF: basic Fibroblast Growth Factor, SDS-PAGE: Sodium Dodecyl Sulphate Polyacrylamide Gel HPLC: High Electrophoresis, PVDF: Polyvinilydenedifluoride, Performance Liquid Chromatography, PTH-aas: Phenylthiohydantoin-amino acids, ECM: Extracellular Matrix.
769
0006-291X/90 $1.50 Copyrighf 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
170,
No.
2, 1990
BIOCHEMICAL
polypeptides. of
This
oncogenes All
(7).
is
int-2 these
processing or
(ll),
with
bFGF
and
no thiol-
due
with
the
FGFs
(6)
to
CUG start of
now
the
products and
proteolytic (5,9)
codon
pituitary kDa
(10). acidic
proteins columns
but
that are
and
that
co-
and
were
antisera.
shown
FGF-6
homology.
glycosylation
bFGF have
COMMUNICATIONS
30-50%
heparin-Sepharose
results
homology
found
28-30
against
microsequencing
FGF-5
protein
detected
from
immunoreactive
share
also
purification
we
and
share
through
the
(2)
(4,5),
in
translation
FGFs
eluted
are
RESEARCH
KGF
components
differences
During
of
h&/KS-3
forms (8),
BIOPHYSICAL
case
FGF-like
alternative
basic
the
(3),
Microheterogenous
AND
However, these
components
related
to
serine-
proteases.
METHODS PURIFICATION OF bFGF AND THE 28-30 kDa the 28-30 kDa components were purified glands as previously described (11).
COMPONENTS from bovine
- bFGF and pituitary
SDS-PAGE AND WESTERN-BLOTTING - SDS-PAGE was carried out according to Laemmli (14) or Moos et al using (13), polyacrylamide gels of different concentrations. Westernblotting was performed according to Burnette (15). The antibFGF antisera were generous gifts from Dr. A. Baird (The Salk Institute, La Jolla, CA, USA) and were raised against synthetic peptides homologous to the sequences 30-50 or 6987 of bFGF (1-146). Details are described elsewhere (11). N-TERMINAL SEQUENCING - Proteins were separated by SDS-PAGE and electroblotted onto a PVDF membrane (Immobilon, Millipore). The bands of interest were visualized by Coomassie Blue staining, cut and sequenced in an Applied Biosystem Model 470A gas-phase sequenator with on-line HPLC detection of PTH-aas (12,13).
RESULTS IMMUNOREACTIVE our
previous
HIGH results
antisera
anti-bFGF
kDa
were
that
columns
MOLECULAR
(Figure
(ll), revealed
eluted 1).
along After
WEIGHT
the
FORMS
Western-blotting
immunoreactive with long 770
bFGF
from
exposure
-
According assays
to using
components
of
30
heparin-Sepharose times,
28
kDa
Vol.
170,
No.
2, 1990
BIOCHEMICAL
AND
a
BIOPHYSICAL
b
RESEARCH
COMMUNICATIONS
c
Fiqure 1 - Western-blotting of pituitary fractions eluted from heparin-Sepharose columns with 1.9M NaCl as previously described (11). (a) Nitrocellulose membrane stained with colloidal gold showing the 28 kDa and 30 kDa components. The arrow indicates two 17-18 kDa forms of bFGF identified by microsequencing; ( b 1 az;z~;;; i~~~ba:~~5~~~~~~~~~~lyA wi;i anti-bFGF (69-87) described (11). The reaction were visualized after exposure to X-ray films: (c) same as in (b) using anti-bFGF (30-50) antiserum.
could
components
clarify
also
whether
forms
or
out,
using
the
either al
the
bands
(Figure
depending
acrylamide shown).
all
bands
same
called sequences
were
order
to
FGF-related was
carried
by Matsuidara
(12)
OF 28 AND 30 kDa COMPONENTS - The la)
can
on
the
the
and the
presence
28
available
the
in
system
length
of
convenience,
the
of
same
NBRF-PIR
771
two (13,
or
all When Protein
28 kDa
or
three
14),
the
gel
the
method
N-terminal
microheterogenous
component.
kDa
into
irrespective
of
For
N-terminus.
be resolved SDS-PAGE
showed
analysed
the
In
analysis
method described
Nevertheless,
suggesting
shown).
kDa components sequencing
concentration
not
(not
(13).
SEQUENCE ANALYSIS component
28-30
N-terminal
not,
by Moos et
be detected
the
(results used,
sequence,
forms
with
the
these
forms
were
compared
with
the
Sequence
Library
Vol.
170,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Table I N-terminal sequence homologies between 28 kDa component and thiol-proteases (A) and 30 kDa component and serine-proteases The identical amino acids are (8) underlined in each case; (*) represents the cysteinyl active site of thiol-proteases
(A)
1
"28 KDa component" Cathepsin L (Chicken) Cathepsin L (Human) Cathepsin L (Bovine) Cathepsin H (Rat) Papain
5
10
1
"30 KDa component" Elastase (Human leukocyte) Cathepsin G (Human) Factor X (Bovine) Chymotrypsin (Bovine)
the
I),
homology
with
N-terminal
cathepsin
L,
a protease
heterogenous SDS-PAGE bands for
and
presented this
component.
The
homologous
to
lysosomes
(17).
in
the
with
described
by
same
Homology (%I 73 73 66 60 53
20
Homology (%I
Table
These results as that
are
Western-blottings
of a
suggest
the
All
these I)
and
the
30
kDa
I
is
highly
serine-proteases,
that
recognized
(Figure
using
protease
proteases,
also
(Table
called
elastase,
are
(13).
sequence
sequences
like
1) bands,
al
et
in
of
(16).
three
Moos
degree
proteases,
(Figure
into
shown
N-terminal
57 47 42 38
high
thiol-
collectively
sequence
a
origin
N-terminal
were
bFGF
of
components
resolved
identified
proteins, epitopes
kDa
leukocyte
particularly
15
showed
lysosomal
be
reason,
10
sequence
of 30
the
5
sequences
can
system
25
IVGGRKARPQELPFLASIQNQ IVGGRRARPHAW~NVSLQLR =~HS~YN@L~I3 -IVGGRDCAEGECPWQALLtiE --IVNGEEAVPGgWFWQ%LQEK ---
consensus
The
20
APDSIDYRKKGYVT-P APRSVDWREKGYVT-PVKDQGICGSCWA ZR&~%&KGY~T-~VKNQGQCGSCWA DDZ~~W&KGGVT-&KYQGACGSCWA -----YPSSMDWRKKGNWSPVKNQGACGSCWT IPEi!VDWRQKGAV!C-i%KNQGSCGSCWA ------*****
(B)
(Table
15
the
the
in
28 and 30 kDa share
may by
found
antisera
similar used
1).
DISCUSSION Here we report and serine-
proteases
that
proteins
by N-terminal 772
identified microsequencing
as thiolcan be
Vol.
170,
No.
BIOCHEMICAL
2, 1990
purified
along
are
These
by
to
internal
results
(not
containing
28-30
kDa It
molecular
weight
components has
of
may play FGFs
suggest
a role
from
process glands.
It
suggested
that
was
liberated
through
matrix the
Cathepsin
L
that
degrade
collagens,
(17,20).
These
enzymes
are
that
the
cells
hydrolysis under
(21,22).
proteases of
normal
pathological
of
they
the
stored
could
be
enzymes lysosomal
and proteoglycans
produced
and
is
described
ECM degrading
elastins
ECM components
action
and
are
are
it
secreted
elastase
described
the
extraction
which
Therefore,
we have
and/or
some
actively
FGFs
we have
are
leukocyte
proteases
transformed
basic
Therefore,
from
of
factor N-terminal
by
the
FGFs
(ECM),
action and
and
during
pituitary
extracellular
-
this
the
proteases
bovine
the
(19)
in
the
high
growth
(8,18,19).
that
serine these
procedures
proteases
to
whether
acidic
purification
tissue-specific
reasonable
in
the
thiol-and
that
by
preparations
exhibit
shown
presented
during
known
resin
Preliminary
bFGF
display
also
the
peptides
bFGF.
that
not
been
microheterogeneity generated
is
to against
of
components
activities.
It
strongly
raised
indicate
COMMUNICATIONS
heparin-Sepharose
sequences
protease
activity.
bind
antisera
shown)
the
RESEARCH
using
proteases
recognized
corresponding
BIOPHYSICAL
bFGF
with
chromatography. and
AND
and secreted
its
is
may play release
conceivable a role
of
by
stored
in
the FGFs
conditions.
ACKNOWLEDGMENTS - P.L.H. and A.G.G. would like to thank Drs. H.A. Armelin and M.C.S. Armelin for helpful discussions P.L.H. is on leave of and comments on the manuscript. absence from Institute Butantan, Sao Paulo, Brasil. This work was supported by the following brazilian research agencies : FINEP, FAPESP and CNPq. REFERENCES 1.
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Fett,
J.W.
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Vol.
170,
No.
2, 1990
BIOCHEMICAL
AND
2.
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
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