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Isolation and characterization of cyanogen bromide peptides from basement membrane collagen
BIOCHEMICAL
Vol. 47, No. 5, 1972
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ISOLATION AND CHARACTERIZATION OF CYANOGEN BROMIDE PEPTIDES FROM BASEMENT MEMBRANE COLLAGEN Nicholas
A. Kefalides
University of Pennsylvania Medical Division at the Philadelphia General Hospital Philadelphia, Pennsylvania 19104
ReceivedApril28,
1972
Summary: Twelve peptides were isolated and characterized after cyanogen bromide cleavage of the a-chain of basement membrane collagen from bovine lens capsule. Glycine accounted for about l/3 of all amino acids in each peptide. 4-Hydroxyproline was present in all; 3-hydroxyproline, in 7, and hydroxylysine, in 9 peptides. 54 to 100% of the hydroxylysine in these peptides was substituted with glucosyl-galactose; only 1 contained galactose. One peptide contained 2 residues of mannose, 1 of glucosamine, 4 of half-cystine, 11 of hydroxylysine, and 7 of glucosyl-galactose. The molecular weight of the chain is 100033 without the hexose and 113600 including the hexose. Introduction:
There
molecule
isolated
three
identical
pears
to contain
is
from a-l
various
chains
been
isolated
tendon,
bone
or cartilage.
include
the plus
nine
high
from
and arginine
and phenylalanine.
to date
contain
form
from
The a-l sues have
The unique
features
content
as well
Basement
chains
been
than
of interstitial
further
1151 Copyright
@ 1972, by Academic
Press, Inc.
membrane
composed
collagen chain
of this as the
for
skin,
high
sum of
amounts
of ala-
leucine,
isoexamined Hexose,
lo-13%,
0.2% by weight
by examining
ap-
collagen
collagens
from
of
which in
of half-cystine.
collagen
characterized
is
found
of valine,
accounts less
collagen
of a-l
decreased
amounts
the
membrane
collagens
the markedly
for
membranes
type
interstitial
4 to 8 residues
account
that
Basement
of glucosyl-galactose,
and hexosamine
indicate
distinct
and the high
leucine
the
(1).
hydroxylysine
hydroxylysine,
to
basement
a genetically
has not
lysine
evidence
while (2,
mannose
3).
a number the
in
of tis-
peptides
ob-
BIOCHEMICAL
Vol. 47, No. 5, 1972
tained
by cleavage
has proven gen
quite
with
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cyanogen
useful
in
bromide
carrying
(4,
out
5, 6),
sequence
a method
studies
which
on colla-
(7). In
the
present
terization
study,
of peptides
of the
cl-1 chain
we report
obtained
on the
isolation
by cleavage
of basement
membrane
with
collagen
and charac-
cyanogen from
bromide
bovine
lens
capsule. Materials
and Methods:
was prepared slight
according
acid
the
column
which
at 4'
C.
Prior
(1).
on CM-cellulose,
collagen
The u-1 chains to the
as described
previously
(1).
to the methods
--et al
(9).
For this
were dissolved with
cyanogen
nitrogen
molar
diluted
relative
lo-fold
one-half dissolved
by rotary in water
evaporation
(6,
material
CNBr to
residues,
followed
described
above.
Small
peptides
9) involved insure
again
were
with
0.1 M acetic
by chromatography
acid, (1.1
Eigner
on
6 Lewis
(8)
essentially
the
of
was incubated water
solution
gm) equal
residues
ac-
following
the
dissolved 30'
volume
which
it
more
treatment
complete
to
separated
the
meth-
lyophilized
of methionyl
and lyophilization
1152
reduced
of the
cleavage
larger
4 hrs,
of the
by evaporation
from
for
was lyophilized,
A modification
a second
flushed
to 150-fold
at
and the
and relyophilized.
ods described with
of CNBr
distilled
on a Bio-Gel
--et al (6) and Epstein of protein weighing 100 mg
samples
The mixture
with
a
by Miller
to methionyl
was added.
purified
was performed
20 ml of 70% formic
and a weight
excess
collagen
in
described
with
and chromatography
of Piez,
bromide
capsules
previously
and eluted
method
purpose,
lens
to denaturation
were prepared
according
cording
described
was equilibrated
with
anterior
was further
CM-cellulose
Cleavage
from
to the method
modification
P-300
The collagen
ones by
as
BIOCHEMICAL
Vol. 47, No. 5, 1972
molecular
sieve
Bio-Gel
P-4
Thirty
chromatography
(100-200
milligrams
out
acid
with
the
peptides
eluted
further
in the
(6).
components tained
small
containing
peptides single
0
Elution
rate
of
of the
on a phosphocellulose peak
acid.
were dissolved
is shown in peaks
retarded
M acetic
column.
at a flow
pattern
three
The most
essentially
on the
x 87 cm) of
0.1
peptides
and applied
elution
(1.5
with
lyophilized
chromatographed
by Miller
equilibrated
same solvent
A representative
hr.
on a column
mesh)
of the
1 ml 0.1 M acetic carried
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
was
24 ml per
Figure
1.
P-4 column
column
the
The were
as described
on P-4 was resolved
1 and 2 while
in
other
into
2
2 peaks
con-
components.
20
40 60 EFFLUENT
60 VOLUME,
100 ML
120
140
Figure 1. Elution pattern of CNBr peptides from the cc-1 chain of bovine lens capsule collagen chromatographed on Bio-Gel P-4. Elution was carried out with 0.1 N acetic acid at a flow rate of 24 ml hr. The peptides column
were
cm) using
Amino previously the
further
the
experiment
Technicon
which
acid
resolved
conditions
appears
eluted
and hexosamine
automatic
methods sugar
the
exclusion
on a CM-cellulose
described
in Figure
described
in
by Miller
volume column (6).
of the (1.5
P-4
x 17.5
A representative
2. analyses (10). analyzer. 1153
were performed
Neutral
sugars
according
were measured
The hydroxylysine
linked
to with
Vol. 47, No. 5,1972
0
BIOCHEMICAL
I 100
50
I 150
AND BiOPHYSlCAL
I 300 250 VOLUME, ML
200 EFFLUENT
RESEARCH COMMUNKATIONS
I 350
400
450
1 550
500
Figure 2. CM-cellulose chromatogram of the large CNBr peptides from the a-l chain of bovine lens capsule collagen which are eluted in the exclusion volume of the P-4 column (Figure 1). Elution was accomplished in 0.02 M (Na ) sodium citrate (pH 3-6) using a linear salt gradient of NaCl from 0.02 to 0.14 M in a total volume of 1000 ml.
glycosides
glucosyl-galactose
to a previous Kefalides
method
(12)
(11)
and Discussion:
collagen
were resolved
Peptides
1 through 1 having
of the
peptides,
between
these
published acid l-4
of Askenasi
peptides
fractions
designated
from
on Bio-Gel
P-4
as such according
molecular according
composition,
appear peptides
all
as well
on Table
I.
position
were resolved
from
accounts
peptides. on P-4
2 contain
no hydroxylysine
1 residue
each of hydroxylysine
as the
No homologies
and peptides
Glycine
4
capsule
(Figure
to their
Peptides
weight. to their
lens
5 through in
the
2).
acid
in
method
according
analyzer.
four
smallest
numbers
so far.
residues which
into
(Figure
The amino
acid
were isolated
by the
The CNBr cleaved
4 are
the
12 were assigned chromatogram
and measured
on an amino
Results
size,
and galactose
for
It
will
are
the
or hexose.
molecular
have been noted
interstitial about
l/3
be noted smallest. Peptides
collagens of the that
amino
peptides
Peptides
1 and
3 and 4 contain
and of glucosyl-galactose.
1154
weights
Pep-
1).
a. b.
1 6 593
i 0 0 0 0
0 1 0 0 0 0 0 1 8 749
0 (034)
11::
11 (0.6) 3 0 11 0 0 0 0 0 11
0
28 2704
i 1
0 1
9 1
2
i 1 1 0 7 60;2
31 4 3 0
4
4
5 3 3
2
1 0 0
2
0
1
z
3 3 0
w19,
8
5 2 3
Cl.11
1 1
:: 03 1 1 60 100 5718 9648
f
0
:
4 20
6
2 2
7
(Ij2)
(7;7)(1?9)
C’$8)
i
4
37 4 2 0 3 6 0 3 1 116 11217
7
7
(:ots) 14 (13.5) 5 2 8
2 2
3
9
9
Chains
1
72 8 5
14
16
9 3 8
31
5 2 ($7)
5
1
8 (8.3) 2 (1.7)
12
are given to nonintegral
54 6 5 4 4 9 2 5 1 176 17196
13
15
(21.3) 9 5 5
Lens
13 (13.4) 3 (2.6)
11
: ; 9 11 0 0 5 6 1 1 176 215 16906 19728
(lF4) 58 7 5
12
8 2 7
27
5 1.5
1
1 (1.3)
8.5
10
of Bovine
whole number. and hydroxyproline giving rise
26 4 2 0 2 3 0 1 1 81 7817
5
6
2 3
(9p
(ii5)
1
7
CNBr Peptides of the a-l Capsule Collagen 5 6 7 8
1
Residues per peptide, rounded off to the nearest Actual values for lysine, hydroxylysine, proline because of the possibility of partial hydroxylation
Glycine Alanine Valine l/2 Cystine Isoleucine Leucine Tyrosine Phenylal. Homoserine Total Mol. Wt.
Prolineb
(003)
1
Glutamic
1
0 1
t3 1
line Aspartic Threonine Serine
2 1 1
(0;6)
1 (0.7)
4-Ho-PI-O-b
(1.2) 1 0 0
3
(Oi2)
line
8
3-Ho-h-O-b
0 0 0
0 0
1 1
0 0
0
Histidine Arginine
(017)
$5)
0
Lysineb
0
4
0 0
0
2
1
3
Compositionaof
Acid
HOLysineb
Amino
‘I‘ABLE
in parentheses values.
E 4 29 54 3 26 7'1 1;;7 100033
348
68
78
5; 42
10 19 9.5 9.5 132
15
Total CNBr Peptides 58.5
BIOCHEMICAL
Vol. 47, No. 5, 1972
tide
5, with
and must
a molecular
represent
weight
the
Hydroxyproline
carboxyl
is present
4-hydroxyproline
to glycine
terminal
where
peptide
hydroxylation varies it
30%.
7 of
proline.
the
peptides
in
peptides
and the
almost
constant
except
significantly
lower.
identical in
the
in
about
42.
membrane
of lysine
lysine
varies
between
degree
of glycosylation
in
in the
C-
about
of
peptides
and
peptide
where in most
is
present
8% of the
hydroxy-
as much 3-hydroxypro-
collagen.
those
glucosyl-galactose
the
12 peptides
hydroxylation
all
3-Hydroxyproline
contains
throughout the
of
of proline
and hydroxylysine-bound
9 of
ratio
The degree
C-terminal
hydroxylation
collagen
to be distributed found
pepticle.
and represents
No interstitial
Hydroxylysine
are
terminal
no homoserine
in all
percent is
as does basement
pear
is
contains
60 and 75%, except
collagens
in only
is
of 6022,
is almost
The average
interstitial
line
it
of proline
between
is
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
pepticle
(Table peptides
50% and 100% with varies
II).
between
chain
ap-
since
they
The degree
containing an average
of
hydroxyof
82%.
50 and 100% with
The
an av-
TABLE II Carbohydrate
Composition Bovine
of CNBr Peptides of the Lens Capsule Collagen
1
2
2
2
2
2
l
0
0
0.85
0.9
0
3
0
0
0
0
0
0
Percent Glycosylation of HOLys
-
-
100
Mannose
00
Glucosamine
0
Glc-Gal-HOLys Gal-HOLys
0
2
of
Total CNBr Pepticles
10
11
12
556
6
7
6
39.5
000
10
0
1
100 - 78 73.5
S
cc-1 Chains
76 78
75
54 87.5
80.2
0
0
0
0
000
0
0
2
2
0
0
0
0
000
0
0
1
1
1156
BIOCHEMICAL
Vol. 47, No. 5, 1972
erage
of 80%.
respect other
Again
to degree
A very
ably
with
non-collagen,
glucosamine
off
(3,
is
linked
14).
it
Other
important
3) and confirmed Although
almost
here
the
of valine,
The molecular amino acid
content
cosyl-galactose, a molecular for newly
intact
a-l
The data from
indicate lens
This
via
with
only
prob-
mannosyl-mannosyl disulfide
pepsin
cleaves-
the linkage
in previous
low arginine
region
l/3
of
of
studies
(1,
and alanine that
present
in most
amino acids in the
the a-l if
chain
calculated
we add the
is
content
mannose and glucosamine, This
by chick
is consistent capsule
are derived
1157
of gluwe obtain
with
collagen
embryo lenses
the peptides
from the
contribution
the weight (1)
and the
(15).
of basement membrane from a genetically
2,
content.
and phenylalanine.
of sheep lens
capsule
the
a peptide,
the marked increases
100033;
that
12).
that
capsule
is about
of
collagen
of all
peptide
polypeptide
noted
the
of 113600.
chains
the
hydroxylysine
the sum of the non-polar
galactose, weight
II,
leaving
leucine
is
synthesized
collagen
include
weight
with
chain.
content
isoleucine,
the
the presence
a trisaccharide
of the lens
same by virtue
that
the mannose and glucosamine
to the collagen
collagens,
correlate
with
(13).
the possibility
observations
the alanine
interstitial
of all
collagen
out
although
(Table
polypeptide
and the
does not
with
observed
was the presence
contains
Digestion
the non-collagen
between
peptide
which
not
be pointed
collagen
observation
suggest
is noted
to glycosylated
skin
the presence
same peptide,
bonds
rat
in a single
along
peptides
vicinal
interesting
half-cystine
in the
for
should
of hydroxylysine,
in a triplet
has been reported
a feature It
in these
of glycosylation
of arginine
among peptides
collagens.
amount of arginine
fact,
a similarity
of glycosylation,
interstitial
degree
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
distinct
Vol.47,No.5,
o-1
On the
chain.
membrane
BIOCHEMICAL
1972
collagens
and interstitial brane
basis
of the
from
the
collagens,
collagens
in
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
general
differences
glomerulus
noted
between
and Descemet's
it
can be stated
form
a genetically
that
basement
membrane
basement
distinct
mem-
group
of
proteins. Acknowledgments: and AM-14526. Mrs.
Mary
This
study
The author
DeMaria
and Mr.
was supported
wishes John
to thank Darnell
for
by NIH Mrs. their
Grants
Emilie
AM-14805
Tomichek,
technical
assis-
tance. References: 1.
2. i: 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
KEFALIDES, N.A., Biochem. Biophys. Res. Comm. 45, 226 (1971). KEFALIDES, N.A., and DENDUCHIS, B. Biochemistry 8, 4613 (1969). KEFALIDES, N.A., Int. Rev. Exp. Pathol. 10, 1 (1971). BUTLER, W.T., PIEZ, K.A., and BORNSTEIN, P., Biochemistry 6, 3771 (1967). KANG, A.H., PIEZ, K.A., and GROSS, J., Biochemistry 8, 1506 (1969). MILLER, E.J., Biochemistry 10, 3030 (1971). KANG, A.H., BORNSTEIN, P., and PIEZ, K.A., Biochemistry, 6, 788 (1967). and LEWIS, M.S., Biochemistry 2, 58 PIEZ, K.A., EIGNER, E.A., (1963). EPSTEIN, E.H., Jr., SCOTT, R.D., MILLER, E.J., and PIEZ, K.A., J. Biol. Chem. 246, 1718 (1971). N.A., Biochim. Biophys. Acta DENDUCHIS, B., and KEFALIDES, 221, 357 (1970). and FORSELL-KNOTT, L., Biochim. Biophys. Acta KEFALIDES, N.A., 203, 62 (1970). XXENASI, R., and KEFALIDES, N.A., Anal. Biochem. 47, 67 (1972). BUTLER, W. T. in E.A.BALAZS (Ed.), Chemistry and Mxecular Biology of Intercellular Matrix, 1, Acad. Press, N.Y.,1970,p.149. KEFALIDES, N.A., Connect. Tissue ‘Res. 1, 3 (1972). and PROCROP, D.J., Fed. Proc. 31, GRANT, M.E., KEFALIDES, N.A., 480 Abs. (1972).
1158
Vol. 47, No. 5, 1972
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ISOLATION AND CHARACTERIZATION OF CYANOGEN BROMIDE PEPTIDES FROM BASEMENT MEMBRANE COLLAGEN Nicholas
A. Kefalides
University of Pennsylvania Medical Division at the Philadelphia General Hospital Philadelphia, Pennsylvania 19104
ReceivedApril28,
1972
Summary: Twelve peptides were isolated and characterized after cyanogen bromide cleavage of the a-chain of basement membrane collagen from bovine lens capsule. Glycine accounted for about l/3 of all amino acids in each peptide. 4-Hydroxyproline was present in all; 3-hydroxyproline, in 7, and hydroxylysine, in 9 peptides. 54 to 100% of the hydroxylysine in these peptides was substituted with glucosyl-galactose; only 1 contained galactose. One peptide contained 2 residues of mannose, 1 of glucosamine, 4 of half-cystine, 11 of hydroxylysine, and 7 of glucosyl-galactose. The molecular weight of the chain is 100033 without the hexose and 113600 including the hexose. Introduction:
There
molecule
isolated
three
identical
pears
to contain
is
from a-l
various
chains
been
isolated
tendon,
bone
or cartilage.
include
the plus
nine
high
from
and arginine
and phenylalanine.
to date
contain
form
from
The a-l sues have
The unique
features
content
as well
Basement
chains
been
than
of interstitial
further
1151 Copyright
@ 1972, by Academic
Press, Inc.
membrane
composed
collagen chain
of this as the
for
skin,
high
sum of
amounts
of ala-
leucine,
isoexamined Hexose,
lo-13%,
0.2% by weight
by examining
ap-
collagen
collagens
from
of
which in
of half-cystine.
collagen
characterized
is
found
of valine,
accounts less
collagen
of a-l
decreased
amounts
the
membrane
collagens
the markedly
for
membranes
type
interstitial
4 to 8 residues
account
that
Basement
of glucosyl-galactose,
and hexosamine
indicate
distinct
and the high
leucine
the
(1).
hydroxylysine
hydroxylysine,
to
basement
a genetically
has not
lysine
evidence
while (2,
mannose
3).
a number the
in
of tis-
peptides
ob-
BIOCHEMICAL
Vol. 47, No. 5, 1972
tained
by cleavage
has proven gen
quite
with
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cyanogen
useful
in
bromide
carrying
(4,
out
5, 6),
sequence
a method
studies
which
on colla-
(7). In
the
present
terization
study,
of peptides
of the
cl-1 chain
we report
obtained
on the
isolation
by cleavage
of basement
membrane
with
collagen
and charac-
cyanogen from
bromide
bovine
lens
capsule. Materials
and Methods:
was prepared slight
according
acid
the
column
which
at 4'
C.
Prior
(1).
on CM-cellulose,
collagen
The u-1 chains to the
as described
previously
(1).
to the methods
--et al
(9).
For this
were dissolved with
cyanogen
nitrogen
molar
diluted
relative
lo-fold
one-half dissolved
by rotary in water
evaporation
(6,
material
CNBr to
residues,
followed
described
above.
Small
peptides
9) involved insure
again
were
with
0.1 M acetic
by chromatography
acid, (1.1
Eigner
on
6 Lewis
(8)
essentially
the
of
was incubated water
solution
gm) equal
residues
ac-
following
the
dissolved 30'
volume
which
it
more
treatment
complete
to
separated
the
meth-
lyophilized
of methionyl
and lyophilization
1152
reduced
of the
cleavage
larger
4 hrs,
of the
by evaporation
from
for
was lyophilized,
A modification
a second
flushed
to 150-fold
at
and the
and relyophilized.
ods described with
of CNBr
distilled
on a Bio-Gel
--et al (6) and Epstein of protein weighing 100 mg
samples
The mixture
with
a
by Miller
to methionyl
was added.
purified
was performed
20 ml of 70% formic
and a weight
excess
collagen
in
described
with
and chromatography
of Piez,
bromide
capsules
previously
and eluted
method
purpose,
lens
to denaturation
were prepared
according
cording
described
was equilibrated
with
anterior
was further
CM-cellulose
Cleavage
from
to the method
modification
P-300
The collagen
ones by
as
BIOCHEMICAL
Vol. 47, No. 5, 1972
molecular
sieve
Bio-Gel
P-4
Thirty
chromatography
(100-200
milligrams
out
acid
with
the
peptides
eluted
further
in the
(6).
components tained
small
containing
peptides single
0
Elution
rate
of
of the
on a phosphocellulose peak
acid.
were dissolved
is shown in peaks
retarded
M acetic
column.
at a flow
pattern
three
The most
essentially
on the
x 87 cm) of
0.1
peptides
and applied
elution
(1.5
with
lyophilized
chromatographed
by Miller
equilibrated
same solvent
A representative
hr.
on a column
mesh)
of the
1 ml 0.1 M acetic carried
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
was
24 ml per
Figure
1.
P-4 column
column
the
The were
as described
on P-4 was resolved
1 and 2 while
in
other
into
2
2 peaks
con-
components.
20
40 60 EFFLUENT
60 VOLUME,
100 ML
120
140
Figure 1. Elution pattern of CNBr peptides from the cc-1 chain of bovine lens capsule collagen chromatographed on Bio-Gel P-4. Elution was carried out with 0.1 N acetic acid at a flow rate of 24 ml hr. The peptides column
were
cm) using
Amino previously the
further
the
experiment
Technicon
which
acid
resolved
conditions
appears
eluted
and hexosamine
automatic
methods sugar
the
exclusion
on a CM-cellulose
described
in Figure
described
in
by Miller
volume column (6).
of the (1.5
P-4
x 17.5
A representative
2. analyses (10). analyzer. 1153
were performed
Neutral
sugars
according
were measured
The hydroxylysine
linked
to with
Vol. 47, No. 5,1972
0
BIOCHEMICAL
I 100
50
I 150
AND BiOPHYSlCAL
I 300 250 VOLUME, ML
200 EFFLUENT
RESEARCH COMMUNKATIONS
I 350
400
450
1 550
500
Figure 2. CM-cellulose chromatogram of the large CNBr peptides from the a-l chain of bovine lens capsule collagen which are eluted in the exclusion volume of the P-4 column (Figure 1). Elution was accomplished in 0.02 M (Na ) sodium citrate (pH 3-6) using a linear salt gradient of NaCl from 0.02 to 0.14 M in a total volume of 1000 ml.
glycosides
glucosyl-galactose
to a previous Kefalides
method
(12)
(11)
and Discussion:
collagen
were resolved
Peptides
1 through 1 having
of the
peptides,
between
these
published acid l-4
of Askenasi
peptides
fractions
designated
from
on Bio-Gel
P-4
as such according
molecular according
composition,
appear peptides
all
as well
on Table
I.
position
were resolved
from
accounts
peptides. on P-4
2 contain
no hydroxylysine
1 residue
each of hydroxylysine
as the
No homologies
and peptides
Glycine
4
capsule
(Figure
to their
Peptides
weight. to their
lens
5 through in
the
2).
acid
in
method
according
analyzer.
four
smallest
numbers
so far.
residues which
into
(Figure
The amino
acid
were isolated
by the
The CNBr cleaved
4 are
the
12 were assigned chromatogram
and measured
on an amino
Results
size,
and galactose
for
It
will
are
the
or hexose.
molecular
have been noted
interstitial about
l/3
be noted smallest. Peptides
collagens of the that
amino
peptides
Peptides
1 and
3 and 4 contain
and of glucosyl-galactose.
1154
weights
Pep-
1).
a. b.
1 6 593
i 0 0 0 0
0 1 0 0 0 0 0 1 8 749
0 (034)
11::
11 (0.6) 3 0 11 0 0 0 0 0 11
0
28 2704
i 1
0 1
9 1
2
i 1 1 0 7 60;2
31 4 3 0
4
4
5 3 3
2
1 0 0
2
0
1
z
3 3 0
w19,
8
5 2 3
Cl.11
1 1
:: 03 1 1 60 100 5718 9648
f
0
:
4 20
6
2 2
7
(Ij2)
(7;7)(1?9)
C’$8)
i
4
37 4 2 0 3 6 0 3 1 116 11217
7
7
(:ots) 14 (13.5) 5 2 8
2 2
3
9
9
Chains
1
72 8 5
14
16
9 3 8
31
5 2 ($7)
5
1
8 (8.3) 2 (1.7)
12
are given to nonintegral
54 6 5 4 4 9 2 5 1 176 17196
13
15
(21.3) 9 5 5
Lens
13 (13.4) 3 (2.6)
11
: ; 9 11 0 0 5 6 1 1 176 215 16906 19728
(lF4) 58 7 5
12
8 2 7
27
5 1.5
1
1 (1.3)
8.5
10
of Bovine
whole number. and hydroxyproline giving rise
26 4 2 0 2 3 0 1 1 81 7817
5
6
2 3
(9p
(ii5)
1
7
CNBr Peptides of the a-l Capsule Collagen 5 6 7 8
1
Residues per peptide, rounded off to the nearest Actual values for lysine, hydroxylysine, proline because of the possibility of partial hydroxylation
Glycine Alanine Valine l/2 Cystine Isoleucine Leucine Tyrosine Phenylal. Homoserine Total Mol. Wt.
Prolineb
(003)
1
Glutamic
1
0 1
t3 1
line Aspartic Threonine Serine
2 1 1
(0;6)
1 (0.7)
4-Ho-PI-O-b
(1.2) 1 0 0
3
(Oi2)
line
8
3-Ho-h-O-b
0 0 0
0 0
1 1
0 0
0
Histidine Arginine
(017)
$5)
0
Lysineb
0
4
0 0
0
2
1
3
Compositionaof
Acid
HOLysineb
Amino
‘I‘ABLE
in parentheses values.
E 4 29 54 3 26 7'1 1;;7 100033
348
68
78
5; 42
10 19 9.5 9.5 132
15
Total CNBr Peptides 58.5
BIOCHEMICAL
Vol. 47, No. 5, 1972
tide
5, with
and must
a molecular
represent
weight
the
Hydroxyproline
carboxyl
is present
4-hydroxyproline
to glycine
terminal
where
peptide
hydroxylation varies it
30%.
7 of
proline.
the
peptides
in
peptides
and the
almost
constant
except
significantly
lower.
identical in
the
in
about
42.
membrane
of lysine
lysine
varies
between
degree
of glycosylation
in
in the
C-
about
of
peptides
and
peptide
where in most
is
present
8% of the
hydroxy-
as much 3-hydroxypro-
collagen.
those
glucosyl-galactose
the
12 peptides
hydroxylation
all
3-Hydroxyproline
contains
throughout the
of
of proline
and hydroxylysine-bound
9 of
ratio
The degree
C-terminal
hydroxylation
collagen
to be distributed found
pepticle.
and represents
No interstitial
Hydroxylysine
are
terminal
no homoserine
in all
percent is
as does basement
pear
is
contains
60 and 75%, except
collagens
in only
is
of 6022,
is almost
The average
interstitial
line
it
of proline
between
is
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
pepticle
(Table peptides
50% and 100% with varies
II).
between
chain
ap-
since
they
The degree
containing an average
of
hydroxyof
82%.
50 and 100% with
The
an av-
TABLE II Carbohydrate
Composition Bovine
of CNBr Peptides of the Lens Capsule Collagen
1
2
2
2
2
2
l
0
0
0.85
0.9
0
3
0
0
0
0
0
0
Percent Glycosylation of HOLys
-
-
100
Mannose
00
Glucosamine
0
Glc-Gal-HOLys Gal-HOLys
0
2
of
Total CNBr Pepticles
10
11
12
556
6
7
6
39.5
000
10
0
1
100 - 78 73.5
S
cc-1 Chains
76 78
75
54 87.5
80.2
0
0
0
0
000
0
0
2
2
0
0
0
0
000
0
0
1
1
1156
BIOCHEMICAL
Vol. 47, No. 5, 1972
erage
of 80%.
respect other
Again
to degree
A very
ably
with
non-collagen,
glucosamine
off
(3,
is
linked
14).
it
Other
important
3) and confirmed Although
almost
here
the
of valine,
The molecular amino acid
content
cosyl-galactose, a molecular for newly
intact
a-l
The data from
indicate lens
This
via
with
only
prob-
mannosyl-mannosyl disulfide
pepsin
cleaves-
the linkage
in previous
low arginine
region
l/3
of
of
studies
(1,
and alanine that
present
in most
amino acids in the
the a-l if
chain
calculated
we add the
is
content
mannose and glucosamine, This
by chick
is consistent capsule
are derived
1157
of gluwe obtain
with
collagen
embryo lenses
the peptides
from the
contribution
the weight (1)
and the
(15).
of basement membrane from a genetically
2,
content.
and phenylalanine.
of sheep lens
capsule
the
a peptide,
the marked increases
100033;
that
12).
that
capsule
is about
of
collagen
of all
peptide
polypeptide
noted
the
of 113600.
chains
the
hydroxylysine
the sum of the non-polar
galactose, weight
II,
leaving
leucine
is
synthesized
collagen
include
weight
with
chain.
content
isoleucine,
the
the presence
a trisaccharide
of the lens
same by virtue
that
the mannose and glucosamine
to the collagen
collagens,
correlate
with
(13).
the possibility
observations
the alanine
interstitial
of all
collagen
out
although
(Table
polypeptide
and the
does not
with
observed
was the presence
contains
Digestion
the non-collagen
between
peptide
which
not
be pointed
collagen
observation
suggest
is noted
to glycosylated
skin
the presence
same peptide,
bonds
rat
in a single
along
peptides
vicinal
interesting
half-cystine
in the
for
should
of hydroxylysine,
in a triplet
has been reported
a feature It
in these
of glycosylation
of arginine
among peptides
collagens.
amount of arginine
fact,
a similarity
of glycosylation,
interstitial
degree
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
distinct
Vol.47,No.5,
o-1
On the
chain.
membrane
BIOCHEMICAL
1972
collagens
and interstitial brane
basis
of the
from
the
collagens,
collagens
in
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
general
differences
glomerulus
noted
between
and Descemet's
it
can be stated
form
a genetically
that
basement
membrane
basement
distinct
mem-
group
of
proteins. Acknowledgments: and AM-14526. Mrs.
Mary
This
study
The author
DeMaria
and Mr.
was supported
wishes John
to thank Darnell
for
by NIH Mrs. their
Grants
Emilie
AM-14805
Tomichek,
technical
assis-
tance. References: 1.
2. i: 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
KEFALIDES, N.A., Biochem. Biophys. Res. Comm. 45, 226 (1971). KEFALIDES, N.A., and DENDUCHIS, B. Biochemistry 8, 4613 (1969). KEFALIDES, N.A., Int. Rev. Exp. Pathol. 10, 1 (1971). BUTLER, W.T., PIEZ, K.A., and BORNSTEIN, P., Biochemistry 6, 3771 (1967). KANG, A.H., PIEZ, K.A., and GROSS, J., Biochemistry 8, 1506 (1969). MILLER, E.J., Biochemistry 10, 3030 (1971). KANG, A.H., BORNSTEIN, P., and PIEZ, K.A., Biochemistry, 6, 788 (1967). and LEWIS, M.S., Biochemistry 2, 58 PIEZ, K.A., EIGNER, E.A., (1963). EPSTEIN, E.H., Jr., SCOTT, R.D., MILLER, E.J., and PIEZ, K.A., J. Biol. Chem. 246, 1718 (1971). N.A., Biochim. Biophys. Acta DENDUCHIS, B., and KEFALIDES, 221, 357 (1970). and FORSELL-KNOTT, L., Biochim. Biophys. Acta KEFALIDES, N.A., 203, 62 (1970). XXENASI, R., and KEFALIDES, N.A., Anal. Biochem. 47, 67 (1972). BUTLER, W. T. in E.A.BALAZS (Ed.), Chemistry and Mxecular Biology of Intercellular Matrix, 1, Acad. Press, N.Y.,1970,p.149. KEFALIDES, N.A., Connect. Tissue ‘Res. 1, 3 (1972). and PROCROP, D.J., Fed. Proc. 31, GRANT, M.E., KEFALIDES, N.A., 480 Abs. (1972).
1158