Vol. 84, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages
October 30, 1978
1038-1044
ISOLATION AND CHARACTERIZATION OF CALCIUM OXALATE CRYSTAL GROWTH INHIBITORS FROM HUMAN URINE*
Y. Nakagawa, E. T. Kaiser and F. L. Coe Michael Reese Hospital and Medical Center Chicago, Illinois 60616; Departments of Chemistry and Biochemistry, University of Chicago, Chicago, Illinois 60637 Received
September
5,1978
Summary: Four acidic polypeptides which inhibit the growth of calcium oxalate crystals have been isolated from normal human urine, and two of these have been characterized with respect to their amino acid and carbohydrate compositions. SDS-Polyacrylamide gel electrophoresis of either of the two latter inhibitors revealed one prominent band that migrated with an apparent molecular weight of 17,500 daltons. y-Carboxyglutamate is present in these inhibitors, and they contain a total of more than 25% glutamic and aspartic acids and less than 10% of basic amino acids. INTRODUCTION Evidence in human urine
supporting appearing
poly-L-aspartic inhibitors hibitory
acid (1).
to be acidic
of calcium
polypeptides
from
to report normal
them with
respect
oxalate
polypeptides
and poly-L-glutamic
We now wish
and characterizing MATERIALS
the presence
acid that
crystal
has been reported
have
been found
we have
succeeded
human urine to their
growth
, purifying amino
acid
inhibitors
recently,
and
to be effective in isolating
two of these and carbohydrate
model four
to homogeneity composition:
AND METHODS
One normal containing sodium in a refrigerator.
donor's azide.
24 urine (about 1.5 During the collection
a) was collected in a plastic bottle of urine, the container was stored
DEAE-Cellulose (Whatman DE-52) was recycled with acid and base, and Biogel P-100 (Bio-Rad) was equilibrated before use as recommended in the accompanying instructions. Preparative polyacrylamide gel electrophoresis was carried out by the use of a Canalco "Prep-Disc" apparatus with an LKB 2103 power supply. The electrophoresis equipment was maintained at 10" during each run by the use of a Lauda low temperature bath. Protein was monitored during column chromatography at 280 or 230 mu employing either LKB recorders (Uvicord II or Uvicord III> or a Beckman DU-2 spectrophotometer.
*
Supported
in-
by SCOR in
Urolithiasis,
USPHS Grant
0006-291X/78/0844-1038$01.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
1038
d lP50
AM 20585.
BIOCHEMICAL
Vol. 84, No. 4, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Calcium oxalate crystal growth inhibition was followed using the assay The molecular weights of the inhibitors method reported by Ito and Coe (1). isolated were determined by SDS polyacrylamide gel electrophoresis, as described by Osborn and Weber (2). Dialysis tubing with a molecular weight cut off of 8,000 was obtained from Arthur Thomas Co. and prepared by boiling in 2% NaHC03, 10 mM EDTA and water for 30 min each, then stored in 50% ethanol at 4". Amino acid analyses were performed by the use of Beckman 121 or Durrum D-500 analyzers after 24 hr acid hydrolysis of samples at 110" in evacuated tubes containing 6 N HCl. y-Carboxyglutamic acid was determined on the amino acid analyzer after 22 hr hydrolysis by 2.0 M KOH, followed by neutralization with HC104 as described by Hauschka (3). Amino-terminal determinations were carried out using dansyl chloride, following the method of Gross and Labouesse (4). The solvent systems used were water:formic acid (100:1.5, v/v> for the first dimension and, subsequently, benzene:acetic acid (9:1, v/v). Carbohydrate analyses were performed by the use of a Perkin-Elmer Model 910 gas chromatograph, and the method described by Clamp et al. (5) was followed. RESULTS The urine at 4" with
three
collected
was dialyzed
against
changes.
The dialyzed
urine
of 1 N NaOH, and NaCl was added of the 7.3,
sample,
l/10
containing
0.05
The resultant with
washed
DEAE-cellulose
was added
was filtered
the same buffer
containing
through
(about
and stirred
a glass
filter
200 ml of 0.6 M NaCl buffer. dialyzed
thoroughly
a concentration
against
of 0.05
to DEAE-cellulose NaCl gradient fractions,
purified using
12.2
separately 0.01
fractions,
0.05
4 II) until
M Tris-HCl, I and II,
urine
filter
by suction,
for
water.
After
collected,
eluting
M Tris-HCl, from
pH 7.3, were
them through containing
isolated
from
M Tris-HCl,
were
was with
combined,
then
was adjusted
eluted
to
subjected
by a linear
Two inhibitory
at conductivities These
P-100
The
was washed
fractions
column
1 M NaCl and 0.02% NaN3. each of the DEAE-cellulose
1039
and
obtained
and then
pH 7.3.
the column
a Biogel
30 min.
pH 7.3,
The slurry
NaCl,
mmho, and 14.6 mmho, respectively.
by passing
for
became colorless.
the solution
The proteins
pH
suction
and wash were
of solid
addition
M Tris-HCl,
gentle
and the cake produced
dialysis,
chromatography.
0.01
24 hr
the volume
and stirred
30 min.
The 0.6 M NaCl filtrates
for
To reduce
with
the filtrate
magnetically
M. with
in 300 ml of 0.01
M to 0.3 M in 0.01
A and B, were
of approximately
a glass
M NaCl by the addition
column from
equilibrated
through
water
to pH 1.3 by the
of 0.05
to the dialyzed
cake was suspended
0.6 M NaCl,
was adjusted
to a concentration
of DRAE-cellulose
M NaCl,
mixture
washed
filtered
volume
12 R of deionized
were
(2 x 110 cm) Two inhibitory fractions
by
Vol. 84, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
7 90
60
_
70
i 0
60
; c
50
2
40
u’ * >
3o
: t
20
m ; z
IO
60 NUM
FRACTION
EER
Figure l.--Elution profile of A-I inhibitor on preparative polyacrylamide gel electrophoresis. The column height was 6 cm and the gel was 10% cross-linked. Electrophoresis was performed at 10°C and 1490 V. Twenty drops/tube were collected. The buffer was 0.04 M Tris-HCl at pH 9.0.
this the
procedure.
The fractions
fractions
eluting
The purification Both
later,
fractions
A-I
and B-I
elution
and the protein
pattern
gel
and B-II,
case one prominent
daltons.
The N-terminal
phenylalanine The results are
purified for
A-I
providing
amino
of amino summarized
and B-I,
were
further is
for acid
with
fractions
are
good evidence
acids A-I
were
1 and 2.
1040
for
identified
and carbohydrate
in Tables
for
1.
both
A-I
pigment.
electrophoresis,
The inhibitory Subsequent and B-I
dansyl
activity SDS-
and revealed
of 17,500 derivatives
B-I.
analyses Five
however,
in progress.
weight
as their
for
a brown
gel
purity.
to a molecular
and serine
colorless;
contaminated
shown in Figure
corresponding
were
by preparative
was performed
band
was found
A-I
of the latter
electrophoresis
in each
fractions
were
peak match,
polyacrylamide
B-I
A-II
first,
and characterization
and a typical
(4);
eluting
performed
residues
on the A-I
of y-carboxyglutamic
and
BIOCHEMICAL
Vol. 84, No. 4, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE 1 AMINO ACID COMPOSITIONS OF THE INHIBITORS ACIDS PRESENT)*
(X OF TOTAL AMINO
25-3-A-I""
25-4-B-I
LYS
3.1%
2.9%
His
1.1
2.1
Arg
2.8
0
ASP Thr
12.9
15.0
a.2
9.5
Ser
7.7
13.5
Glu
16.9
17.0
Pro
2.1 13.6
13.1
6.3
7.5
5.9
6.6
Ile
1.7
2.2
Leu
6.1
5.5
W Phe
2.8
GUY Ala Val Met
a.2
4.4
*
Because of the limited amounts of the inhibitors which have isolated,Cys and Trp have not been determined. ** Employing alkaline hydrolysis following the method of Hauschka, 5.2 residues of y-carboxyglutamic acid/l,000 residues of glutamic acid were detected for the A-I sample. y-Carboxyglutamate acid was detected for the B-I fraction but was not determined quantitatively. been
acid
per
glutamic not
1,000 acid
sufficient
the A-I
residues was detected for
fraction
molecule.
decarboxylation
also
quantitative corresponds
Whether
y-carboxyglutamate
of glutamic
this
of this
residue
were
found
in the A-I
in the B-I
fraction;
analysis.
The y-carboxyglutamate
to considerably
means that
or whether
acid
not
the low during
all
less
however,
than
of the A-I
y-carboxyglutamate isolation
1041
fraction. the sample
one residue inhibitor content
of the inhibitor
y-Carboxy used was
content per
found
for
inhibitor
molecules observed remains
contain is
due to
to be de-
BIOCHEMICAL
Vol. 84, No. 4, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE 2 CARBOHYDRATE COMPOSITIONS
OF THE INHIBITORS
(WEIGHT X)
25-3-A-I
termined. with
Mannose,
than
2.0
1.2
Gal
16.8
3.1
Gal NAc
1.2
1.7
Glu NAc
1.0
1.0
Neu AC
2.2
3.2
amount
10% of basic
galactose,
aminic
acid
tially
greater
A-I,
Man
The total
less
were
of glutamic
and aspartic
amino
present
N-acetyl detected
amount
arginine
25-4-B-I
acids
galactosamine, in both
was not
found
in
than
these
N-acetyl
inhibitors.
of galactose
acids
the
crystal
B-I
was more than
growth
glucosamine
The A-I
did
measured
inhibitors.
and N-acetyl
fraction
contained
fraction;
also,
25%
neur-
a substan-
in contrast
to
in B-I.
DISCUSSION That been known been
calcium for
a long
the subject
composition of a non-stone
which
is
aspect that
in
to remove
they
Two of
determined.
of the
growth
four
inhibitory
the proteins we are
y-carboxyglutamic
acid. (9),
fractions,
A-I
by us,
has
(8).
and B-I of free
has also
their in the urine
and their
the brown
in some cases
proteins
The presence
and the acid
1042
have been detected
to remove
has
materials
have been made concerning
now attempting
proteins
in human urine
of the inhibitory
proteins
from urinary
of the purified
present
have been purified
two protein
such contaminants
are
the nature
and many proposals
Although
has been reported
inhibitors
However,
the remaining
composition
contain
in human urine
(6).
In our work, former.
present
possible
time
crystal
of controversy,
(7).
compositions
oxalate
it
chemical pigment
has not
A particularly we have
isolated
been
intriguin is
y-carboxyglutamic
been detected
in kidney
acid
BIOCHEMICAL
Vol. 84, No. 4, 1978
stones
The amino
(10).
different
from
phosphoprotein containing
ing
latter
of a number
(II),
statherin isolated
proteins
to be unique
tigating hibit
not only
proteins
binding
from saliva
from various
types
reported
oxalate
which
involve
vitamin
of bone
by which
(13-15).
growth
but
K in
their
also
are
bovine
dentin
Furthermore, a feature
glycoproteins in plasma
(13-16).
the urinary
and B-I
including
carbohydrate,
formation ins.tances
A-I
and y-carboxyglutamate-
acid-containing
in several
crystal
(12),
to contain
of y-carboxyglutamate
the mechanism
proteins
proteins
isolated
to the y-carboxyglutamic
K has been shown
calcium
of the urinary
of calcium
has been
The dependence on vitamin
compositions
those
proteins
of the
acid
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
appear-
A-I
and B-I.
and bone proteins
We are
proteins
none
currently
inves-
we have
isolated
of our
inhibitors
the relationship
into
biosynthesis.
ACKNOWLEDGMENTS We wish assistance
to thank
Mr.
and Professor
M. Zehfus
G. Dawson
and Mrs.
for
E. Kot for
useful
their
suggestions
expert
technical
on the carbohydrate
analyses.
REFERENCES 1)
Ito,
H. and Coe, F. L.,
2)
Weber,
3)
Hauschka,
4)
Gross,
5)
Clamp, J. R., (1967).
6)
Hodgkinson,A.,
Oxalic
7)
Howard,
The Johns Hopkins
8)
Ekstrsm,
9)
Fernlund,
K.,
Pringle, P. V.,
Am. J. Physiol.,
J. R. and Osborn, Anal.
Biochem.,
C. and Labouesse,
J. E.,
10)
Nelsetuen, Biophys.
11)
Lee,
12)
Schlessinger,
Dawson,
B.,
B. and Berggard,
S. L.,
P.,
Clin.
Chim.
212-223
C.,
in Biology
I., Acta,
J. Biol. 2,
1,
Biochim.
J.,
139,
Chem., 147-155
Enzymol.,
a,
463-470
(1969).
Biophys.
and Medicine,
Med.
(1977).
Academic
239-252 252,
Acta,
A. and Glonek,
T.,
D. H. and Hay, D. I.,
1043
148,
342-349 N. Y. (1977).
(1976).
8048-8057
(1977).
(1976).
Biochemistry, .I. Biol.
(1972).
Press,
G. L., Broderus, M., Zytokovica, T. H. and Howard, Res. Commun., 65, 233-240 (1975). Veis,
3-27
(1977).
J. Biochem.,
G. and Hough, Acid
455-463
M., Methods
so,
Eur.
233,
Chem.,
16, 252,
J. B.,
2971-2979 1689-1695
Biochem.
(1977). (1977).
Vol. 04, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
13)
Price, P. A., Proc. Natl.
14)
Lian, J. B., Prien, E. L., Jr., Glimcher, Invest., 59-, 1151-1157 (1977).
15)
Hauschka, P. V., 22, 3925-3929
16)
Stenflo,
J.,
Otsuka, A, S., Poser, Acad. Sci. U.S., 12,
Lian, J. (1975).
J. Biol.
J. W., Kristaponis, 1447-1451 (1976).
B. and Gallop,
Chem.,
241,
355-363
1044
J. and Ramen,
M. J. and Gallop, P. M., (1976).
Proc.
Natl.
P. M., Acad.
N.,
J. Clin. Sci.
U.S.,
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages
October 30, 1978
1038-1044
ISOLATION AND CHARACTERIZATION OF CALCIUM OXALATE CRYSTAL GROWTH INHIBITORS FROM HUMAN URINE*
Y. Nakagawa, E. T. Kaiser and F. L. Coe Michael Reese Hospital and Medical Center Chicago, Illinois 60616; Departments of Chemistry and Biochemistry, University of Chicago, Chicago, Illinois 60637 Received
September
5,1978
Summary: Four acidic polypeptides which inhibit the growth of calcium oxalate crystals have been isolated from normal human urine, and two of these have been characterized with respect to their amino acid and carbohydrate compositions. SDS-Polyacrylamide gel electrophoresis of either of the two latter inhibitors revealed one prominent band that migrated with an apparent molecular weight of 17,500 daltons. y-Carboxyglutamate is present in these inhibitors, and they contain a total of more than 25% glutamic and aspartic acids and less than 10% of basic amino acids. INTRODUCTION Evidence in human urine
supporting appearing
poly-L-aspartic inhibitors hibitory
acid (1).
to be acidic
of calcium
polypeptides
from
to report normal
them with
respect
oxalate
polypeptides
and poly-L-glutamic
We now wish
and characterizing MATERIALS
the presence
acid that
crystal
has been reported
have
been found
we have
succeeded
human urine to their
growth
, purifying amino
acid
inhibitors
recently,
and
to be effective in isolating
two of these and carbohydrate
model four
to homogeneity composition:
AND METHODS
One normal containing sodium in a refrigerator.
donor's azide.
24 urine (about 1.5 During the collection
a) was collected in a plastic bottle of urine, the container was stored
DEAE-Cellulose (Whatman DE-52) was recycled with acid and base, and Biogel P-100 (Bio-Rad) was equilibrated before use as recommended in the accompanying instructions. Preparative polyacrylamide gel electrophoresis was carried out by the use of a Canalco "Prep-Disc" apparatus with an LKB 2103 power supply. The electrophoresis equipment was maintained at 10" during each run by the use of a Lauda low temperature bath. Protein was monitored during column chromatography at 280 or 230 mu employing either LKB recorders (Uvicord II or Uvicord III> or a Beckman DU-2 spectrophotometer.
*
Supported
in-
by SCOR in
Urolithiasis,
USPHS Grant
0006-291X/78/0844-1038$01.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
1038
d lP50
AM 20585.
BIOCHEMICAL
Vol. 84, No. 4, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Calcium oxalate crystal growth inhibition was followed using the assay The molecular weights of the inhibitors method reported by Ito and Coe (1). isolated were determined by SDS polyacrylamide gel electrophoresis, as described by Osborn and Weber (2). Dialysis tubing with a molecular weight cut off of 8,000 was obtained from Arthur Thomas Co. and prepared by boiling in 2% NaHC03, 10 mM EDTA and water for 30 min each, then stored in 50% ethanol at 4". Amino acid analyses were performed by the use of Beckman 121 or Durrum D-500 analyzers after 24 hr acid hydrolysis of samples at 110" in evacuated tubes containing 6 N HCl. y-Carboxyglutamic acid was determined on the amino acid analyzer after 22 hr hydrolysis by 2.0 M KOH, followed by neutralization with HC104 as described by Hauschka (3). Amino-terminal determinations were carried out using dansyl chloride, following the method of Gross and Labouesse (4). The solvent systems used were water:formic acid (100:1.5, v/v> for the first dimension and, subsequently, benzene:acetic acid (9:1, v/v). Carbohydrate analyses were performed by the use of a Perkin-Elmer Model 910 gas chromatograph, and the method described by Clamp et al. (5) was followed. RESULTS The urine at 4" with
three
collected
was dialyzed
against
changes.
The dialyzed
urine
of 1 N NaOH, and NaCl was added of the 7.3,
sample,
l/10
containing
0.05
The resultant with
washed
DEAE-cellulose
was added
was filtered
the same buffer
containing
through
(about
and stirred
a glass
filter
200 ml of 0.6 M NaCl buffer. dialyzed
thoroughly
a concentration
against
of 0.05
to DEAE-cellulose NaCl gradient fractions,
purified using
12.2
separately 0.01
fractions,
0.05
4 II) until
M Tris-HCl, I and II,
urine
filter
by suction,
for
water.
After
collected,
eluting
M Tris-HCl, from
pH 7.3, were
them through containing
isolated
from
M Tris-HCl,
were
was with
combined,
then
was adjusted
eluted
to
subjected
by a linear
Two inhibitory
at conductivities These
P-100
The
was washed
fractions
column
1 M NaCl and 0.02% NaN3. each of the DEAE-cellulose
1039
and
obtained
and then
pH 7.3.
the column
a Biogel
30 min.
pH 7.3,
The slurry
NaCl,
mmho, and 14.6 mmho, respectively.
by passing
for
became colorless.
the solution
The proteins
pH
suction
and wash were
of solid
addition
M Tris-HCl,
gentle
and the cake produced
dialysis,
chromatography.
0.01
24 hr
the volume
and stirred
30 min.
The 0.6 M NaCl filtrates
for
To reduce
with
the filtrate
magnetically
M. with
in 300 ml of 0.01
M to 0.3 M in 0.01
A and B, were
of approximately
a glass
M NaCl by the addition
column from
equilibrated
through
water
to pH 1.3 by the
of 0.05
to the dialyzed
cake was suspended
0.6 M NaCl,
was adjusted
to a concentration
of DRAE-cellulose
M NaCl,
mixture
washed
filtered
volume
12 R of deionized
were
(2 x 110 cm) Two inhibitory fractions
by
Vol. 84, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
7 90
60
_
70
i 0
60
; c
50
2
40
u’ * >
3o
: t
20
m ; z
IO
60 NUM
FRACTION
EER
Figure l.--Elution profile of A-I inhibitor on preparative polyacrylamide gel electrophoresis. The column height was 6 cm and the gel was 10% cross-linked. Electrophoresis was performed at 10°C and 1490 V. Twenty drops/tube were collected. The buffer was 0.04 M Tris-HCl at pH 9.0.
this the
procedure.
The fractions
fractions
eluting
The purification Both
later,
fractions
A-I
and B-I
elution
and the protein
pattern
gel
and B-II,
case one prominent
daltons.
The N-terminal
phenylalanine The results are
purified for
A-I
providing
amino
of amino summarized
and B-I,
were
further is
for acid
with
fractions
are
good evidence
acids A-I
were
1 and 2.
1040
for
identified
and carbohydrate
in Tables
for
1.
both
A-I
pigment.
electrophoresis,
The inhibitory Subsequent and B-I
dansyl
activity SDS-
and revealed
of 17,500 derivatives
B-I.
analyses Five
however,
in progress.
weight
as their
for
a brown
gel
purity.
to a molecular
and serine
colorless;
contaminated
shown in Figure
corresponding
were
by preparative
was performed
band
was found
A-I
of the latter
electrophoresis
in each
fractions
were
peak match,
polyacrylamide
B-I
A-II
first,
and characterization
and a typical
(4);
eluting
performed
residues
on the A-I
of y-carboxyglutamic
and
BIOCHEMICAL
Vol. 84, No. 4, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE 1 AMINO ACID COMPOSITIONS OF THE INHIBITORS ACIDS PRESENT)*
(X OF TOTAL AMINO
25-3-A-I""
25-4-B-I
LYS
3.1%
2.9%
His
1.1
2.1
Arg
2.8
0
ASP Thr
12.9
15.0
a.2
9.5
Ser
7.7
13.5
Glu
16.9
17.0
Pro
2.1 13.6
13.1
6.3
7.5
5.9
6.6
Ile
1.7
2.2
Leu
6.1
5.5
W Phe
2.8
GUY Ala Val Met
a.2
4.4
*
Because of the limited amounts of the inhibitors which have isolated,Cys and Trp have not been determined. ** Employing alkaline hydrolysis following the method of Hauschka, 5.2 residues of y-carboxyglutamic acid/l,000 residues of glutamic acid were detected for the A-I sample. y-Carboxyglutamate acid was detected for the B-I fraction but was not determined quantitatively. been
acid
per
glutamic not
1,000 acid
sufficient
the A-I
residues was detected for
fraction
molecule.
decarboxylation
also
quantitative corresponds
Whether
y-carboxyglutamate
of glutamic
this
of this
residue
were
found
in the A-I
in the B-I
fraction;
analysis.
The y-carboxyglutamate
to considerably
means that
or whether
acid
not
the low during
all
less
however,
than
of the A-I
y-carboxyglutamate isolation
1041
fraction. the sample
one residue inhibitor content
of the inhibitor
y-Carboxy used was
content per
found
for
inhibitor
molecules observed remains
contain is
due to
to be de-
BIOCHEMICAL
Vol. 84, No. 4, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE 2 CARBOHYDRATE COMPOSITIONS
OF THE INHIBITORS
(WEIGHT X)
25-3-A-I
termined. with
Mannose,
than
2.0
1.2
Gal
16.8
3.1
Gal NAc
1.2
1.7
Glu NAc
1.0
1.0
Neu AC
2.2
3.2
amount
10% of basic
galactose,
aminic
acid
tially
greater
A-I,
Man
The total
less
were
of glutamic
and aspartic
amino
present
N-acetyl detected
amount
arginine
25-4-B-I
acids
galactosamine, in both
was not
found
in
than
these
N-acetyl
inhibitors.
of galactose
acids
the
crystal
B-I
was more than
growth
glucosamine
The A-I
did
measured
inhibitors.
and N-acetyl
fraction
contained
fraction;
also,
25%
neur-
a substan-
in contrast
to
in B-I.
DISCUSSION That been known been
calcium for
a long
the subject
composition of a non-stone
which
is
aspect that
in
to remove
they
Two of
determined.
of the
growth
four
inhibitory
the proteins we are
y-carboxyglutamic
acid. (9),
fractions,
A-I
by us,
has
(8).
and B-I of free
has also
their in the urine
and their
the brown
in some cases
proteins
The presence
and the acid
1042
have been detected
to remove
has
materials
have been made concerning
now attempting
proteins
in human urine
of the inhibitory
proteins
from urinary
of the purified
present
have been purified
two protein
such contaminants
are
the nature
and many proposals
Although
has been reported
inhibitors
However,
the remaining
composition
contain
in human urine
(6).
In our work, former.
present
possible
time
crystal
of controversy,
(7).
compositions
oxalate
it
chemical pigment
has not
A particularly we have
isolated
been
intriguin is
y-carboxyglutamic
been detected
in kidney
acid
BIOCHEMICAL
Vol. 84, No. 4, 1978
stones
The amino
(10).
different
from
phosphoprotein containing
ing
latter
of a number
(II),
statherin isolated
proteins
to be unique
tigating hibit
not only
proteins
binding
from saliva
from various
types
reported
oxalate
which
involve
vitamin
of bone
by which
(13-15).
growth
but
K in
their
also
are
bovine
dentin
Furthermore, a feature
glycoproteins in plasma
(13-16).
the urinary
and B-I
including
carbohydrate,
formation ins.tances
A-I
and y-carboxyglutamate-
acid-containing
in several
crystal
(12),
to contain
of y-carboxyglutamate
the mechanism
proteins
proteins
isolated
to the y-carboxyglutamic
K has been shown
calcium
of the urinary
of calcium
has been
The dependence on vitamin
compositions
those
proteins
of the
acid
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
appear-
A-I
and B-I.
and bone proteins
We are
proteins
none
currently
inves-
we have
isolated
of our
inhibitors
the relationship
into
biosynthesis.
ACKNOWLEDGMENTS We wish assistance
to thank
Mr.
and Professor
M. Zehfus
G. Dawson
and Mrs.
for
E. Kot for
useful
their
suggestions
expert
technical
on the carbohydrate
analyses.
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