/. Embryol. exp. Morph. Vol. 28, 1, pp. 77-86, 1972
Printed in Great Britain
77
Inhibitors in amphibian
morphogenesis: Enzymic degradation of an
inhibitor for the vegetalizing factor
By J. BORN, H. TIEDEMANN 1 AND
H. TIEDEMANN 1
From the Freie Universitdt Berlin, Institut fur Biochemie und
Molekularbiologie
SUMMARY
A naturally occurring inhibitor for the vegetalizing inducing factor has been incubated
with different enzymes. Pancreatic ribonuclease, ribonuclease-Tx, deoxyribunuclease, neuraminidase as well trypsin and papain diminish the inhibitor only very slightly or not at all.
Pronase, a proteolytic enzyme from Streptomyces griseus inactivates the inhibitor completely.
INTRODUCTION
A morphogenetic factor, which induces mesodermal and endodermal tissues
in the gastrula ectoderm of Triturus alpestris has been isolated from chicken
embryos (Tiedemann, 1968; Kocher-Becker & Tiedemann, 1971). The factor is
protein in nature and has been called the vegetalizing factor, because in normogenesis the Anlage for mesoderm and endoderm is located in the vegetal half of
the embryo. A protein fraction with the same biological activity, which is, however, not as highly purified, has been extracted from gastrula stages of Xenopus
laevis (Faulhaber, 1970).
In amphibian embryos a similar factor is distributed in an active form in the
mesoderm as well as the endoderm Anlage. Both these Anlagen induce mesodermal and endodermal tissues, if tested by the implantation (Mangold, 1924)
or sandwich (Holtfreter, 1933) method on gastrula ectoderm. The gastrula
ectoderm itself does not contain active vegetalizing or neuralizing factor. The
ectoderm contains however, factors in a masked form, which can be unmasked
by treatment with ethanol or with phenol. After treatment with ethanol, ectoderm induces neural structures and after treatment with phenol to a small
extent also mesodermal structures (Tiedemann, Becker & Tiedemann, 1961).
These results may indicate that inactive inducer-inhibitor complexes exist in
the ectoderm which can be activated by treatment with ethanol or phenol.
1
Authors'' address: Institut fur Biochemie und Molekularbiologie, 1 Berlin 33, Arnimallee
22, Germany.
78
J. BORN, H. TIEDEMANN AND H. TIEDEMANN
Other explanations are not yet completely excluded. The inhibitor hypothesis is
supported by recent experiments which have shown that inhibitory fractions can
actually be isolated from chicken as well as amphibian embryos. After extraction
of 105000 g supernatant from chick embryo homogenates with phenol at
60 °C the inhibitor is found in the aqueous phase. If the inducing protein fraction (isolated from the phenol layer) is recombined with the inhibitory aqueous
layer in 6 M urea and dialysed against 0-5 % NaCl solution a re-inhibition of the
vegetalizing factor occurs (Tiedemann, 1968).
In this paper we describe the incubation of the inhibitory fraction with enzymes
which degrade different types of macromolecules. The results of these experiments - whether the inhibitory activity remains or whether it is lost after enzymic
incubation - are discussed together with experiments on the partial purification
(Born, Tiedemann & Tiedemann, 1971) of the inhibitor.
MATERIALS AND METHODS
Recombination of'inhibitor'' and 'induced. Preparation for biological testing
The inhibitory fraction was prepared by homogenizing 20 g portions of
11-day chicken trunks with 2 vol. 0-5% NaCl intensively in a Potter homogenizer with Teflon pestle (about 5 min at maximal 3000 rev/min in an ice
bath), treatment of the supernatant (2h at 105000 g) with redistilled phenol
and precipitation of the material in the aqueous phase by 2 vol. ethanol
according to step 1 of the procedure of Born, Tiedemann & Tiedemann
(1971). The precipitate which contains the inhibitor was dissolved in 0-5 %
NaCl. In some experiments the inhibitor fraction was centrifuged in a
Spinco Ti 50 rotor (50 min, 155000 g) and the supernatant used for the recombination experiments. No significant differences of the inhibitory activity
between centrifuged and uncentrifuged supernatant have been observed. In a
few experiments the inhibitor was further purified by treatment with hydroxylapatite to remove most of the RNA (Born et ah 1971).
The inhibitor fractions were incubated with or without enzyme and recombined with 2-5 mg of the inducing protein fraction (isolated from the phenol
+ middle layer by precipitation with 4 vol. ethanol). Urea (Merck p. A.) which
was filtered through anion (IRA 400) and cation (IR 120) exchangers in 7 M
solution in the cold room to remove isocyanate and then freeze dried was added
to 6 M final concentration (total volume 1-2-1-5 ml) and dialysed for 20-24 h
against 3 x 5 1 . water. NaCl (final cone. 0-5 % or 5 %) was then added and the
mixture precipitated with 2 vol. ethanol (ice bath or - 2 5 °C bath). After being
washed twice with 66 % ethanol, the precipitate was dried for 2 h at 20 °C on a
suction pump. Almost identically sized pieces of the pellet were tested by
implanting them into the blastocoel of an early gastrula of Triturus alpestris
(Implantation method, Mangold, 1924). The embryos were cultured for about
14 days in Holtfreter solution with 0-1 % Cibazol or 0-1 % Gantrisin.
Inhibitors in amphibian morphogenesis
79
The amount of inhibitor is standardized by' g-equivalents'. One' g-equivalent'
is the amount of inhibitor fraction derived from 1 g wet tissue.
One 'g-equivalent' centrifuged inhibitory aqueous phase from 11-day chicken
trunks contains about 150 fig RNA and 50 pig glucose-equivalents (measured by
the anthron method, Spiro, 1966).
Incubation with DNase (E.C. 3.1.4.5; Worthington DPFF, electrophoretically
purified).
1/10 vol. of 0-1 M-MgCl2 and the amount of DNase stated in Table 1 were
added. After incubation for 60 min at 37 °C, 2-5 mg protein and urea were
added, dialysed and prepared for testing as described.
Incubation with pancreatic RNase (E.C. 2.7.7.16; Worthington RASE).
RNase dissolved in 002ml-005 ml 0 1 M phosphate buffer (pH 7-4) was
added and the pH controlled during incubation (60 min at 37 °C). If larger
amounts of RNase were added, the samples were extracted after enzymic
incubation with 1 vol. 80 % phenol (5 min, 60 °C), the phenol layer and the
middle layer re-extracted with 1 ml 0-5 % NaCl (5 min, 20 °C), the combined
aqueous layers precipitated by 2 vol. ethanol (15 min, - 2 5 ° C ) and washed
twice with 96 % ethanol. The precipitate was dissolved in 0-5 ml 0-5 % NaCl and
recombined with inducer protein as described.
In experiment H 125 the inhibitor was incubated with RNase, extracted with
phenol and then subjected to gel chromatography on Sephadex G 50 (medium)
in 6 M urea-0-5 M - N H 4 H C O 3 . 900 mg urea and 98-8 mg NH 4 HCO 3 were added
to 2 ml inhibitor. The mixture was applied to a 1-7 cm x 54 cm Sephadex G 50
column and eluted at 8 ml/h in the cold room. •} of the exclusion peak (~ 7-5 ml)
was adjusted to pH 7, dialysed against 6 M urea - 0-5 % NaCl (6 h) and 500 pig
tRNA (Boehringer, Tutzing) from baker's yeast added. The fraction was then
recombined as described.
Separate experiments have shown that the inhibitor fraction does not contain
an inhibitor for pancreatic RNase.
Incubation with RNase-Tx (from Aspergillus oryzae; E.C. 2 . 7 . 7 . 2 6 ; Worth-
ington RTX). The RNase was diluted and aliquots dialyzed for 3 h against water.
To about 0 4 ml inhibitor fraction, 0-1 ml 0-1 M Tris (pH 7-5) and 6000 U RNase-^
(~ 10 jug) were added, incubated for 60 min at 37 °C and then recombined
with inducer protein as described.
Incubation with trypsin (E.C. 3.4.4.4; Worthington 2xcryst. TRL). After
incubation with trypsin for 60 min at 37 °C, 2-5 peg trypsin inhibitor (Worthington) were added for each jag trypsin, incubated for 5 min at 20 °C and recombined with inducer protein.
Incubation withpapain (E.C. 3.4.4.10; Boehringer, Mannheim). Papain was
activated with cystein (Smyterman, 1967; Kimmel & Smith, 1954). To 0-5 ml
(5 mg) papain 007 ml 005 M cystein-Hl and 007 ml 001 M - E D T A were added,
the pH adjusted to 6 and the mixture subjected to chromatography on Sephadex
G 50 (0-9 x 30 cm) in 0-02 M-Na-acetate pH 5 in N2-atmosphere (elution rate
—
Inducer control: * H 87/3
29
29
30
147
102
RNase (10 fig)
RNase-Tj (6000 U)
RNase-Ti (6000 U)
—
—
40
—
H 125/3; H 125/6
Inducer control*: H 1969;
H 126/2; H 134/1; H 136/1
|J/-»C 1 'f l\7f±
81
13
51
31
37
54
21
60
77
69
31
80
40
46
80
22
54
33
.rUMUVC
A
38
—
9
—
4
—
—
2
36
24
3
23
2
8
10
4
7
24
—
7
7
15
4
5
9
28
17
—
30
7
8
4
2
18
7
Large Medium
r
v /o)
f Extraction with phenol after incubation at 37 °C.
45
RNase (30 ^g)t
58
RNase (30 /tg)t,
5 % NaCl
62
RNase (60/*g)|,
Sephadex G 50,
Chromat. 5 % NaCl
—.
135
5 % NaCl
* Not incubated at 37 °C.
30
30
27
28
38
53
109
30
54
28
27
cases
INO. OI
RNase (15 fig)
DNase (10-20 fig)
DNase (20 fig)
DNase (20 fig) +
RNase (15 fig)
DNase (20 fig)
Enzyme
30
RNase (100/ig)f
30
-t
2-5 (110 °C) RNase (15 fig)
—
2-5 (110 °C)
H 122/1; H 122/3
H 122/2; H 122/4
H98/1
H98/3
H83/2
Inhibitor control: H 83/1 +
H 83/3
H 70/6; H 74/2; H 7 4 / 3 ;
2-5-3-0
H 90/1 %
Inducer control*: H 70/7
—.
H81/1
2-5
Inducer control*: H 81/2
—
Inhibitor control*: H 67/4;
2-5
H 71/3; H 73/3; H 79/1; H 79/2
—
Inducer control:* H 1968;
H 7 1 / 1 ; H 79/3 ; H 99/2
2-5
1-25
1-25
H 87/1+2
H90/2
H90/5
Fraction
gequivalents
inhibitor
—
—
11
—
4
5
11
—
—
4
—
—
—
—
2
3
14
10
3
10
2
8
71
—
7
13
27
21
26
54
18
47
—
—
3
11
20
2
14
—
24
7
32
27
27
20
50
26
fied
11CU.
60
62
7
70
5
16
50
2
—
4
.
4
4
7
tail
1 1 UlliV"
Trunk
HinH
head
JTL111U.
.—
•—
•—
—
1
—
1
—
—
head
JT orc~
(
Induced region (%)
J 1-5 g-equivalents.
19
13
36
24
18
50
16
50
13
28
28
27
31
29
10
20
32
19
Small
Size of inductions
Induction
not
z
Z
O
m
H
oX
-^
>
O
a
H
to
n
i"
Table 1. Incubation of the inhibitor for the vegetalizing factor with DNase, pancreatic RNase and RNase-Tx: very high doses of
RNase, melting of RNA at 110 °C before incubation, extraction of RNase by phenol at 60 °C and removal of polynucleotides by
Sephadex G 50 chromatography did not alter the results. The unincubated inhibitory fractions (' inhibitor control') and the enzymically
incubated inhibitory fractions were combined with 2-5 mg inducer protein. The amount of inhibitory fraction is given in g-equivalents oo
{see Methods)
H 70/1; H 74/4
H80/1
Inhibitor control: H 70/4;
H 70/8; H 74/1 ; H 80/2
Inhibitor control*: H 67/4;
H 73/3; H 71/3
Protein control*: H 1968;
H 71/1; H 79/3 ; H 99/2
H 207/3 ; H 207/4
Inhibitor control; H 207/1
Protein control H 190/2*
Fraction
—
Papain (100 fig)
—
60
60
—
A
6
3
4
2
36
6
—
45
49
77
50
29
90
86
102
62
17
20
8
—
20
28
9
8
3
6
Large Medium
52
40
32
62
30
108
cases
"No of P<JolllVC
* Not incubated at 37°C.
—
2-5
—
—
—
Trypsin (20 /*g)
Trypsin (100/tg)
Enzyme
2-5
2-5
2-5
equivalents
inhibitor
CT—
&
36
29
25
13
37
37
33
22
Small
Size of induction
—
—
—
—
1
—
—
head
11
—
5
14
2
5
—
5
head
HinH
XJ.111U-
A
14
35
29
15
7
—
75
37
8
60
29
33
23
fi
PH
liCVJ.
Induction not
21
7
5
tail
Tmnk"XA H1JJV
Induced region (%)
Table 2. Incubation oj the inhibitor Jor the vegetalizing Jactor with trypsin and papain: the amount oj inhibitory Jraction is given
in g-equivalents (see Methods). All combinations have been carried out with 2-5 mg inducer protein. Trypsin activity was stopped after
incubation by addition oj trypsin inhibitor. Papain was removed ajter incubation by extraction with phenol (Jor more details see
Methods).
oo
I
100
21
90
91
71
21
72
107
135
21
19
20
22
21
28
72
—
—
Pronase P (3/*g)t
Pronase P (30/tg)t
81
80
—
33
20
18
25
45
36
19
24
23
22
19
Medium
38
38
9
46
19
Large
29
19
25
36
71
21
28
19
19
14
31
Small
—
—
—
—
—
2
—
—
18
5
36
29
11
15
26
30
62
5
75
59
10
0
60
71
12
30
63
m
I-H
H
d
d
m
H
X
H 142/1; H 142/2; H 142/3;
H 166/1 ; H 166/2
Inhibitor control: H 142/4; H 166/3
Protein control: H 157; H 172/1
Fraction
149
33
72
20-300 U
—
—
2-5
—
Enyzme
No. of
cases
2-5
equivalents
inhibitor
46
72
25
9
19
5
15
25
5
21
28
15
Size of induction
(%)
Positive
,
*
,
Large Medium Small
(%)
—
—
1
Forehead
18
18
2
Hindhead
12
60
3
>
Trunktail
Induced region ( %)
24
7
20
Induction not
specified
(%)
Table 4. Incubation of the inhibitor for the vegetalizing factor with neuraminidase: the amount of inhibitory fraction is given in d
w
g-equivalents {see Methods). All combinations have been carried out with 2-5 mg inducer protein
24
16
15
14
57
21
7
7
27
25
9
InducInduced region (%)
tion not
/
*
^
speciFore- Hind- Trunkfied
head
head
tail
(%)
f Pronase preincubated 2 h at 37 °C % Treated with hydroxylapatite.
51
72
80
79
No. of Positive
cases
(%)
Pronase P (20 /tg)
Enzyme
* Not incubated at 37 °C and not extracted with phenol,
H 108/1; H 118/1; H 118/3
2-5-30
Inhibitor control: H 108/2;
2-5-3-0
H 118/2; H 118/4
Inhibitor control *: H 102;
2-5
H 1 0 2 A ; H 101/2; H 115/4
Inducer control*: H 1969
—
H 126/2; H 134/1; H 136/1;
H 141/4
H 230/2
40
Inhibitor control: H 230/3
40
Protein control*: H 190/2
—
H173/1
6-0*
Inhibitor control: H 173/2
6-01
Inhibitor control*: H 173/3
6-0J
Inducer control*: H 157; H 172/1 —
Fraction
gequivalents
inhibitor
Size of induction
Table 3. Incubation of the inhibitor for the vegetalizing factor with pronase: the amount of inhibitory fraction is given in g-equivalents {see Methods). All combinations have been carried out with 2-5 mg inducer protein. Pronase was removed after incubation by
extraction with phenol
oo
Inhibitors in amphibian morphogenesis
83
4-5 ml/10 min). The papain peak was separated from the peak of self-digested
papain and adjusted to pH 5-5. The amount of papain was calculated from
A280 nm = 24 for a 1 % solution. The inhibitor was incubated with aliquots of
the papain solution for 60 min at 37 °C, adjusted to pH 7 and extracted twice
with phenol as described (see incubation with RNase).
Incubation with pronase (pronase P; Serva Heidelberg). The inhibitor was
incubated with pronase for 60 min at 30 °C, extracted twice with phenol as
described and the combined aqueous layers recombined with inducer protein.
Incubation with neuraminidase (from Vibrio cholerae; 3.2.1.18; Behringwerke, Marburg), y^vol. 0-5 M-Na-acetate (pH5-5); TV vol. 0-1 M-CaCl2 and
20-300 U neuraminidase (1 U ~ 1 /ig N-acetylneuraminic acid split off from
o^-glvco-protein in 15 min at 37 °C) were added to the inhibitor fraction. The
mixture was incubated for 30 min at 37 °C, adjusted to pH 6-7 and then recombined with 2-5 mg inducer protein.
Neuraminic acid was measured after hydrolysis (1 h. 70 °C at pH 2) with the
thiobarbituric acid procedure according to Aminoff (1961).
RESULTS
In the first series of experiments the inhibitor was incubated with deoxyribonuclease or with a combination of deoxyribonuclease and ribonuclease
(Table 1). A loss of inhibitory activity was not observed. This is not surprising
because no DNA can be detected in the inhibitor fraction. In a control series
deoxyribonuclease but no inhibitor was added to the inducing protein fraction
(H 87/3) to show whether DNase has an effect on gastrula ectoderm in the biological test. In this control series the inducing capacity is not reduced. Obviously
the responsiveness of gastrula ectoderm is not diminished by deoxyribonuclease
added to the inducing protein fraction.
Pancreatic ribonuclease as well as ribonuclease-Tx do not inactivate the inhibitor
either (Table 1). The inducing capacity for trunk and tail structures is somewhat
higher (16 %) as compared to the inhibitor control without RNase (5 %). But
more than one half of these inductions are small mesenchymatic tails. Addition
of pancreatic RNase (H 70/7) or ribonuclease-Tj (H 81/2) to the inducer protein does not weaken the inductive response. Treatment of the inhibitor with
large amounts of pancreatic RNase (up to 100 jug) also did not diminish the
inhibitory activity. In these experiments RNase has been removed after incubation by extraction with phenol at 60 °C (H 98/1; control without RNase
H 98/3). The extraction with phenol must be carried out at 60 °C. Extraction at
20 °C did lead to a loss of inhibitory activity. In another experiment (H 83/2,
Table 1) the RNA was heated to 110 °C and rapidly cooled to melt helical
structures which may be present in the RNA and then incubated with RNase.
No loss of the inhibitory activity was observed.
The RNase treated inhibitor was slightly more active when the recombination
6-2
84
J. BORN, H. TIEDEMANN AND H. TIEDEMANN
was carried out at a concentration of 5 % NaCl. This result was also obtained in
other experiments where the inhibitor was not incubated with RNase.
The inhibitory activity is not lost when, after incubation with RNase the
polynucleotides are separated by chromatography on Sephadex G50 (H 125).
The inhibitory activity is found in the exclusion peak, which contains glycoproteins and mucopolysaccharides.
After incubation with trypsin the inhibitory activity was diminished either to
a very small extent (H 70/1, H 74/4; Table 2) or not at all (H 80/1; Table 2).
The inductions obtained were mostly small mesenchymatic tails. The same result
was obtained when the inhibitor was incubated with papain (H 207, Table 2).
In both series of experiments it is excluded that the proteolytic enzymes destroy
the biological activity of the inducing protein. The activity of trypsin was completely stopped by addition of soy bean trypsin inhibitor before combination of
the incubated inhibitory fraction with the inducing protein. Addition of trypsin
together with trypsin inhibitor to the protein fraction does not affect its inductive ability (Tiedemann, Tiedemann & Kesselring, 1960). Papain was removed
from the inhibitory fraction by extraction with phenol before combination
with the inducing protein fraction (for details see Methods).
Incubation with pronase on the other hand leads to a complete inactivation of
the inhibitor (Table 3), even when only very small amounts of pronase (predigested to reduce contaminating enzymes) are used (H 230/2). In one control
series of experiments (Inhibitor control, incubated at 37 °C) the percentage of
trunk-tail induction was relatively high (30 %) compared to the pronase incubated series (63 %). The trunk-tail inductions in the control series were however mostly small. The difference between the pronase incubated series and the
incubated control is statistically significant (x2 method, P < 0001).
In all experiments pronase has been removed by extraction with phenol
(see Methods) before combination of the incubated inhibitory fraction with the
inducing protein fraction. It can therefore be excluded that pronase itself induces
trunk and tail structures.
The inhibitor does not lose its biological activity after incubation with protease-free neuraminidase (Table 4). The ultracentrifuged inhibitor fraction contains about 6 /tg neuraminic acid per g-equivalent. Most of the neuraminic acid
is enzymically split off after incubation with 300 units neuraminidase for 30 min
at 37 °C.
DISCUSSION
The experiments show that the inhibitor for the vegetalizing factor is not inactivated by either pancreatic ribonuclease or ribonuclease-Ti. A small loss of
inhibitory activity which was observed in some experiments with pancreatic
RNase is probably not caused by a degradation of the inhibitor. It is more
likely that the efficiency of recombination with the inhibitor has somewhat changed. The polynucleotides which arise by RNase incubation are not
Inhibitors in amphibian morphogenesis
85
inhibitory. After separation of the polynucleotides on Sephadex G 50 full
inhibitory activity is found in the exclusion peak which consists mostly of
glycoproteins. It can also be excluded that RNase resistant RNA-RNA helix
structures are responsible for the inhibition, because the inhibitor is not inactivated by heating to 110 °C, followed by RNase incubation. By this treatment
such helix structures would be denatured and then degraded. Incubation with
DNase had also no effect on the inhibitory activity. This was to be expected
because the inhibitory fraction contains, if any, only traces of DNA.
When the inhibitor is incubated with proteolytic enzymes the result varies
with different proteases. The outcome of the incubation may depend on the
specificity of a certain enzyme for peptide bond splitting. It may be also important whether a peptide chain in a glycoprotein molecule is accessible to a certain
enzyme. Trypsin and papain. inactivate the inhibitor only to a very small extent
or not at all. Pronase, a proteolytic enzyme isolated from Streptomyces griseus,
inactivates the inhibitor completely. Trypsin splits only peptide bonds in which
carboxylgroups of basic amino acids are involved, papain has a broader
specificity whereas pronase splits all peptide bonds. The inhibitor probably contains a protein which is indispensable for its biological activity. Preliminary data
in the amino acid composition of the protein portion of the inhibitor fraction
have shown that the percentage of basic amino acids is very low.
After partial purification the inhibitor is found in the glycoprotein fraction
(Born, Tiedemann & Tiedemann, 1971); polysaccharide splitting enzymes should
therefore be a useful tool to show whether the polysaccharide part is necessary
for the inhibitory activity. The enzymes used must not contain proteolytic
contaminants. So far only experiments with protease-free neuraminidase have
been carried out. The inhibitory activity is not lost, when neuraminic acid is
split off. This does not, however, exclude that neuraminic acid is part of the
inhibitor molecule. Neuraminic acid seems, however, not to be essential for the
inhibitory activity.
Our investigations were aided by grants from the Deutsche Forschungsgemeinschaft
(Sonderforschungsbereich Embryonale Entwicklung und Differenzierung).
REFERENCES
D. (1961). Methods for quantitative estimation of N-acetylneuraminic acid and
their application to hydrolysates in sialomucoids. Biochem. J. 81, 384-392.
BORN, J., TIEDEMANN, H. & TIEDEMANN, H. (1972). The mechanism of embryonic induction:
Isolation of an inhibitor for the vegetalizing factor. Biochim. biophys. Acta. (In Press.)
FAULHABER, I. (1970). Anreicherung des vegetalisierenden Induktionsfaktors aus der Gastrula des Krallenfrosches (Xenopus laevis) und Abgrenzung des Molekulargewichtsbereiches
durch Gradientenzentrifugation. Hoppe-Seyler's Z. physiol. Chem. 351, 588.
HOLTFRETER, J. (1933). Nachweis der Induktionsfahigkeit abgetoteter Keimteile. Isolationsund Transplantationsversuche. Wilhelm Roux Arch. EntwMech. Org. 128, 584-633.
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TIEDEMANN, H., TIEDEMANN, H. & KESSELRING, K. (1960). Versuche zur Kennzeichnung von
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