A naturally occurring inhibitor for the vegetalizing inducing factor has been incubated with different enzymes. Pancreatic ribonuclease, ribonuclease-T1, deoxyribunuclease, neur-aminidase 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.

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.

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.

Recombination of ‘inhibitor’ and ‘inducer’. 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 re-combination 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 hydroxyl-apatite to remove most of the RNA (Born et al. 1971).

The inhibitor fractions were incubated with or without enzyme and re-combined 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 3×51. water. NaCl (final cone. 0·5 % or 5 %) was then added and the mixture precipitated with 2 vol. ethanol (ice bath or –25 °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.

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 μg RNA and 50 μ g 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.

Table 1.

Incubation of the inhibitor for the vegetalizing factor with DNase, pancreatic RNase and RNase-T\: 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)

Incubation of the inhibitor for the vegetalizing factor with DNase, pancreatic RNase and RNase-T\: 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)
Incubation of the inhibitor for the vegetalizing factor with DNase, pancreatic RNase and RNase-T\: 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)

Incubation with pancreatic RNase (E.C. 2.7.7.16; Worthington RASE)

RNase dissolved in 0· 02 ml–0· 05 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, –25 °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-NH4HCO3. 900 mg urea and 98·8 mg NH4HCO3 were added to 2 ml inhibitor. The mixture was applied to a 1·7 cm × 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 μ g 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-T1 (from Aspergillus oryzae; E.C. 2.7.7.26; Worthington RT1). 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-T1 (~ 10 μg) 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 2×cryst. TRL). After incubation with trypsin for 60 min at 37 °C, 2 ·5 μg trypsin inhibitor (Worthington) were added for each μg trypsin, incubated for 5 min at 20 °C and recombined with inducer protein.

Incubation with papain (E.C. 3.4.4.10; Boehringer, Mannheim). Papain was activated with cystein (Sluyterman, 1967; Kimmel & Smith, 1954). To 0·5 ml (5 mg) papain 0· 07 ml 0· 05 M cystein-Hl and 0· 07 ml 0· 01 M-EDTA were added, the pH adjusted to 6 and the mixture subjected to chromatography on Sephadex G 50 (0·9 × 30 cm) in 0·02 M-Na-acetate pH 5 in N2-atmosphere (elution rate 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), vol. 0·5 M-Na-acetate (pH 5·5); vol. 0·1 M-CaCl2 and 20–300 U neuraminidase (1 U ~ 1 μg N-acetylneuraminic acid split off from α 1 -glyco-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).

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-T1 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-T1 (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 μg) 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 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 G 50 (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).

Table 2.

Incubation of the inhibitor for the vegetalizing factor with trypsin and papain: the amount of inhibitory fraction 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 of the inhibitor for the vegetalizing factor with trypsin and papain: the amount of inhibitory fraction 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 of the inhibitor for the vegetalizing factor with trypsin and papain: the amount of inhibitory fraction 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 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 < 0 ·001).

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

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
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

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 μg 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.

Table 4.

Incubation of the inhibitor for the vegetalizing factor with neuraminidase: the amount of inhibitory fraction is given in g-equivalents (see Methods). All combinations have been carried out with 2–5 mg inducer protei

Incubation of the inhibitor for the vegetalizing factor with neuraminidase: the amount of inhibitory fraction is given in g-equivalents (see Methods). All combinations have been carried out with 2–5 mg inducer protei
Incubation of the inhibitor for the vegetalizing factor with neuraminidase: the amount of inhibitory fraction is given in g-equivalents (see Methods). All combinations have been carried out with 2–5 mg inducer protei

The experiments show that the inhibitor for the vegetalizing factor is not inactivated by either pancreatic ribonuclease or ribonuclease-T1 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 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).

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