552
E T I E N N E
W O L F F
perchloric acid (Kuusi, 1953), nor by ribonuclease (Yamada and
Takata, 1955; Hayashi, 1959a). In fact, the idea that ribonucleic acids
have no inducing effect whatsoever in their pure state seems to be very
well documented.
Inducing substances are, on the other hand, rapidly inactivated by
proteolytic enzymes. This has been demonstrated by the Japanese
school (Yamada and Takata, 1955; Hayashi, 1958, 1959b) and by the
German authors (Tiedemann and Tiedemann, 1956; Tiedemann, 1959).
Pepsin inactivates the spino-caudal inducers of a fraction of chick
embryo extracted with phenol. The deuterencephalic inducing action
of a nucleoprotein extracted from guinea-pig liver is rapidly inactivated,
but the archencephalic inducer appears to be much more resistant. The
same thing occurs in the case of a purified preparation of nucleoproteins
from guinea-pig kidney; its principal effect—which is spino-caudal and
deuterencephalic—is suppressed, and its secondary effect—which is
archencephalic—is diminished.
In contrast, crystalline trypsin and chymotrypsin inactivate both the
substances which give rise to the spino-caudal effect and those which
induce the archencephalon (extracts from guinea-pig liver and kidney
and active fractions of the extract from chick embryo).
One may conclude from these investigations that the specific inducing
substances are proteins, and not ribonucleic acids.
3. Further Separation of the Specific Inducing Substances
Beginning with the extract from chick embryo, Tiedemann and
Tiedemann isolated three fractions corresponding to archencephalic,
deuterencephalic and spino-caudal inducers. Starting with the fraction
inducing spino-caudal formations, they obtained by purification a
substance inducing mesoderm. This result was obtained by phenol
extraction of proteins present in the granular layer of mitochondria and
of microsomes, followed by methanol precipitation. After further
purification, the authors obtained a protein fraction which had an u.v.
absorption-maximum at 279 m/z (at p H 3-6). Electrophoresis at p H 3-6
permitted the isolation of a principal component whose mesodermizing
activity was strong, and of a slower component. Further electrophoresis
at p H 8-6 in veronal buffer still permitted the isolation ofthe principal
component, and of two other minor components. The principal component, submitted to tests of viscosity and sedimentation, probably has
a molecular weight of about 50,000. The sedimentation and diffusion
constants gave a molecular weight of about 43,000. The same result was
achieved by extraction of acetone powder, followed by purification.
From the viscosity coefficient it is likely that it is the partially denatured
proteins, that are here concerned.
E T I E N N E
W O L F F
perchloric acid (Kuusi, 1953), nor by ribonuclease (Yamada and
Takata, 1955; Hayashi, 1959a). In fact, the idea that ribonucleic acids
have no inducing effect whatsoever in their pure state seems to be very
well documented.
Inducing substances are, on the other hand, rapidly inactivated by
proteolytic enzymes. This has been demonstrated by the Japanese
school (Yamada and Takata, 1955; Hayashi, 1958, 1959b) and by the
German authors (Tiedemann and Tiedemann, 1956; Tiedemann, 1959).
Pepsin inactivates the spino-caudal inducers of a fraction of chick
embryo extracted with phenol. The deuterencephalic inducing action
of a nucleoprotein extracted from guinea-pig liver is rapidly inactivated,
but the archencephalic inducer appears to be much more resistant. The
same thing occurs in the case of a purified preparation of nucleoproteins
from guinea-pig kidney; its principal effect—which is spino-caudal and
deuterencephalic—is suppressed, and its secondary effect—which is
archencephalic—is diminished.
In contrast, crystalline trypsin and chymotrypsin inactivate both the
substances which give rise to the spino-caudal effect and those which
induce the archencephalon (extracts from guinea-pig liver and kidney
and active fractions of the extract from chick embryo).
One may conclude from these investigations that the specific inducing
substances are proteins, and not ribonucleic acids.
3. Further Separation of the Specific Inducing Substances
Beginning with the extract from chick embryo, Tiedemann and
Tiedemann isolated three fractions corresponding to archencephalic,
deuterencephalic and spino-caudal inducers. Starting with the fraction
inducing spino-caudal formations, they obtained by purification a
substance inducing mesoderm. This result was obtained by phenol
extraction of proteins present in the granular layer of mitochondria and
of microsomes, followed by methanol precipitation. After further
purification, the authors obtained a protein fraction which had an u.v.
absorption-maximum at 279 m/z (at p H 3-6). Electrophoresis at p H 3-6
permitted the isolation of a principal component whose mesodermizing
activity was strong, and of a slower component. Further electrophoresis
at p H 8-6 in veronal buffer still permitted the isolation ofthe principal
component, and of two other minor components. The principal component, submitted to tests of viscosity and sedimentation, probably has
a molecular weight of about 50,000. The sedimentation and diffusion
constants gave a molecular weight of about 43,000. The same result was
achieved by extraction of acetone powder, followed by purification.
From the viscosity coefficient it is likely that it is the partially denatured
proteins, that are here concerned.