288
JEAN BRÄCHET
lower (M/300-M/1,000) concentrations of mercaptoethanol, at the same
time that similar results were obtained with chick embryos by Pohl
(unpublished). Under these experimental conditions, mercaptoethanol
markedly stimulates the incorporation of thymidine into DNA and that
of uridine and cytidine into RNA. It has no effect, or exerts a moderate
inhibition, on DNA synthesis at the expense of the ribonucleosides
(uridine, cytidine). Lipoic acid exerts the same effects, except that, as
mentioned before, it inhibits to a much greater extent the synthesis of
DNA from ribonucleosides (see p. 251). These observations suggest that
mercaptoethanol (and lipoic acid) treatment might induce the rapid
synthesis of some abnormal type of RNA with, as a consequence, the
production of abnormal proteins. In fact, preliminary experiments of
Quertier and Pohl on the incorporation of various amino-acids in the
proteins of mercaptoethanol and lipoic acid-treated eggs seem to support
this hypothesis : apparently, the incorporation of certain amino-acids is
stimulated, while that of others is inhibited.
There is other evidence that one of the targets of mercaptoethanol is
the egg protein. A negative finding should first be mentioned: we have
seen that one of the possible explanations for the favourable effects of
ATP on mercaptoethanol-treated eggs is that mercaptoethanol might
inhibit or destroy a contractile protein having ATPase activity. As
described above, the independence of the ATP effect on the concentration of Mg
2+ ion speaks against this hypothesis. Moreover, our chemical
studies have brought no support for the hypothesis. We were unable
(Brächet, 1962) to detect any ecto-ATPase
(surface ATPase) in frog
eggs ; this means that they are unable to hydrolyze ATP added to the
medium. Endo-ATPase
is very active, but remains unchanged in
mercaptoethanol-treated eggs.
But experiments with
Z5
S4abelled mercaptoethanol
(Brächet, 1962;
Decroly et al., 1961 ; Brächet et al., 1963) have yielded positive results.
They first showed that the increase in radioactivity in the acid-soluble
fraction is in good agreement with the chemical measurement of the
—SH content of this fraction: there is a moderate increase, but no
flooding, of mercaptoethanol in the treated eggs. It is more important
that 25% of the total radioactivity can be recovered in the protein
fraction, a fact which suggests that mercaptoethanol might form mixed
disulphides with the egg proteins. When homogenates of treated eggs
are submitted to differential centrifugation, it is found that the major
part of the proteins which combine with labelled mercaptoethanol is in
the yolk and pigment fractions. It is of interest that 65% of the total
radioactivity is present in the dorsal half, i.e., in the region of the
embryos which is most susceptible to mercaptoethanol, when young
neurulae treated with mercaptoethanol are dissected into dorsal and
JEAN BRÄCHET
lower (M/300-M/1,000) concentrations of mercaptoethanol, at the same
time that similar results were obtained with chick embryos by Pohl
(unpublished). Under these experimental conditions, mercaptoethanol
markedly stimulates the incorporation of thymidine into DNA and that
of uridine and cytidine into RNA. It has no effect, or exerts a moderate
inhibition, on DNA synthesis at the expense of the ribonucleosides
(uridine, cytidine). Lipoic acid exerts the same effects, except that, as
mentioned before, it inhibits to a much greater extent the synthesis of
DNA from ribonucleosides (see p. 251). These observations suggest that
mercaptoethanol (and lipoic acid) treatment might induce the rapid
synthesis of some abnormal type of RNA with, as a consequence, the
production of abnormal proteins. In fact, preliminary experiments of
Quertier and Pohl on the incorporation of various amino-acids in the
proteins of mercaptoethanol and lipoic acid-treated eggs seem to support
this hypothesis : apparently, the incorporation of certain amino-acids is
stimulated, while that of others is inhibited.
There is other evidence that one of the targets of mercaptoethanol is
the egg protein. A negative finding should first be mentioned: we have
seen that one of the possible explanations for the favourable effects of
ATP on mercaptoethanol-treated eggs is that mercaptoethanol might
inhibit or destroy a contractile protein having ATPase activity. As
described above, the independence of the ATP effect on the concentration of Mg
2+ ion speaks against this hypothesis. Moreover, our chemical
studies have brought no support for the hypothesis. We were unable
(Brächet, 1962) to detect any ecto-ATPase
(surface ATPase) in frog
eggs ; this means that they are unable to hydrolyze ATP added to the
medium. Endo-ATPase
is very active, but remains unchanged in
mercaptoethanol-treated eggs.
But experiments with
Z5
S4abelled mercaptoethanol
(Brächet, 1962;
Decroly et al., 1961 ; Brächet et al., 1963) have yielded positive results.
They first showed that the increase in radioactivity in the acid-soluble
fraction is in good agreement with the chemical measurement of the
—SH content of this fraction: there is a moderate increase, but no
flooding, of mercaptoethanol in the treated eggs. It is more important
that 25% of the total radioactivity can be recovered in the protein
fraction, a fact which suggests that mercaptoethanol might form mixed
disulphides with the egg proteins. When homogenates of treated eggs
are submitted to differential centrifugation, it is found that the major
part of the proteins which combine with labelled mercaptoethanol is in
the yolk and pigment fractions. It is of interest that 65% of the total
radioactivity is present in the dorsal half, i.e., in the region of the
embryos which is most susceptible to mercaptoethanol, when young
neurulae treated with mercaptoethanol are dissected into dorsal and