278
JEAN BRÄCHET
ATP on morphogenesis in the mercaptoethanol-treated eggs are thus
strictly limited to neural plate closure. The fact that, in our experiments,
there is no Mg
2+
requirement for the ATP effect speaks against the
hypothesis that mercaptoethanol acts, in a more or less specific manner,
on a contractile protein having ATPase activity. We shall return later
to this question.
Another possible mechanism for the action of mercaptoethanol is that
it acts as a chemical analog of thiols normally found in cells, such as
cysteine or glutathione (Bäckström, 1958, 1959). However, it was found
that these thiols also inhibit development (as already observed by
Barbieri and Braukmann, 1961) although they are much less effective
than mercaptoethanol. When cysteine or glutathione are added together
with mercaptoethanol, the effects on morphogenesis are at first additive,
an observation which eliminates the 'competition' hypothesis. But, after
one or two days, the embryos treated with mixtures of sulphydryl
compounds develop much better than those placed in mercaptoethanol
alone. The reasons for this phenomenon became obvious when it was
noticed that, in a mercaptoethanol-cysteine mixture, crystals of cystine
formed within a few days (even in the absence of the embryos). Measurements of the redox potential of the external media have provided an
explanation of the biological results : when cysteine or glutathione are
added to mercaptoethanol, the latter remains initially in the reduced
form. But, when cysteine or glutathione undergo autoxidation,
mercaptoethanol also becomes oxidized, so that its concentration in the
medium decreases.
These studies on the change in the redox potential undergone by
mercaptoethanol-containing solutions raised another possibility: since
the autoxidation of thiols often leads to the production of hydrogen
peroxide (H 2 0 2 ), perhaps the inhibition of morphogenesis could be a
consequence of H 2 0 2 formation during mercaptoethanol autoxidation.
It has been possible to rule out this hypothesis : firstly, the addition of
hydrogen peroxide, except at relatively high concentrations (10~
4 M or
more), has no effect on morphogenesis; secondly, we were unable to
detect the production of H 2 0 2 when eggs were treated with mercaptoethanol, even with the use of a very sensitive colorimetric method;
finally, the addition of heavy metals (Fe, Co) which should promote the
autoxidation of thiols and the production of H 2 0 2 , did not enhance the
inhibitory effects on morphogenesis of such substances as cysteine,
glutathione or mercaptoethanol.
2. Regeneration in Acetabularia and other Organisms
Mercaptoethanol (M/300, M/1,000) exerts a striking inhibitory effect
on cap formation in Acetabularia,
especially in anucleate fragments
JEAN BRÄCHET
ATP on morphogenesis in the mercaptoethanol-treated eggs are thus
strictly limited to neural plate closure. The fact that, in our experiments,
there is no Mg
2+
requirement for the ATP effect speaks against the
hypothesis that mercaptoethanol acts, in a more or less specific manner,
on a contractile protein having ATPase activity. We shall return later
to this question.
Another possible mechanism for the action of mercaptoethanol is that
it acts as a chemical analog of thiols normally found in cells, such as
cysteine or glutathione (Bäckström, 1958, 1959). However, it was found
that these thiols also inhibit development (as already observed by
Barbieri and Braukmann, 1961) although they are much less effective
than mercaptoethanol. When cysteine or glutathione are added together
with mercaptoethanol, the effects on morphogenesis are at first additive,
an observation which eliminates the 'competition' hypothesis. But, after
one or two days, the embryos treated with mixtures of sulphydryl
compounds develop much better than those placed in mercaptoethanol
alone. The reasons for this phenomenon became obvious when it was
noticed that, in a mercaptoethanol-cysteine mixture, crystals of cystine
formed within a few days (even in the absence of the embryos). Measurements of the redox potential of the external media have provided an
explanation of the biological results : when cysteine or glutathione are
added to mercaptoethanol, the latter remains initially in the reduced
form. But, when cysteine or glutathione undergo autoxidation,
mercaptoethanol also becomes oxidized, so that its concentration in the
medium decreases.
These studies on the change in the redox potential undergone by
mercaptoethanol-containing solutions raised another possibility: since
the autoxidation of thiols often leads to the production of hydrogen
peroxide (H 2 0 2 ), perhaps the inhibition of morphogenesis could be a
consequence of H 2 0 2 formation during mercaptoethanol autoxidation.
It has been possible to rule out this hypothesis : firstly, the addition of
hydrogen peroxide, except at relatively high concentrations (10~
4 M or
more), has no effect on morphogenesis; secondly, we were unable to
detect the production of H 2 0 2 when eggs were treated with mercaptoethanol, even with the use of a very sensitive colorimetric method;
finally, the addition of heavy metals (Fe, Co) which should promote the
autoxidation of thiols and the production of H 2 0 2 , did not enhance the
inhibitory effects on morphogenesis of such substances as cysteine,
glutathione or mercaptoethanol.
2. Regeneration in Acetabularia and other Organisms
Mercaptoethanol (M/300, M/1,000) exerts a striking inhibitory effect
on cap formation in Acetabularia,
especially in anucleate fragments