Reversal of Ethionine Inhibition by Methionine — Hohl and Hamamoto 
537 
effect is still very strong. Once the stalk is being 
formed the mass rises above the substrate and 
the further effects of ethionine are difficult to 
assess since the substances in the filter pad are 
no longer in direct contact with the cell mass. 
If ethionine is added for a certain period of 
time before aggregation, aggregation itself is 
also disturbed as described previously, i.e., only 
vaguely defined clumps form as the result of 
some streaming, or there is no reaction at all. 
Also, if ethionine is added after aggregation, 
then the proper polarization of the aggregated 
mass is disturbed and many small tips are 
formed as long as the contact with ethionine is 
maintained. The results of this experiment show 
that ethionine acts on several phases of the 
developmental sequence as well as on the vege- 
tative growth. Most significantly, however, it 
acts directly on the last stage, culmination. 
DISCUSSION 
Ethionine interferes with various stages of 
the life cycle of Dictyostelium, such as vege- 
tative growth, aggregation, and culmination. 
We have been able to show that the inhibition 
of the later stages of development is not neces- 
sarily a consequence of inhibition of earlier 
ones, since addition of ethionine as late as just 
prior to culmination is still inhibitory to a large 
extent. 
Spore differentiation is most sensitive to the 
action of ethionine, followed by cellulose sheath 
formation, stalk cell differentiation, and finally, 
morphogenetic movement. A similar sequence 
of sensitivity has been observed by Gerisch 
(1961) in the case of mercaptoethanol. Ethio- 
nine, then, seems to be primarily an inhibitor 
of differentiation. In turn, several aspects of 
disturbed morphogenesis, such as lack of a 
sorus or production of a short bulky stalk, can 
be indirectly attributed to this effect. More- 
over, how can the cell mass build a slender, 
evenly tapered stalk in the absence of any cellu- 
lose component for structural support? 
We have established that ethionine acts as a 
competitive inhibitor of methionine. This con- 
clusion is based on the observations that (1) 
methionine is able to reverse the inhibitory 
effect of ethionine when the two are added to 
the cultures simultaneously, and (2) the final 
product of development is a function of the 
ratio of the two components rather than of 
their absolute amounts. The proteins incorporat- 
ing ethionine instead of methionine are rendered 
biologically inactive and, therefore, normal 
development cannot proceed. It may be imag- 
ined that the various disturbances effected by 
ethionine have a common cause, but this is 
unlikely since ethionine is probably incorporated 
into a variety of proteins normally containing 
methionine. One point deserves mentioning: 
the ethionine has to be in contact with the cell 
population for at least 4-8 hours to produce 
any persisting inhibitory effect. At least two 
possible explanations might account for this: 
(1) ethionine slows down protein synthesis 
as long as it is in contact with the cells, so that 
only a small amount of ethionine-containing 
material is formed; and (2) the protein turn- 
over is high, so that the ethionine-containing 
proteins are rapidly broken down and replaced 
by the normal methionine-containing ones, as 
soon as the cells are switched from ethionine 
to methionine. 
The fate of the ethionine in the cell popula- 
tion has not been directly traced. However, 
from the work of Wright and Anderson (I960) 
on methionine metabolism in Dictyostelium we 
are able to get some information directly ap- 
plicable to our situation. These authors have 
shown that the endogenous "free” amino acid 
pool is a function of the stage of development 
only and is not influenced by exogenous methio- 
nine. However, exogenous methionine can ex- 
change with endogenous methionine, and the 
extent of this exchange is a linear function of 
the exogenous methionine present. This means 
that increasing the external methionine con- 
centration does not alter the size of the internal 
pool, but does increase the rate of exchange 
between exogenous and endogenous methionine. 
If we assume that ethionine behaves similarly 
in this respect, it then becomes clear that in- 
creasing the amount of ethionine in the medium 
does not simply add more ethionine to the 
existing endogenous amino acid pool, but re- 
sults in a higher exchange of ethionine for 
methionine between this pool and the environ- 
ment. In this way we have changed the ratio 
of methionine to ethionine, which is the essen- 
