66
EDWARD C. CANTINO AND JAMES S. LOVETT
enzymes residing in the 'soluble' fraction (Lehninger, 1959). In another
system (Marcus and Velasco, 1960), the glyoxylic cycle appeared to be
localized in the mitochondria while isocitric dehydrogenase was outside ;
it was suggested that isocitric metabolism might have been occurring
mainly in the mitochondria, thus by-passing the ketoglutaric acid stage
(also, see report by Lowenstein and Smith, 1962, on the occurrence of
immunologically and electrophoretically distinct intra- and extramitochondrial isocitric dehydrogenases).
In Blastocladiella, therefore, it is possible that ketoglutaric dehydrogenase, isocitric dehydrogenase, and isocitritase may be distributed in
the cell so as to be isolated from one another. Indeed, the isocitric
dehydrogenase in homogenates is always found in supernatants from
20,000g* centrifugations and never in the pellets (Cantino and Horenstein,
1955) while isocitritase is always found in the pellets and never in the
supernatants (McCurdy and Cantino, 1960).
In any event, if two basic premises are accepted for the sake of the
argument—(a) that succinate inhibits or represses both isocitritase and
ketoglutaric dehydrogenase, and (b) that these two systems are isolated
from one another in the cell—then the speculations outlined in Fig. 6
become attractive. The normal sequence of events, occurring in young
OC cells in the absence of bicarbonate, is pictured in solid lines. During
normal flow from isocitrate to ketoglutarate and thence to succinate,
the concentration of the steady-state pool of succinate is insufficient to
affect the ketoglutaric dehydrogenase. So long as it is turning over, some
of this succinate continuously 'leaks' out of the organelle in which it is
formed and accumulates in sufficient quantity at the isocitritase 'site' to
repress its synthesis or inhibit its activity by feedback.
The altered picture, induced in young RS cells by exogenous bicarbonate, is shown in dotted lines. Bicarbonate (or C0 2 ) inhibits the
succinic dehydrogenase system (perhaps as described by Bendall et al.,
1960) and thus slows down the flow of molecules from isocitrate to
ketoglutarate. Concomitantly, the pool of succinate increases to the
point where synthesis of ketoglutaric dehydrogenase is gradually
repressed and perhaps its activity inhibited as well. By virtue of these
effects, as well as the addition of bicarbonate and the transient pool of
ketoglutarate which accumulated, reductive carboxylation of ketoglutarate*)* back to isocitrate commences. Additional ketoglutarate is
•f Indeed, when exogenous ketoglutarate itself, or metabolic inhibitors such as arsenite
which presumably interfere with its further oxidative decarboxylation are fed to
Blastocladiella, they induce RS cell formation under conditions where bicarbonate,
alone, will not suffice (Cantino, 1961a). Thus, while ketoglutarate has not been substituted
directly for bicarbonate to test its effect upon the synthesis of enzymes during exponential
growth of synchronized cells, it seems most likely that it would duplicate some of the
effects of bicarbonate upon the enzymes we have been discussing.
EDWARD C. CANTINO AND JAMES S. LOVETT
enzymes residing in the 'soluble' fraction (Lehninger, 1959). In another
system (Marcus and Velasco, 1960), the glyoxylic cycle appeared to be
localized in the mitochondria while isocitric dehydrogenase was outside ;
it was suggested that isocitric metabolism might have been occurring
mainly in the mitochondria, thus by-passing the ketoglutaric acid stage
(also, see report by Lowenstein and Smith, 1962, on the occurrence of
immunologically and electrophoretically distinct intra- and extramitochondrial isocitric dehydrogenases).
In Blastocladiella, therefore, it is possible that ketoglutaric dehydrogenase, isocitric dehydrogenase, and isocitritase may be distributed in
the cell so as to be isolated from one another. Indeed, the isocitric
dehydrogenase in homogenates is always found in supernatants from
20,000g* centrifugations and never in the pellets (Cantino and Horenstein,
1955) while isocitritase is always found in the pellets and never in the
supernatants (McCurdy and Cantino, 1960).
In any event, if two basic premises are accepted for the sake of the
argument—(a) that succinate inhibits or represses both isocitritase and
ketoglutaric dehydrogenase, and (b) that these two systems are isolated
from one another in the cell—then the speculations outlined in Fig. 6
become attractive. The normal sequence of events, occurring in young
OC cells in the absence of bicarbonate, is pictured in solid lines. During
normal flow from isocitrate to ketoglutarate and thence to succinate,
the concentration of the steady-state pool of succinate is insufficient to
affect the ketoglutaric dehydrogenase. So long as it is turning over, some
of this succinate continuously 'leaks' out of the organelle in which it is
formed and accumulates in sufficient quantity at the isocitritase 'site' to
repress its synthesis or inhibit its activity by feedback.
The altered picture, induced in young RS cells by exogenous bicarbonate, is shown in dotted lines. Bicarbonate (or C0 2 ) inhibits the
succinic dehydrogenase system (perhaps as described by Bendall et al.,
1960) and thus slows down the flow of molecules from isocitrate to
ketoglutarate. Concomitantly, the pool of succinate increases to the
point where synthesis of ketoglutaric dehydrogenase is gradually
repressed and perhaps its activity inhibited as well. By virtue of these
effects, as well as the addition of bicarbonate and the transient pool of
ketoglutarate which accumulated, reductive carboxylation of ketoglutarate*)* back to isocitrate commences. Additional ketoglutarate is
•f Indeed, when exogenous ketoglutarate itself, or metabolic inhibitors such as arsenite
which presumably interfere with its further oxidative decarboxylation are fed to
Blastocladiella, they induce RS cell formation under conditions where bicarbonate,
alone, will not suffice (Cantino, 1961a). Thus, while ketoglutarate has not been substituted
directly for bicarbonate to test its effect upon the synthesis of enzymes during exponential
growth of synchronized cells, it seems most likely that it would duplicate some of the
effects of bicarbonate upon the enzymes we have been discussing.