8
Ν. G. PON
to be detected were triose phosphate and hexose monophosphate, a finding that immediately suggested the reduction of PGA and the coupling
of this reduction product, head to head, as in the reversal of the wellknown Embden-Meyerhof-Parnas sequence. Degradation of these two
sugar phosphates gave distribution of radioactivity which was consistent
with this notion.
The relationship of the observed labeling of the pentose phosphates
and of sedoheptulose phosphate remained obscure even after repeated
kinetic studies (10). Degradation of these radioactive compounds gave
a complicated and confusing picture also, even when the algae were
exposed for a very short time to labeled bicarbonate. By calling on logical
arithmetic, the investigators deduced that the heptulose must have
originated from the hexoses and trioses by the actions of transketolase
and aldolase. The sedoheptulose phosphate is converted by transketolase, with labeled triose phosphate from the pool as the acceptor, to two
different pentose phosphates, which, when mixed together, had the same
distribution of radiocarbon as that observed in the pentose monophosphates. Thus the pathway from PGA to hexose phosphates, to sedoheptulose phosphates, and to pentose phosphates was provided.
Despite all this progress, however, the identity of the two-carbon
C0 2 acceptor posed the most difficult problem. Here a rather unusual
approach was employed, involving the study of the transient changes
immediately after interruption of a steady-state condition. In this method
the biological sample was exposed to radioactive bicarbonate or carbon
dioxide for a long period of time, until the intermediates under steady
state had become uniformly labeled. If the assimilation of C
14 from C
14 0 2
into these intermediates is plotted as a function of time, this assimilation
will rise from zero and level off after a certain time of exposure to radiocarbon. (The incorporation of non-steady-state intermediates does not
level off with increasing time.) The specific activity of the initial C
14 0 2
being known, one can calculate the concentration of each of these intermediates in the cell.
When this "steady-state" method was applied to Scenedesmus in
C
14 0 2 and in light, followed by a dark period after the photostationary
state was reached, a marked change occurred (54). Thus with the onset
of the dark period, the phosphoglycerate concentration suddenly rose
while the diphosphate (mostly ribtilose diphosphate) concentration decreased. These results not only confirmed that PGA was the primary
product of carboxylation, but also strongly suggested that ribulose-1,5diphosphate (RuDP) was the C0 2 acceptor.
This idea was strengthened by another experiment in which the C
14 0 2
pressure was reduced suddenly from 1% to 0.003% over the algae after
steady-state conditions were established (55). The transient changes in
Ν. G. PON
to be detected were triose phosphate and hexose monophosphate, a finding that immediately suggested the reduction of PGA and the coupling
of this reduction product, head to head, as in the reversal of the wellknown Embden-Meyerhof-Parnas sequence. Degradation of these two
sugar phosphates gave distribution of radioactivity which was consistent
with this notion.
The relationship of the observed labeling of the pentose phosphates
and of sedoheptulose phosphate remained obscure even after repeated
kinetic studies (10). Degradation of these radioactive compounds gave
a complicated and confusing picture also, even when the algae were
exposed for a very short time to labeled bicarbonate. By calling on logical
arithmetic, the investigators deduced that the heptulose must have
originated from the hexoses and trioses by the actions of transketolase
and aldolase. The sedoheptulose phosphate is converted by transketolase, with labeled triose phosphate from the pool as the acceptor, to two
different pentose phosphates, which, when mixed together, had the same
distribution of radiocarbon as that observed in the pentose monophosphates. Thus the pathway from PGA to hexose phosphates, to sedoheptulose phosphates, and to pentose phosphates was provided.
Despite all this progress, however, the identity of the two-carbon
C0 2 acceptor posed the most difficult problem. Here a rather unusual
approach was employed, involving the study of the transient changes
immediately after interruption of a steady-state condition. In this method
the biological sample was exposed to radioactive bicarbonate or carbon
dioxide for a long period of time, until the intermediates under steady
state had become uniformly labeled. If the assimilation of C
14 from C
14 0 2
into these intermediates is plotted as a function of time, this assimilation
will rise from zero and level off after a certain time of exposure to radiocarbon. (The incorporation of non-steady-state intermediates does not
level off with increasing time.) The specific activity of the initial C
14 0 2
being known, one can calculate the concentration of each of these intermediates in the cell.
When this "steady-state" method was applied to Scenedesmus in
C
14 0 2 and in light, followed by a dark period after the photostationary
state was reached, a marked change occurred (54). Thus with the onset
of the dark period, the phosphoglycerate concentration suddenly rose
while the diphosphate (mostly ribtilose diphosphate) concentration decreased. These results not only confirmed that PGA was the primary
product of carboxylation, but also strongly suggested that ribulose-1,5diphosphate (RuDP) was the C0 2 acceptor.
This idea was strengthened by another experiment in which the C
14 0 2
pressure was reduced suddenly from 1% to 0.003% over the algae after
steady-state conditions were established (55). The transient changes in