RESPIRATION RATE IN PLANTS
223
trophenol, a brief discussion of what is known of the mechanism of action
of dinitrophenol may help to evaluate the significance of such experiments. The early work of Loomis and Lipmann (1948) suggested dinitrophenol to be a substitute for phosphate, since in the presence of
dinitrophenol appreciable respiratory activity was manifested by isolated
cellular particles in the absence of added inorganic phosphate. Cross
et al. (1949) and Teply (1949) subsequently maintained that the action
of dinitrophenol was to cleave inorganic phosphate from a primary phosphorylated form on the enzyme itself, thereby returning phosphate to
solution, and simultaneously freeing an acceptor site on the enzyme for
the next phosphorylative reaction. In this view the small quantity of
endogenous phosphate in the particles was deemed enough to sustain
respiration in the presence of dinitrophenol. Until recently, therefore,
it seemed likely that phosphorylation occurred even in the presence of
dinitrophenol, and that the failure to observe any net phosphorylation
was due to the dinitrophenol-mediated hydrolysis of a primary phosphorylated intermediate.
The elegant work of Cohn (1953) and Cohn and Drysdale (1955) has
thrown serious doubt upon this interpretation. Cohn observed that the
O
18
of isotopically enriched inorganic phosphate was diluted with O
16
from water during active phosphorylation. Such a dilution is the consequence of the hydrolytic cleavage of a P-O bond between phosphate
and some component to which phosphate is linked during phosphorylation. Since, with dinitrophenol present, any isotope exchange between
water and 0
18
-labeled phosphate was completely prevented, while
respiration proceeded unabated, it was concluded that in the presence
of dinitrophenol there is no phosphorylation whatsoever.
How then does dinitrophenol exert its uncoupling action? The accumulated evidence suggests a mechanism such as that depicted in
Fig. 4. The symbols A, B, and C have the same significance as in
Slater s equation 1 presented above. Dinitrophenol is thought to cause
the hydrolysis of a high-energy bond formed early in the oxidative
process, before the participation of inorganic phosphate. Presumably
dinitrophenol may bring about the cleavage of the primary phosphorylated intermediate (Phos ~ C) as well, but in the normal course of oxidative metabolism in the presence of dinitrophenol the hydrolysis of the
bond A ~ C precludes the formation of the bond Phos ~ C.
Although the scheme for the manner in which oxidative phosphorylation takes place, represented by equations 1 and 2 above, is general,
applying both to substrate-level phosphorylation and to phosphorylation
during electron transport, the uncoupling action of dinitrophenol applies
only to phosphorylation during electron transport. Substrate-level phos-
223
trophenol, a brief discussion of what is known of the mechanism of action
of dinitrophenol may help to evaluate the significance of such experiments. The early work of Loomis and Lipmann (1948) suggested dinitrophenol to be a substitute for phosphate, since in the presence of
dinitrophenol appreciable respiratory activity was manifested by isolated
cellular particles in the absence of added inorganic phosphate. Cross
et al. (1949) and Teply (1949) subsequently maintained that the action
of dinitrophenol was to cleave inorganic phosphate from a primary phosphorylated form on the enzyme itself, thereby returning phosphate to
solution, and simultaneously freeing an acceptor site on the enzyme for
the next phosphorylative reaction. In this view the small quantity of
endogenous phosphate in the particles was deemed enough to sustain
respiration in the presence of dinitrophenol. Until recently, therefore,
it seemed likely that phosphorylation occurred even in the presence of
dinitrophenol, and that the failure to observe any net phosphorylation
was due to the dinitrophenol-mediated hydrolysis of a primary phosphorylated intermediate.
The elegant work of Cohn (1953) and Cohn and Drysdale (1955) has
thrown serious doubt upon this interpretation. Cohn observed that the
O
18
of isotopically enriched inorganic phosphate was diluted with O
16
from water during active phosphorylation. Such a dilution is the consequence of the hydrolytic cleavage of a P-O bond between phosphate
and some component to which phosphate is linked during phosphorylation. Since, with dinitrophenol present, any isotope exchange between
water and 0
18
-labeled phosphate was completely prevented, while
respiration proceeded unabated, it was concluded that in the presence
of dinitrophenol there is no phosphorylation whatsoever.
How then does dinitrophenol exert its uncoupling action? The accumulated evidence suggests a mechanism such as that depicted in
Fig. 4. The symbols A, B, and C have the same significance as in
Slater s equation 1 presented above. Dinitrophenol is thought to cause
the hydrolysis of a high-energy bond formed early in the oxidative
process, before the participation of inorganic phosphate. Presumably
dinitrophenol may bring about the cleavage of the primary phosphorylated intermediate (Phos ~ C) as well, but in the normal course of oxidative metabolism in the presence of dinitrophenol the hydrolysis of the
bond A ~ C precludes the formation of the bond Phos ~ C.
Although the scheme for the manner in which oxidative phosphorylation takes place, represented by equations 1 and 2 above, is general,
applying both to substrate-level phosphorylation and to phosphorylation
during electron transport, the uncoupling action of dinitrophenol applies
only to phosphorylation during electron transport. Substrate-level phos-