460
ERIC E. CONN
the difference between the standard potentials for cytochromes a and c
is only 0.03 volts (13, 14). This suggests that the one phosphorylation known to occur between cytochrome c and 0 2 may occur between
cytochromes a and a 3 or between cytochrome a 3 and 0 2 . Chance (13)
has recently emphasized that calculations of this type must take into
account the actual concentrations of products and reactants. Such information has been provided by these authors, and the E h for the observed steady-state ratios are also given in Table I. However, since the
ratios are not far from one, except for cytochrome a 3 , the differences
between E 0 and E h are small.
C. CELLULAR LOCALIZATION OF OXIDATIVE PHOSPHORYLATION
The earlier studies on oxidative phosphorylation were carried out
with muscle minces (134, 138), "extracts" of kidney (139) or heart
(135), and brain (140) homogenates. These types of preparations had
to be freshly prepared each day, and little success was observed in reactivating preparations which had lost their activity. With the development of methods of isolation of mitochondria and examination of their
enzymatic properties, these particles were shown to contain the necessary enzymes to support oxidative phosphorylation (141-144). As a result of using mitochondria, the phosphorylation processes were separated
from side reactions catalyzed by soluble enzymes present in the cell
cytoplasm.
Recently phosphorylative submitochondrial fragments have been
obtained by treating mitochondria with digitonin (75) or alcohol (78),
or by sonic vibrations (79, 80). Some of these particles will carry out
phosphorylation during the oxidation of ß-hydroxybutyrate, DPNH, succinate, and other Krebs cycle acids with P/O ratios in the range of 1 to
2. The digitonin and alcohol particles however support phosphorylation
only during oxidation of ß-hydroxybutyrate. The phosphorylation reactions are affected by uncoupling agents in a manner similar to that exhibited by the intact mitochondrion. However, oxidation of DPNH by
the particles obtained by sonic treatment is much more sensitive to the
inhibitor, antimycin A. All of these particles, regardless of manner of
preparation, would seem to constitute simpler systems in which to
study oxidative phosphorylation.
In bacteria the phosphorylation processes are associated with an
even smaller particle (107).
D. METHODOLOGY
To determine the P/O ratio associated with the oxidation of an
organic substrate, one wishes to measure the amount of inorganic phosphate which is esterified and the amount of 0 2 uptake which occurs
ERIC E. CONN
the difference between the standard potentials for cytochromes a and c
is only 0.03 volts (13, 14). This suggests that the one phosphorylation known to occur between cytochrome c and 0 2 may occur between
cytochromes a and a 3 or between cytochrome a 3 and 0 2 . Chance (13)
has recently emphasized that calculations of this type must take into
account the actual concentrations of products and reactants. Such information has been provided by these authors, and the E h for the observed steady-state ratios are also given in Table I. However, since the
ratios are not far from one, except for cytochrome a 3 , the differences
between E 0 and E h are small.
C. CELLULAR LOCALIZATION OF OXIDATIVE PHOSPHORYLATION
The earlier studies on oxidative phosphorylation were carried out
with muscle minces (134, 138), "extracts" of kidney (139) or heart
(135), and brain (140) homogenates. These types of preparations had
to be freshly prepared each day, and little success was observed in reactivating preparations which had lost their activity. With the development of methods of isolation of mitochondria and examination of their
enzymatic properties, these particles were shown to contain the necessary enzymes to support oxidative phosphorylation (141-144). As a result of using mitochondria, the phosphorylation processes were separated
from side reactions catalyzed by soluble enzymes present in the cell
cytoplasm.
Recently phosphorylative submitochondrial fragments have been
obtained by treating mitochondria with digitonin (75) or alcohol (78),
or by sonic vibrations (79, 80). Some of these particles will carry out
phosphorylation during the oxidation of ß-hydroxybutyrate, DPNH, succinate, and other Krebs cycle acids with P/O ratios in the range of 1 to
2. The digitonin and alcohol particles however support phosphorylation
only during oxidation of ß-hydroxybutyrate. The phosphorylation reactions are affected by uncoupling agents in a manner similar to that exhibited by the intact mitochondrion. However, oxidation of DPNH by
the particles obtained by sonic treatment is much more sensitive to the
inhibitor, antimycin A. All of these particles, regardless of manner of
preparation, would seem to constitute simpler systems in which to
study oxidative phosphorylation.
In bacteria the phosphorylation processes are associated with an
even smaller particle (107).
D. METHODOLOGY
To determine the P/O ratio associated with the oxidation of an
organic substrate, one wishes to measure the amount of inorganic phosphate which is esterified and the amount of 0 2 uptake which occurs