462
ERIC E. CONN
cytochrome respiratory pathway have stimulated development of methods for measuring extremely small amounts of phosphate and respiration. In the former case, Lehninger (153) developed a method in which
oxidative phosphorylation is carried out in the presence of radioactive
inorganic phosphate. The inorganic phosphate is then extracted quantitatively as the phosphomolybdate in isobutanol-benzene and the radioactivity in the esterfied fraction is determined.
For determining the amount of oxidation which has occurred, Lehninger followed the reduction and oxidation of cytochrome c spectrophotometrically. In measuring phosphorylation occurring at oxidation
levels between DPNH and cytochrome c, the reduction of the hemeprotein was measured and its reoxidation prevented by cyanide. For
phosphorylation occurring between cytochrome c and oxygen, the
amount of ferrocytochrome c oxidized was determined.
Krebs et al. (154) also employed P
32 in measurements on oxidative
phosphorylation. In their studies these authors measured the rate of incorporation of P
32 into ATP under conditions where steady state concentrations of the inorganic phosphate and ATP existed.
Chance and Williams (67) have also introduced a new method for
measuring the amount of phosphorylation associated with 0 2 uptake.
In their studies they employed mitochondria in which the phosphorylation process is tightly coupled to the oxidation processes, and they followed the concentration of oxygen in the reaction mixture with a
vibrating platinum electrode. In the presence of excess substrate (ßhydroxybutyrate) the ADP present in the particles was phosphorylated
and the 0 2 consumption was retarded. Then, when small amounts of
ADP were added, the oxygen content, as measured by the 0 2 electrode,
decreased a definite amount. The P/O ratio (or more accurately the
ADP/O ratio) was then calculated from the ADP added and 0 2
consumed.
E. P/O RATIOS
1. Oxidation of Krebs Cycle Substrates
Much of the energy available in organic metabolites is released to the
organism when acetyl-CoA is oxidized through the reactions of the Krebs
cycle. Because Ochoa's (135) studies on rat heart extracts suggested
that the average number of phosphorylation steps per atom of oxygen
consumed during pyruvate oxidation was 3.0, it was of interest to determine which of the reactions of the cycle gave rise to oxidative phosphorylation and the number of phosphorylation. steps which could be
obtained.
ERIC E. CONN
cytochrome respiratory pathway have stimulated development of methods for measuring extremely small amounts of phosphate and respiration. In the former case, Lehninger (153) developed a method in which
oxidative phosphorylation is carried out in the presence of radioactive
inorganic phosphate. The inorganic phosphate is then extracted quantitatively as the phosphomolybdate in isobutanol-benzene and the radioactivity in the esterfied fraction is determined.
For determining the amount of oxidation which has occurred, Lehninger followed the reduction and oxidation of cytochrome c spectrophotometrically. In measuring phosphorylation occurring at oxidation
levels between DPNH and cytochrome c, the reduction of the hemeprotein was measured and its reoxidation prevented by cyanide. For
phosphorylation occurring between cytochrome c and oxygen, the
amount of ferrocytochrome c oxidized was determined.
Krebs et al. (154) also employed P
32 in measurements on oxidative
phosphorylation. In their studies these authors measured the rate of incorporation of P
32 into ATP under conditions where steady state concentrations of the inorganic phosphate and ATP existed.
Chance and Williams (67) have also introduced a new method for
measuring the amount of phosphorylation associated with 0 2 uptake.
In their studies they employed mitochondria in which the phosphorylation process is tightly coupled to the oxidation processes, and they followed the concentration of oxygen in the reaction mixture with a
vibrating platinum electrode. In the presence of excess substrate (ßhydroxybutyrate) the ADP present in the particles was phosphorylated
and the 0 2 consumption was retarded. Then, when small amounts of
ADP were added, the oxygen content, as measured by the 0 2 electrode,
decreased a definite amount. The P/O ratio (or more accurately the
ADP/O ratio) was then calculated from the ADP added and 0 2
consumed.
E. P/O RATIOS
1. Oxidation of Krebs Cycle Substrates
Much of the energy available in organic metabolites is released to the
organism when acetyl-CoA is oxidized through the reactions of the Krebs
cycle. Because Ochoa's (135) studies on rat heart extracts suggested
that the average number of phosphorylation steps per atom of oxygen
consumed during pyruvate oxidation was 3.0, it was of interest to determine which of the reactions of the cycle gave rise to oxidative phosphorylation and the number of phosphorylation. steps which could be
obtained.