10. ELECTRON TRANSPORT AND OXIDATIVE PHOSPHORYLATION
461
simultaneously. In the absence of complicating factors, the former is
equivalent to the amount of inorganic phosphate which disappears from
the reaction mixture during a given period of time. This quantity can
be determined by measuring the difference between the inorganic phosphate present at the beginning and at the end of the experiment. 0 2 uptake during this period can be observed manometrically. Phosphatases
which catalyze the hydrolysis of ATP will interfere, and lower P/O
ratios will be observed. Ochoa (140) introduced the use of glucose and
hexosemonophosphate as acceptors of phosphate from the adenylic acid
system, since the phosphorylated sugars appeared to be less susceptible to phosphatase action. Fluoride was also added to the reaction
mixtures to inhibit phosphatase action.
With the use of mitochondria (145, 146) and other particulate
preparations (141, 143) in studies on oxidative phosphorylation, side reactions that are due to soluble enzymes present in homogenates become
less significant. However, it is necessary to add hexokinase to the reaction mixtures (146-149) if glucose is employed as a phosphate acceptor from ATP (141,142). ADP can serve as acceptor and under these
conditions ATP will accumulate. In experiments of this sort fluoride addition inhibits ATP-ase activity and higher P/O ratios are obtained
(135, 145).
A typical reaction mixture for studying oxidative phosphorylation
would therefore contain mitochondria or some other source of the necessary enzymes, inorganic phosphate, Mg
2+ , AMP or ADP, F", and substrate. Depending on the type of preparation or the substrate being
studied, DPN
+ and/or cytochrome c might be added. In those experiments where the final phosphate acceptor is glucose, both glucose and
hexokinase would be added to the reaction mixture. Only catalytic
amounts of ADP are required.
The substrates most frequently employed in studies of this sort are
the intermediates of the Krebs cycle. In addition the oxidation of
glutamate (141, 146), butyrate (141), other fatty acids (150), and
proline (141) are known to support oxidative phosphorylation. Another
substrate which has been extensively used is /?-hydroxybutyrate (145).
Slater introduced a valuable modification of the procedure for determining P/O ratios (151). He proposed measuring the amount of glucose6-phosphate formed in the presence of a glucose-and-hexokinase trap
rather than the disappearance of inorganic phosphate. Since sensitive
and precise methods are available for determining extremely small
amounts of glucose-6-phosphate (152), the phosphorylation process can
be followed accurately at the beginning of the experiments when the
disappearance of inorganic phosphate is small.
Studies on the localization of the sites of phosphorylation in the
461
simultaneously. In the absence of complicating factors, the former is
equivalent to the amount of inorganic phosphate which disappears from
the reaction mixture during a given period of time. This quantity can
be determined by measuring the difference between the inorganic phosphate present at the beginning and at the end of the experiment. 0 2 uptake during this period can be observed manometrically. Phosphatases
which catalyze the hydrolysis of ATP will interfere, and lower P/O
ratios will be observed. Ochoa (140) introduced the use of glucose and
hexosemonophosphate as acceptors of phosphate from the adenylic acid
system, since the phosphorylated sugars appeared to be less susceptible to phosphatase action. Fluoride was also added to the reaction
mixtures to inhibit phosphatase action.
With the use of mitochondria (145, 146) and other particulate
preparations (141, 143) in studies on oxidative phosphorylation, side reactions that are due to soluble enzymes present in homogenates become
less significant. However, it is necessary to add hexokinase to the reaction mixtures (146-149) if glucose is employed as a phosphate acceptor from ATP (141,142). ADP can serve as acceptor and under these
conditions ATP will accumulate. In experiments of this sort fluoride addition inhibits ATP-ase activity and higher P/O ratios are obtained
(135, 145).
A typical reaction mixture for studying oxidative phosphorylation
would therefore contain mitochondria or some other source of the necessary enzymes, inorganic phosphate, Mg
2+ , AMP or ADP, F", and substrate. Depending on the type of preparation or the substrate being
studied, DPN
+ and/or cytochrome c might be added. In those experiments where the final phosphate acceptor is glucose, both glucose and
hexokinase would be added to the reaction mixture. Only catalytic
amounts of ADP are required.
The substrates most frequently employed in studies of this sort are
the intermediates of the Krebs cycle. In addition the oxidation of
glutamate (141, 146), butyrate (141), other fatty acids (150), and
proline (141) are known to support oxidative phosphorylation. Another
substrate which has been extensively used is /?-hydroxybutyrate (145).
Slater introduced a valuable modification of the procedure for determining P/O ratios (151). He proposed measuring the amount of glucose6-phosphate formed in the presence of a glucose-and-hexokinase trap
rather than the disappearance of inorganic phosphate. Since sensitive
and precise methods are available for determining extremely small
amounts of glucose-6-phosphate (152), the phosphorylation process can
be followed accurately at the beginning of the experiments when the
disappearance of inorganic phosphate is small.
Studies on the localization of the sites of phosphorylation in the