448
VERNON H. CHELDELIN, CHIH H. WANG, AND TSOO E. KING
chemical yields of pyruvate from the specifically labeled glucose substrates for a given extent of pentose cycle participation.
From the operation of the pentose cycle in Fig. 3, it can be readily
visualized that the interfering eflFect of regenerated fructose-6-phosphate
should be more pronounced when glucose-2-C
14 is used as the tracing
substrate and essentially insignificant when glucose-1-C
14 is used (136).
It should be pointed out that the equations devised by these authors
are valid only if no drainage exists of intermediates of the pentose
cycle for biosynthetic functions, a condition that cannot be fulfilled even
in studies with resting cells. It is suspected that with organisms in which
phosphogluconate cleavage amounts to a minor portion of glucose
catabolized, this pathway may function mainly for the synthesis of
pentoses, heptoses, and tetroses.
3. The Radiorespirornetric Method Based on Yields of C
14 0 2
Observed in Time-Course Experiments
The confusion that often arises from dealing with the uncontrollable
dilution of isotopic substrates with unlabeled ones of endogenous origin
prompted the development of method that is based on total yield of
C
14 0 2 , without regard for specific activities (137a). In addition, the
method developed by Wang and co-workers (15, 138, 139) permits a
kinetic study of the oxidation sequences involved.
The radiorespirornetric method for pathway studies calls for the
collection as well as the determination of C
14 0 2 produced at regular
short intervals from a bank of concurrent experiments. [For convenience
a new unit for measuring time is introduced here, called the relative
time unit (RTU). This is defined as the time required to complete the
initial catabolic reaction with respect to the administered substrate.*
This unit is employed since rates of metabolism vary among organisms,
and this device tends to relate the data from one organism to another
more effectively.] Each represents the incubation of the test organism
with a substrate labeled with C
14 at a specific carbon atom. Currently,
possible labelings are C-l or C-2 of acetate; C-l, C-2, or C-3 of pyruvate;
and C-l, C-2, C-3, C-3,4, C-6, or C-U of glucose or gluconate. These
substrates represent all major carbon sources in the currently recognized
* For many organisms and substrates, this is equivalent to the time required
for complete removal of the substrate from the medium. Sometimes this is difficult
to diagnose, as in the conversion of gluconate to ketogluconate. Often the cessation
of appearance of the initially oxidized carbon atom (e.g., C-l or C-3,4) is used
to denote the duration of 1 RTU. It should be stressed that the introduction of
the RTU concept is made mainly to focus attention on the optimal duration of the
experiment, when primary catabolic reactions tend to be completed. Variations in
reaction rates among concurrent pathways are thus minimized.
VERNON H. CHELDELIN, CHIH H. WANG, AND TSOO E. KING
chemical yields of pyruvate from the specifically labeled glucose substrates for a given extent of pentose cycle participation.
From the operation of the pentose cycle in Fig. 3, it can be readily
visualized that the interfering eflFect of regenerated fructose-6-phosphate
should be more pronounced when glucose-2-C
14 is used as the tracing
substrate and essentially insignificant when glucose-1-C
14 is used (136).
It should be pointed out that the equations devised by these authors
are valid only if no drainage exists of intermediates of the pentose
cycle for biosynthetic functions, a condition that cannot be fulfilled even
in studies with resting cells. It is suspected that with organisms in which
phosphogluconate cleavage amounts to a minor portion of glucose
catabolized, this pathway may function mainly for the synthesis of
pentoses, heptoses, and tetroses.
3. The Radiorespirornetric Method Based on Yields of C
14 0 2
Observed in Time-Course Experiments
The confusion that often arises from dealing with the uncontrollable
dilution of isotopic substrates with unlabeled ones of endogenous origin
prompted the development of method that is based on total yield of
C
14 0 2 , without regard for specific activities (137a). In addition, the
method developed by Wang and co-workers (15, 138, 139) permits a
kinetic study of the oxidation sequences involved.
The radiorespirornetric method for pathway studies calls for the
collection as well as the determination of C
14 0 2 produced at regular
short intervals from a bank of concurrent experiments. [For convenience
a new unit for measuring time is introduced here, called the relative
time unit (RTU). This is defined as the time required to complete the
initial catabolic reaction with respect to the administered substrate.*
This unit is employed since rates of metabolism vary among organisms,
and this device tends to relate the data from one organism to another
more effectively.] Each represents the incubation of the test organism
with a substrate labeled with C
14 at a specific carbon atom. Currently,
possible labelings are C-l or C-2 of acetate; C-l, C-2, or C-3 of pyruvate;
and C-l, C-2, C-3, C-3,4, C-6, or C-U of glucose or gluconate. These
substrates represent all major carbon sources in the currently recognized
* For many organisms and substrates, this is equivalent to the time required
for complete removal of the substrate from the medium. Sometimes this is difficult
to diagnose, as in the conversion of gluconate to ketogluconate. Often the cessation
of appearance of the initially oxidized carbon atom (e.g., C-l or C-3,4) is used
to denote the duration of 1 RTU. It should be stressed that the introduction of
the RTU concept is made mainly to focus attention on the optimal duration of the
experiment, when primary catabolic reactions tend to be completed. Variations in
reaction rates among concurrent pathways are thus minimized.