446
VERNON H. CHELDELIN, CHIH H. WANG, AND TSOO E. KING
nature, since the labeling pattern of the regenerated hexose phosphates
is far different from that in the administered hexose. The administered
hexose is therefore subject to drastic changes in specific activity of its
carbon atoms, particularly in carbons 1, 2, and 3. In order to evaluate
the extent of dilution of these carbon atoms, the authors carried out
comprehensive calculations to account for the effect of recycling via the
pentose cycle on the specific activity of glucose-6-phosphate derived
from glucose-1-, -2-, and -3-C
14 , respectively.
It is noted, however, that these calculations are based on the assumptions that fructose-6-phosphate derived from the pentose cycle operation
is in perfect isotopic equilibrium with glucose-6-phosphate, derived from
the administered labeled glucose, and that there exists no drainage of
pentose cycle intermediates for other cellular functions, such as the
use of pentose for nucleotide and nucleoside synthesis. Both these
assumptions, especially the latter, represent limiting situations that may
not be realized in vivo. Although knowledge is incomplete on the
equilibrium: glucose-6-phosphate ^ fructose-6-phosphate ^± fructose-1,6diphosphate in animal tissue, there exists evidence that in microorganisms, the fructose-6-phosphate derived from the pentose cycle can be
readily converted to fructose-l,6-diphosphate, and hence trioses, via
glycolysis. The portion of fructose-6-phosphate so catabolized will then
interrupt the operation of the pentose cycle in the strict cyclic sense,
making it impossible to calculate the exact dilution effect of regenerated
fructose-6-phosphate upon the substrate glucose. The detailed experiments bearing on this point will be discussed later. As to the drainage
of the pentose cycle intermediates into biosynthetic pathways, one may
rationalize that it may not be important in isolated tissues, but it is
certain that such processes do occur extensively in living systems.
2. Methods Based on the Comparison of the Specific Activity of
Metabolic Intermediates or Products Derived from
C
14 Specifically Labeled Substrates
Methods of this type have been employed by many workers since the
late 1940's. Thus, the Entner-Doudoroff pathway in Pseudomonas saccharophila was derived from the observation that C-l of glucose or gluconate was converted to C-l of the immediate catabolic product,
pyruvate (114). However, it was 1954 before a workable method was
developed by Blumenthal, Lewis, and Weinhouse (132-134) to correlate
the specific activity of glucose-1-C
14 or glucose-6-C
14 to that of acetate,
or some other metabolic product, for the estimation of pathway participation in yeast, Pseudomonas fiuorescens, and liver tissue. The successful application of this method relies on the following characteristics:
VERNON H. CHELDELIN, CHIH H. WANG, AND TSOO E. KING
nature, since the labeling pattern of the regenerated hexose phosphates
is far different from that in the administered hexose. The administered
hexose is therefore subject to drastic changes in specific activity of its
carbon atoms, particularly in carbons 1, 2, and 3. In order to evaluate
the extent of dilution of these carbon atoms, the authors carried out
comprehensive calculations to account for the effect of recycling via the
pentose cycle on the specific activity of glucose-6-phosphate derived
from glucose-1-, -2-, and -3-C
14 , respectively.
It is noted, however, that these calculations are based on the assumptions that fructose-6-phosphate derived from the pentose cycle operation
is in perfect isotopic equilibrium with glucose-6-phosphate, derived from
the administered labeled glucose, and that there exists no drainage of
pentose cycle intermediates for other cellular functions, such as the
use of pentose for nucleotide and nucleoside synthesis. Both these
assumptions, especially the latter, represent limiting situations that may
not be realized in vivo. Although knowledge is incomplete on the
equilibrium: glucose-6-phosphate ^ fructose-6-phosphate ^± fructose-1,6diphosphate in animal tissue, there exists evidence that in microorganisms, the fructose-6-phosphate derived from the pentose cycle can be
readily converted to fructose-l,6-diphosphate, and hence trioses, via
glycolysis. The portion of fructose-6-phosphate so catabolized will then
interrupt the operation of the pentose cycle in the strict cyclic sense,
making it impossible to calculate the exact dilution effect of regenerated
fructose-6-phosphate upon the substrate glucose. The detailed experiments bearing on this point will be discussed later. As to the drainage
of the pentose cycle intermediates into biosynthetic pathways, one may
rationalize that it may not be important in isolated tissues, but it is
certain that such processes do occur extensively in living systems.
2. Methods Based on the Comparison of the Specific Activity of
Metabolic Intermediates or Products Derived from
C
14 Specifically Labeled Substrates
Methods of this type have been employed by many workers since the
late 1940's. Thus, the Entner-Doudoroff pathway in Pseudomonas saccharophila was derived from the observation that C-l of glucose or gluconate was converted to C-l of the immediate catabolic product,
pyruvate (114). However, it was 1954 before a workable method was
developed by Blumenthal, Lewis, and Weinhouse (132-134) to correlate
the specific activity of glucose-1-C
14 or glucose-6-C
14 to that of acetate,
or some other metabolic product, for the estimation of pathway participation in yeast, Pseudomonas fiuorescens, and liver tissue. The successful application of this method relies on the following characteristics: