1. PENTOSE PHOSPHATE CYCLE
15
from a medium of low ionic strength (90). This enzyme could subsequently be released from these particles upon addition of salt.
The problem posed by the compartmentalization of the enzymes has
in part been solved. Progress toward this end was first achieved by Arnon
and his co-workers, who showed that chloroplasts contain the enzymes
necessary to fix carbon dioxide (91). Whether all these enzymes are
located entirely in the chloroplasts or some in the cytoplasm was not
certain. However, a novel method was independently elaborated by two
investigators (92, 93), who obtained chloroplasts from lyophilized leaves,
ground and fractionated in a nonaqueous medium. One of them (93)
showed that aldolase normally thought to be associated with the cytoplasm (i.e., to exist in low concentrations within the chloroplasts), was
in fact localized predominantly in the chloroplasts. Later, Smillie and
Fuller showed that there is a parallel relationship between the percentage distribution of carboxydismutase and the percentage distribution
of chlorophyll in chloroplast-containing fractions derived by the nonaqueous method (94). Finally, Heber et al. (95), using the same type
of chloroplasts, showed that all the carboxydismutase and the TPNdependent triose phosphate dehydrogenase is located in the chloroplasts,
whereas the DPN-linked triose phosphate dehydrogenase is distributed
between both the chloroplasts and the cytoplasm.
F. METABOLISM OF C
1 4
-LABELED SUBSTRATES
A specifically labeled substrate is incubated with the tissue or organism, and the distribution of the label in the products and/or the
specific activities of the products are measured. An application of this
method is the study of the heterolactic fermentation of glucose-l-C
14 and
glucose-3,4-C
14 by Leuconostoc mesenteroides (50). The products, lactate, carbon dioxide, and ethanol, were degraded chemically. The label
was found to be in the carboxyl group of lactate, in C0 2 , and in the C-l
of ethanol, indicating that a new pathway is in operation. Another pioneering work was done by Bloom et al. (96), who determined the
catabolic routes of glucose in mammalian tissues. They fed glucose-l-C
14
and glucose-6-C
14 and specifically labeled lactate to these tissues and
then measured the C
14 incorporated into the C0 2 . The assumption underlying this procedure is that the pentose phosphate cycle would yield
C
14 0 2 from glucose carbon atom 1 at a greater rate than that from carbon
atom 6, whereas glycolysis plus the operation of the Krebs cycle would
yield carbon dioxide derived from carbon atoms 1 and 6 at the same rate.
The ratio of the C
14 0 2 derived from glucose-6-C
14 to the C
14 0 2 derived
from glucose-l-C
14 would be a measure of relative participation of the
15
from a medium of low ionic strength (90). This enzyme could subsequently be released from these particles upon addition of salt.
The problem posed by the compartmentalization of the enzymes has
in part been solved. Progress toward this end was first achieved by Arnon
and his co-workers, who showed that chloroplasts contain the enzymes
necessary to fix carbon dioxide (91). Whether all these enzymes are
located entirely in the chloroplasts or some in the cytoplasm was not
certain. However, a novel method was independently elaborated by two
investigators (92, 93), who obtained chloroplasts from lyophilized leaves,
ground and fractionated in a nonaqueous medium. One of them (93)
showed that aldolase normally thought to be associated with the cytoplasm (i.e., to exist in low concentrations within the chloroplasts), was
in fact localized predominantly in the chloroplasts. Later, Smillie and
Fuller showed that there is a parallel relationship between the percentage distribution of carboxydismutase and the percentage distribution
of chlorophyll in chloroplast-containing fractions derived by the nonaqueous method (94). Finally, Heber et al. (95), using the same type
of chloroplasts, showed that all the carboxydismutase and the TPNdependent triose phosphate dehydrogenase is located in the chloroplasts,
whereas the DPN-linked triose phosphate dehydrogenase is distributed
between both the chloroplasts and the cytoplasm.
F. METABOLISM OF C
1 4
-LABELED SUBSTRATES
A specifically labeled substrate is incubated with the tissue or organism, and the distribution of the label in the products and/or the
specific activities of the products are measured. An application of this
method is the study of the heterolactic fermentation of glucose-l-C
14 and
glucose-3,4-C
14 by Leuconostoc mesenteroides (50). The products, lactate, carbon dioxide, and ethanol, were degraded chemically. The label
was found to be in the carboxyl group of lactate, in C0 2 , and in the C-l
of ethanol, indicating that a new pathway is in operation. Another pioneering work was done by Bloom et al. (96), who determined the
catabolic routes of glucose in mammalian tissues. They fed glucose-l-C
14
and glucose-6-C
14 and specifically labeled lactate to these tissues and
then measured the C
14 incorporated into the C0 2 . The assumption underlying this procedure is that the pentose phosphate cycle would yield
C
14 0 2 from glucose carbon atom 1 at a greater rate than that from carbon
atom 6, whereas glycolysis plus the operation of the Krebs cycle would
yield carbon dioxide derived from carbon atoms 1 and 6 at the same rate.
The ratio of the C
14 0 2 derived from glucose-6-C
14 to the C
14 0 2 derived
from glucose-l-C
14 would be a measure of relative participation of the