1. PENTOSE PHOSPHATE CYCLE
13
E. PRESENCE OF ENZYMES OF THE PENTOSE PHOSPHATE PATHWAY
This method involves the assay of one or more of the pentose phosphate pathway enzymes by adding the required substrate(s) to a crude
extract or a purified fraction from the tissue. Control experiments have
to be performed in order to ensure that there are no inhibitors of the
enzyme in the crude extract. The latter experiments are done by adding
the fraction to be tested to a purified enzyme preparation from another
organism. If the activity of this enzyme is not affected by this addition,
then the test fraction does not contain an inhibitor of the enzyme. Such
an approach was used by Richter (70) in his studies of carbohydratemetabolizing enzymes in two photosynthetic algae.
The interpretation of this method is fraught with many reservations.
Even when an enzyme is found to be present, this does not necessarily
mean that the organism is using the pentose phosphate cycle for the
catabolism of carbohydrates. There are already a number of cases where
enzymes of the pentose phosphate cycle are found in extracts of the
organism, but where the main pathway in operation (greater than 90%)
is the Embden-Meyerhof-Parnas sequence. Among the many examples
that contain shunt enzymes, but metabolize glucose chiefly via glycolysis,
are Avena coleoptiles (71), Ehrlich ascites mouse tumors (72), and yeast
(73, 74).
Even if we grant that glucose is not metabolized mainly by the
Embden-Meyerhof-Parnas route, and assuming that we find one or two
enzymes normally associated with the pentose phosphate pathway, we
are still uncertain as to the interpretation of the results. As already
pointed out by Axelrod (75), the assignment of a given enzyme to a
particular pathway of metabolism is purely arbitrary and, to add to the
confusion, some enzymes initially thought to belong to the glycolytic
pathway were later found to belong also to the oxidative pathway (as
well as to the reductive cycle). These enzymes are aldolase, triose phosphate isomerase, triose phosphate dehydrogenase, and others. It should
be noted that some differences exist between triose phosphate dehydrogenase derived from plants and animals. In contrast to the DPN-linked
dehydrogenase from animal tissues, the corresponding plant enzymes
are of three types: (a) DPN-linked (76,77),
(b) TPN-linked
(77-80),
and (c) TPN-linked, but not coupled with phosphorylation (81, 82).
In spite of the reservations mentioned above, it is nevertheless useful
to obtain information as to whether a tissue or organism has a particular
"indicator" enzyme of the pentose phosphate cycle. Most prevalently
used are glucose-6-phosphate dehydrogenase, transketolase, transaldolase,
13
E. PRESENCE OF ENZYMES OF THE PENTOSE PHOSPHATE PATHWAY
This method involves the assay of one or more of the pentose phosphate pathway enzymes by adding the required substrate(s) to a crude
extract or a purified fraction from the tissue. Control experiments have
to be performed in order to ensure that there are no inhibitors of the
enzyme in the crude extract. The latter experiments are done by adding
the fraction to be tested to a purified enzyme preparation from another
organism. If the activity of this enzyme is not affected by this addition,
then the test fraction does not contain an inhibitor of the enzyme. Such
an approach was used by Richter (70) in his studies of carbohydratemetabolizing enzymes in two photosynthetic algae.
The interpretation of this method is fraught with many reservations.
Even when an enzyme is found to be present, this does not necessarily
mean that the organism is using the pentose phosphate cycle for the
catabolism of carbohydrates. There are already a number of cases where
enzymes of the pentose phosphate cycle are found in extracts of the
organism, but where the main pathway in operation (greater than 90%)
is the Embden-Meyerhof-Parnas sequence. Among the many examples
that contain shunt enzymes, but metabolize glucose chiefly via glycolysis,
are Avena coleoptiles (71), Ehrlich ascites mouse tumors (72), and yeast
(73, 74).
Even if we grant that glucose is not metabolized mainly by the
Embden-Meyerhof-Parnas route, and assuming that we find one or two
enzymes normally associated with the pentose phosphate pathway, we
are still uncertain as to the interpretation of the results. As already
pointed out by Axelrod (75), the assignment of a given enzyme to a
particular pathway of metabolism is purely arbitrary and, to add to the
confusion, some enzymes initially thought to belong to the glycolytic
pathway were later found to belong also to the oxidative pathway (as
well as to the reductive cycle). These enzymes are aldolase, triose phosphate isomerase, triose phosphate dehydrogenase, and others. It should
be noted that some differences exist between triose phosphate dehydrogenase derived from plants and animals. In contrast to the DPN-linked
dehydrogenase from animal tissues, the corresponding plant enzymes
are of three types: (a) DPN-linked (76,77),
(b) TPN-linked
(77-80),
and (c) TPN-linked, but not coupled with phosphorylation (81, 82).
In spite of the reservations mentioned above, it is nevertheless useful
to obtain information as to whether a tissue or organism has a particular
"indicator" enzyme of the pentose phosphate cycle. Most prevalently
used are glucose-6-phosphate dehydrogenase, transketolase, transaldolase,