1. PENTOSE PHOSPHATE CYCLE
9
the PGA level and in the RuDP level were measured. The results were a
decrease in the concentration of PGA and a concomitant increase in the
concentration of RuDP.
These data naturally suggested a search for a cell-free extract of algae
containing this type of carboxylase activity (56). Such a cell-free extract
was obtained by sonically rupturing Chlorella pyrenoidosa and then centrifuging down the whole cells. To the supernatant liquid were added
labeled bicarbonate and unlabeled ribulose diphosphate (isolated in a
separate experiment by paper chromatography of an aqueous extract of
algae). Experiments were also carried out with this cell-free extract
using unlabeled bicarbonate and labeled ribulose diphosphate. In both
cases, the product obtained was radioactive PGA and to a lesser extent,
organic acids. Where labeled bicarbonate plus unlabeled ribulose diphosphate were used, all the tracer was located in the carboxyl group of PGA.
Meanwhile, Weissbach et al. found in a soluble spinach extract an
enzyme system which when incubated with radioactive carbonate and
ribose-5-phosphate in the presence of ATP and TPN
+ , formed carboxyllabeled PGA (57). Furthermore, if ribose-5-phosphate-l-C
14 was incubated with the extract in the presence of unlabeled carbonate, over 70%
of the tag was in the beta carbon of PGA, the rest being in the carboxyl
carbon.
Finally, Racker (58) has constructed an artificial cell-free system by
mixing together enzymes from various sources—enzymes necessary for
the operation of the carbon reduction cycle plus ATP, hydrogenase for
generating reduced pyridine nucleotide and metal ion. In the presence of
C0 2 , H 2 , and a catalytic amount of ribose-5-phosphate, this system
yielded a net synthesis of carbohydrate. One difference should be indicated: the in vivo scheme elaborated by Calvin and his co-workers uses
aldolase to produce sedoheptulose diphosphate and then a phosphatase
to form sedoheptulose monophosphate, whereas Racker's reconstructed
system uses transaldolase to yield sedoheptulose monophosphate directly.
This difference would not alter substantially the over-all labeling pattern
and the distribution labeling in sedoheptulose.
V. Criteria for the Presence of the Pentose Phosphate Cycle
The sequences of reactions have already been defined in the beginning paragraphs of this review. It should be clearly stated, however, that
it is not the author's intent to stipulate what are the standards for justifying the existence of any given pathway. Rather, it should be noted that
different investigators use different indicators for suggesting the operation of a particular pathway in a tissue, but any definite conclusion must
take into account the criticism and restrictions inherent in the method.
Axelrod and Beevers (5) have indicated, in their review article of
9
the PGA level and in the RuDP level were measured. The results were a
decrease in the concentration of PGA and a concomitant increase in the
concentration of RuDP.
These data naturally suggested a search for a cell-free extract of algae
containing this type of carboxylase activity (56). Such a cell-free extract
was obtained by sonically rupturing Chlorella pyrenoidosa and then centrifuging down the whole cells. To the supernatant liquid were added
labeled bicarbonate and unlabeled ribulose diphosphate (isolated in a
separate experiment by paper chromatography of an aqueous extract of
algae). Experiments were also carried out with this cell-free extract
using unlabeled bicarbonate and labeled ribulose diphosphate. In both
cases, the product obtained was radioactive PGA and to a lesser extent,
organic acids. Where labeled bicarbonate plus unlabeled ribulose diphosphate were used, all the tracer was located in the carboxyl group of PGA.
Meanwhile, Weissbach et al. found in a soluble spinach extract an
enzyme system which when incubated with radioactive carbonate and
ribose-5-phosphate in the presence of ATP and TPN
+ , formed carboxyllabeled PGA (57). Furthermore, if ribose-5-phosphate-l-C
14 was incubated with the extract in the presence of unlabeled carbonate, over 70%
of the tag was in the beta carbon of PGA, the rest being in the carboxyl
carbon.
Finally, Racker (58) has constructed an artificial cell-free system by
mixing together enzymes from various sources—enzymes necessary for
the operation of the carbon reduction cycle plus ATP, hydrogenase for
generating reduced pyridine nucleotide and metal ion. In the presence of
C0 2 , H 2 , and a catalytic amount of ribose-5-phosphate, this system
yielded a net synthesis of carbohydrate. One difference should be indicated: the in vivo scheme elaborated by Calvin and his co-workers uses
aldolase to produce sedoheptulose diphosphate and then a phosphatase
to form sedoheptulose monophosphate, whereas Racker's reconstructed
system uses transaldolase to yield sedoheptulose monophosphate directly.
This difference would not alter substantially the over-all labeling pattern
and the distribution labeling in sedoheptulose.
V. Criteria for the Presence of the Pentose Phosphate Cycle
The sequences of reactions have already been defined in the beginning paragraphs of this review. It should be clearly stated, however, that
it is not the author's intent to stipulate what are the standards for justifying the existence of any given pathway. Rather, it should be noted that
different investigators use different indicators for suggesting the operation of a particular pathway in a tissue, but any definite conclusion must
take into account the criticism and restrictions inherent in the method.
Axelrod and Beevers (5) have indicated, in their review article of