1. The distributions and rates of transfer of carbon isotopes from a selection of specifically labelled ketosugar-phosphate substrates by exchange reactions catalyzed by the pentose and photosynthetic carbon-reduction-pathway group-transferring enzymes transketolase, transaldolase and aldolase have been measured using I3C-NMR spectroscopy.2. The rates of these exchange reactions were 5, 4 and 1.5 pmol min-' mg-' for transketolase exchange, transaldolase exchange and aldolase exchange, respectively.3. A comparison of the exchange capacities contributed by the activities of these enzymes in three in v i m liver preparations with the maximum non-oxidative pentose pathway flux rates of the preparations shows that transketolase and aldolase exchanges exceeded flux by 9-19 times in liver cytosol and acetone powder enzyme preparations and by 5 times in hepatocytes. Transaldolase was less effective in the comparison of exchange versus flux rates: transaldolase exchange exceeded flux by 1.6 and 5 in catalysis by liver cytosol and acetone powder preparations, respectively, but was only 0.6 times the flux in hepatocytes.4. Values of group enzyme exchange and pathway flux rates in the above three preparations are important because of the feature role of liver and of these particular preparations in the establishment, elucidation and measurement of a proposed reaction scheme for the fat-cell-type pentose pathway in biochemistry.5. It is the claim of this paper that the excess of exchange rate activity (particularly transketolase exchange) over pathway flux will overturn attempts to unravel, using isotopically labelled sugar substrates, the identity, reaction sequence and quantitative contribution of the pentose pathway to glucose metabolism.6. The transketolase exchange reactions relative to the pentose pathway flux rates in normal, regenerating and foetal liver, Morris hepatomas, mammary carcinoma, melanoma, colonic epithelium, spinach chloroplasts and epididymal fat tissue show that transketolase exchange may exceed flux in these tissues by factors ranging over 5 -600 times.7. The confusion of pentose pathway theory by the effects of transketolase exchange action is illustrated by the 13C-NMR spectrum of the hexose 6-phosphate products of ribose 5-phosphate dissimilation, formed after 30 min of liver enzyme action, and shows I3C-labelling in carbons 1 and 3 of glucose 6-phosphate with ratios which range over 2.1 -6.4 rather than the mandatory value of 2 which is imposed by the theoretical mechanism of the pathway.

The electron impact mass spectrometric fragmentation patterns of the per-0-trimethylsilyl (TMS) and alkoxime-TMS derivatives of D-glyCerO-D-UftrO and D-glycero-D-ido-octulo~ were fully analysed using seven specifically labelled 13C analogues. Many of the more intense ions in the spectra of the per&-TMS furanose and pyranose derivatives have more than one origin. On the other hand, the mass spectra of the straight-chain methoxime and ethoxime hepta-0-TMS derivatives contain ions each of which in general originates from a single cleavage process. This makes gas chromatography/mass spectrometry of the alkoxime-TMS derivatives a suitable method for monitoring incorporations at individual carbon atoms in the corresponding octulose phosphates during photosynthesis experiments with "CO, . Synthesis of 3C-labelled sugar phosphatesLabelled hexose &phosphates. [ 1-' 3C]Glucose 6phosphate and [l-' 3C]allose 6-phosphate were prepared as described previou~ly.~ The synthesis of [3-13C]-,

The (EIMS) electron ionization mass spectrometric fragmentation patterns of the methoxime- and ethoxime-trimethylsilyl (TMS) derivatives of C(4) to C(7) sugars involved as phosphates in the Calvin pathway of photosynthesis in plants were analysed by gas chromatography/EIMS using specifically labelled (13)C analogs. In general, most but not all of the major ions in the mass spectra arise from single carbon-carbon bond cleavages of the straight-chain derivatives. The results confirm that GC/MS of the alkoxime-TMS derivatives is a viable method for measuring (13)C incorporations at individual carbon atoms in each of the sugar phosphates during photosynthetic experiments with (13)CO(2).

A GC/EIMS/SIM methodology has been developed to re-examine the path of carbon in photosynthesis. Exposing isolated spinach chloroplasts to 13 CO 2 on a solid support for a defined period followed by quenching and work-up provided a mixture of labelled sugar phosphates. After enzymatic dephosphorylation and derivatization, the Mox-TMS sugars were analysed using the above method. The purpose of the study was to try to calculate the atom% enrichment of 13 C in as many of the individual carbons in each of the derivatized sugars as was practical using diagnostic fragment ions. In the event, only one 45 s experiment provided sufficient data to enable a range of enrichment values to be calculated. This confirmed that D-glycero-D-altrooctulose phosphate was present in the chloroplasts and was heavily labelled in the C4, C5 and C6 positions, in keeping with the hypothesis that it had an inclusive role and a labelling pattern consistent with a new modified pathway of carbon in photosynthesis.

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