These results indicated that the availability of NADPH played a very important role in fatty acid production. Keywords: E. Glycolysis and the pentose phosphate pathway are differentially associated with the dichotomous regulation of glioblastoma cell migration versus proliferation. Kirby, B. Impaired skeletal muscle blood flow control with advancing age in humans: attenuated atp release and local vasodilation during erythrocyte deoxygenation.
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Last, the double bond is hydrogenated to yield a saturated intermediate. The process cycles with the addition of another malonyl-ACP to the growing chain until ultimately an intermediate with 16 carbons is produced palmitoyl-CoA. At this point, the cytoplasmic synthesis ceases.
Acetyl-CoA carboxylase , which catalyzes synthesis of malonyl-CoA, is the only regulated enzyme in fatty acid synthesis.
Its regulation involves both allosteric control and covalent modification. Dephosphorylation is stimulated by phosphatases activated by insulin binding. Dephosphorylation activates the enzyme and favors its assembly into a long polymer, while phosphorylation reverses the process. Citrate acts as an allosteric activator and may also favor polymerization. Palmitoyl-CoA allosterically inactivates it. In all cases, controls were included in which osmotically lysed plastids were used in place of intact organelles.
The evolution of 14 CO 2 was measured under four sets of conditions. Figure 1 shows that flux through the OPPP was linear with time up to 60 min and was measurable even when plastids were supplied only with Glc6P, although the rate of evolution of 14 CO 2 was lowest in this case.
A greater than 3-fold stimulation was seen in the presence of the substrates and cofactors required for fatty acid synthesis. Previous experiments have shown that, in the absence of these cofactors and substrates, plastids isolated from B.
In the present experiment, the carbon skeletons for fatty acid synthesis would have been supplied by the oxidation of Glc6P by plastidial glycolysis, since no other carbon precursor was present. However, by far the largest stimulation of OPPP flux was seen when methyl viologen was supplied to the organelles where the 14 CO 2 released after 60 min was almost 8-fold greater than when Glc6P was supplied alone. Oxidation of Glc6P by B. The data are corrected for 14 CO 2 evolution from osmotically-lysed plastids and are expressed relative to the maximum value with methyl viologen 1.
These interactions were studied in more detail to determine the degree to which OPPP flux would be limited by the supply of Glc6P as opposed to the sink for reductant. Figure 2 demonstrates the increase in CO 2 evolution as a function of Glc6P concentration, in the absence or presence of methyl viologen or the substrates and cofactors for fatty acid synthesis. The relationship between the three conditions remains the same at all concentrations of Glc6P, with methyl viologen providing by far the greatest stimulation.
The data presented in Fig. In both cases, the calculated apparent K m for Glc6P was approximately 1 mM. The relationship between fatty acid synthesis and OPPP flux when Glc6P was the sole source of acetyl units was investigated by simultaneously measuring the incorporation of 14 C into fatty acids and the release of 14 CO 2 Table 2.
The relative rates of carbon flux into fatty acids and into CO 2 were 2. The latter ratio was very similar to that shown previously for plastids isolated from B. The amount of carbon incorporated into fatty acids from all atoms of the hexose molecule when plastids were fed [U- 14 C]Glc6P was almost 8-fold greater than that from the C-1 position when [1- 14 C]Glc6P was supplied Table 2. This value is larger than the expected 4-fold increase based upon the difference in conservation of 14 C in the actetate that is derived, via glycolysis and decarboxylation of pyruvate by PDC, from 1- or U-labelled Glc6P.
Almost 2. When plastids were supplied with [6- 14 C]Glc6P, or with [6- 14 C]Glc added as a control for any contamination of the in vitro -synthesized [6- 14 C]Glc6P by [6- 14 C]Glc, in the presence of the cofactors necessary for fatty acid synthesis, the evolution of 14 CO 2 was negligible Table 2. Synthesis of fatty acids and evolution of CO 2 by isolated plastids. Isolated plastids were incubated with 5 mM [1- 14 C], [U- 14 C], [6- 14 C] glucose 6-phosphate or [6- 14 C] glucose 1.
Incorporation of 14 C into fatty acids and the evolution of 14 CO 2 were determined during 1 h incubation. ND, not determined. When embryo plastids were incubated in darkness, G6PDH activity in lysates from them was not significantly changed over 90 min Fig.
This DTT-dependent loss of activity closely resembled that caused by light Fig. To provide a comparison of the sensitivity of the embryo plastid G6PDH activity with that of the chloroplast enzyme, chloroplasts isolated from B. Effect of light on the activity of glucose 6-phosphate dehydrogenase in isolated plastids.
Plastids were incubated in isolation medium in the light or dark, lysed and the enzyme activity was expressed relative to that at zero time Effect of DTT on the activity of glucose 6-phosphate dehydrogenase in isolated plastids.
Plastids were incubated in the dark in isolation medium with or without 5 mM DTT, lysed and the enzyme activity was expressed relative to that at zero time These studies of the subcellular localization of all of the OPPP enzymes in the developing embryos of Brassica napus have added to other studies that have questioned a dual localization of the complete OPPP in both the cytosol and the plastid Schnarrenberger et al.
For example, biochemical evidence for the confinement of the TK and TA activities to the plastid fraction is provided here. This observation for B. Thom et al. Two assays were used to determine RPE activity. Although the percentage recovery of RPE activity in the plastid fraction was no higher than that of contaminating cytosolic enzymes, which implies a cytosolic localization of RPE activity, the total RPE activity in homogenates was, on average, an order of magnitude greater than that of the other OPPP enzymes Table 1.
If, as the data imply, this activity is mostly cytosolic, it would be difficult to detect a small proportion of the total RPE that was within the plastids because of cytosolic contamination. Therefore, although the large majority of RPE is localized in the cytosol, the possibility that there is RPE activity in the plastid cannot be ruled out. Ensuring export of Ru5P to the cytosol would allow a balance between the production of Xu5P for re-entry into the plastid and the cytosolic synthesis of R5P.
This model of partitioning of pentose phosphates for different purposes is reinforced if TK and TA are confined to the plastid. Eicks et al. This translocator would enable metabolic interaction between cytosolic oxidative and plastidial non-oxidative reactions of the OPPP. Whilst the dehydrogenases of the OPPP are generally accepted as having a dual location in both cytosol and plastids, as reported here for B. This disparity may reflect genuine species differences and also the possibility that, within a species, there is tissue-specific expression of particular isozymes in different subcellular locations.
In leaves and roots of spinach, pea, and maize, the non-oxidative steps in roots and leaves are largely, if not entirely, plastid localized Schnarrenberger et al. The reasons for these different distributions of OPPP enzymes remain to be elucidated. As the major function of the OPPP in plastids is likely to be the generation of reducing power for biosynthesis, the ability of different metabolic sinks for electrons to stimulate flux through the pathway were examined. The first point to note is that it appears that even when Glc6P is supplied without any additional supplement to intact plastids, it can be metabolized via the OPPP Fig.
The small stimulation of OPPP flux by the addition of glutamine and 2-oxoglutarate suggests that there is a functional glutamate synthase reaction within the plastids of B. A much greater stimulation of the OPPP was seen when cofactors for fatty acid synthesis were supplied, enabling the utilization of Glc6P as the source of acetyl units as well as for the generation of reductant.
Previous studies Eastmond and Rawsthorne, have shown that i Glc6P is the cytosolic precursor best able to supply carbon skeletons for fatty acid synthesis by plastids isolated from embryos at the same developmental stage used in this present study and ii that fatty acid synthesis by these plastids absolutely requires the added cofactors and substrates Kang, When a non-physiological electron acceptor, methyl viologen, was provided to the isolated plastids metabolizing Glc6P, the stimulation of the OPPP was more than 2-fold greater than when the substrates for fatty acid synthesis were supplied.
This suggests that there is ample capacity for increasing the flux through the OPPP and that the generation of reductant via this route is unlikely to be limiting with respect to fatty acid synthesis. The apparent affinity for Glc6P of Glc6P-dependent fatty acid synthesis and Glc6P-dependent methyl viologen reduction K m of 1 mM in embryo plastids is close to the estimated concentration of Glc6P in whole embryos Eastmond and Rawsthorne, As the K m for the Glc6P transporter is approximately 0.
These observations suggest that generation of acetyl units and their subsequent utilization is more of a limitation to the production of fatty acids than the uptake of Glc6P or its oxidation in the OPPP. Given that CO 2 in this equation arises from carbon atoms 3 and 4, as a result of pyruvate dehydrogenase activity, this would imply that for every 16 carbon atoms synthesized as fatty acids palmitate , 4 would arise from carbon atom 1. Therefore, when feeding [U- 14 C]Glc6P it would be expected that the incorporation of labelled carbon into fatty acid would be approximately 4-fold that from [1- 14 C]Glc6P, not 8-fold.
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