Labelled pathways hypothesis

The labelled pathways hypothesis, developed by Goodman and co-workers to explain the specific choices of axonal pathways made by growth cones in insect embryos (see Section 3.3.1), predicts the existence of a set of axon guidance molecules with a highly specific and restricted distribution on central and peripheral axonal pathways. 

However, the linear bond cleavage hypothesis of the firefly bioluminescence was made invalid in 1977. It was clearly shown by Shimomura et al. (1977) that one O atom of the CO2 produced is derived from molecular oxygen, not from the solvent water, using the same 180-labeling technique as used by DeLuca and Dempsey. The result was verified by Wannlund et al. (1978). Thus it was confirmed that the firefly bioluminescence reaction involves the dioxetanone pathway. Incidentally, there is currently no known bioluminescence system that involves a splitting of CO2 by the linear bond cleavage mechanism. 

At the moment, experimental data with labeled precursors are still lacking and the proposed pathways are to be considered as "working hypothesis". Nevertheless, the partly unexpected results, which were obtained in these investigation, show that there must be a competition of pathways and enzymes in the biogenesis of chiral aroma constituents in tropical fruits. 

The results of incorporation experiments using tritiated thymidine lend support to the hypothesis that thymidine rhamnosyl pyrophosphate in Lactobacillus acidophilus is an intermediate on the pathway to deoxyribonucleic acid synthesis. When cells were grown in a medium containing tritiated thymidine, labeled thymidine rhamnosyl pyrophosphate constituted two-thirds of the total label in the acid-soluble fraction. When these cells were transferred to a medium containing 2-deoxyadenosine, 2-deoxy-guanosine, and 2-deoxycytidine as the nucleosides, 99 % of the label initially present was transferred to deoxyribonucleic acid after incubation for 50 minutes. Therefore, almost complete incorporation (into deoxyribonucleic acids) of the isotope from tritiated th3anidine had occurred. 

In this way Solomon and coworkers attempted to explain the conversion of lactate, pyruvate and carbon dioxide to glycogen. According to the proposed mechanism, carboxyl-labelled pyruvate must first undergo the Wood-Werkman reaction to form oxalacetate before carboxyl-labelled phosphopyruvate can be formed by way of fumarate, phosphomalate and phospho-oxalacetate. This hypothesis is no longer necessary in view of Lardy and Ziegler s results described above. It is possible, however, that both pathways are utilized. 

Tyrosine was indeed found to be a precursor for cephalotaxine (122) but the labelling pattern precluded a phenethylisoquinoline intermediate. Thus [3- C]tyrosine gave cephalotaxine with 68% of the activity at C-16 and 32% at C-9 (Scheme 14), whereas [2- C]tyrosine labelled C-17 to the extent of 37% with no label at C-2 or C-3. It is to be noted that both samples of tyrosine were incorporated to similar extents and yet the expected correlation of activity between C-16 and C-17 is poor. This was attributed to some scatter of the label from C-2 of tyrosine. [l- CjTyrosine was only incorporated at a very low level and the activity appeared to be scattered. The results clearly implicate two molecules of tyrosine with loss of the carboxy-functions in a novel pathway to cephalotaxine. A scheme which is consistent with these results has been tentatively advanced (Scheme 15). This hypothesis is capable of further testing, which is in progress. 

The existence of two separate mechanisms for the synthesis of sucrose raises the question of their respective roles in vivo. Experiments in which C Mabeled n-glucose was supplied to plants have shown that the d-fructofuranosyl moiety of sucrose becomes highly labeled before any label appears in the free D-fructose pool, suggesting that this monosaccharide is not an intermediate in sucrose synthesis. On the other hand, d-fructose 6-phosphate becomes labeled before sucrose, and small proportions of sucrose 6 -phosphate have also been detected among the labeled pro-ducts. These results are compatible with the hypothesis that sucrose 6 -phosphate is synthesized first and is subsequently hydrolyzed to free sucrose. Such a pathway would be clearly irreversible, since it includes a hydrolytic step, and could account for the large accumulation of sucrose in many plants. 

Exposure of N. glutinosa to C02 resulted in a labeling pattern in the pyrrolidine ring which is not consistent with the symmetrical intermediate hypothesis 138, 139). C-2 and C-5 were found to be equally labeled, but the activity at C-3 was much lower than predicted on the basis of a symmetrical intermediate. Although there appears to be little doubt that ornithine is incorporated into nicotine via a symmetrical intermediate, the work with 002 raises the question as to whether this may be a minor or aberrant pathway to nicotine in the plant 139). 

Incorporation of label from [2- " C]mevalonate into the ergoline ring system supported the hypothesis that the pathway involved aromatic prenylation 68). This was further supported by the identification and purification of tryptophan dimethylallyltransferase 69,70), which transferred the dimethylallyl moiety from dimethylallyl-diphosphate (DMAPP) to the 4-position on the six-membered ring of L-tryptophan. Molecular genetic studies confirmed the role of this enzyme in EA biosynthesis 39,54). 

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