PubMed · 1152052
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R J Sheehy, R P Novick. 1975-04-05. Studies on plasmid replication. V Replicative intermediates.. https://doi.org/10.1016/0022-2836(75)90130-8
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Evidence is presented that intermediates of the oxidative pentose phosphate pathway (OPPP) are channeled from one pathway enzyme to the next. CO2 produced from [1-14C]glucose in the presence of unlabelled pathway intermediates contained much more radioactivity than predicted by a model in which pathway-produced intermediates are in equilibrium with identical molecules in the bulk phase. This was the case whether glucose 6-phosphate (Glc6P), 6-phosphogluconolactone, or 6-phosphogluconate was added. Assumptions involved in calculating the amount of 14CO2 predicted for free mixing of 14C-labelled and unlabelled intermediates are discussed, together with the following results. (a) 14CO2 production by pea nodules in the presence of 3 mM 6-phosphogluconate was higher than in its absence. (b) Apparent channeling of intermediates was much higher for purified yeast enzymes than for yeast extract. (c) 6-Phosphogluconate and 6-phosphogluconolactone were channeled between yeast Glc6P dehydrogenase and 6-phosphogluconate dehydrogenase despite the absence of 6-phosphogluconolactonase in the purified yeast enzyme mixture. (d) When purified yeast hexokinase was physically separated from Glc6P dehydrogenase and 6-phosphogluconate dehydrogenase by a dialysis membrane, there was no apparent channeling. (e) Poly(ethylene glycol), high salt and detergents had little effect on apparent channeling of OPPP intermediates, which is consistent with a stable complex of enzymes. On the other hand, density gradient centrifugation experiments suggested a more transient interaction between the enzymes. Taken together, the results support channeling of OPPP pathway intermediates.
A method is developed and described for analysis of [11C]-meta-hydroxyephedrine, [11C]MHED, a tracer of cardiac function, and its metabolites in plasma samples. The method combines on-column solid-phase extraction and separation on a single weak cation-exchange column. Phenylethanolamines were used to develop the separation method that concentrates the analytes on-column from physiological saline and then elutes them by changing to an acidic mobile phase. Hydrophobic interactions determine the selectivity, and elution order is the same as for reversed-phase liquid chromatography on a C1 stationary phase. The mechanism of separation is mixed mode, with ion-exchange coupled with a reversed-phase liquid chromatography mechanism. Each sample analysis requires only 10 min and does not require deproteinization or the use of organic solvents. In human samples, a single plasma metabolite of [11C]MHED along with the parent compound were observed using this method. The method was sufficiently rapid so that in 70 min seven samples were assayed, providing a well-defined time course for MHED and its metabolites in blood. The metabolite concentration increased with time to approximately 85% of the plasma activity 50 min after administration. The results with the developed method are comparable to those described for reversed-phase separations, with the advantage that our method does not require deproteinization, reducing sample analysis time by a factor of two.