When [4-13C]glutamate is incubated with yeast cells, the [13C]aspartate formed shows a [2-13C]/[3-13C] ratio that is greater than 1. If the conversion occurs through the Krebs TCA cycle with the symmetrical intermediates succinate and fumarate, the ratio is expected to be 1. The addition of fluoroacetate to the incubation increases the ratio further. When the mutants Aco1- or CS1- are used, little or no aspartate is formed. When [2-13C]acetate is incubated with yeast, the C2-/C3-aspartate ratio is 1.5 at 2 min and decreases to 1.0 after 10 min of incubation. These results indicate that orientation-conserved transfer occurs at the succinate thiokinase and succinate dehydrogenase steps of the Krebs TCA cycle.
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