A Highly Sensitive Method for Detection of 32P Incorporation into Acid‐Soluble Compounds and Its Application to Evaluate ATP‐Forming Ability of Bacillus megaterium QM B1551 Spores at the Very Early Stage of Germination

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Abstract
A method for specific removal of [32P]orthophosphate (Pi) as phosphomolybdate-triethylamine complex was slightly modified by repeating the Pi precipitation procedures in the presence of unlabeled Pi, which resulted in a complete removal of 32Pi (greater than 99.98%). Using this modified method, we determined 32P incorporation into acid-soluble compounds in order to evaluate the ATP-forming ability of Bacillus megaterium spores at the very early stage of germination. Addition of fructose as a substrate started the 32P incorporation later than a few min after triggering germination. This delay of a few min was well coincident with the onset of 3-phosphoglycerate (3PGA) breakdown, indicating that fructose metabolism and the accompanying aerobic ATP formation were initiated only after fructose phosphorylation by the ATP derived from anaerobic breakdown of endogenous 3PGA. In contrast, addition of glucose started incorporation of 32P into acid-soluble compounds immediately after germination. In the latter case, NADH generated by glucose oxidation to gluconate (catalyzed by glucose dehydrogenase) might serve as an initial ATP source without depending on 3PGA breakdown and glucose metabolism via the Embden-Meyerhof pathway.