The occurrence of a stepwise-decreasing respiration rate during oxidative assimilation of different substrates by resting Klebsiella aerogenes in a system open to oxygen

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Abstract
The addition of an oxidizable substrate to a continuous culture of Klebsiella aerogenes is known to cause an increased respiration rate that decreases in discrete steps as the added substrate is being exhausted. We have used a simple new technique to show that this phenomenon is also produced by washed, resting cells harvested from batch or continuous growth culture. The stepwise-decreasing respiration rate is caused by the exhaustion of different pools of intermediates. Each plateau of respiration rate is a measure of the activity of one or more enzymes that are rate limiting in the exhaustion of pools of intermediates. If the identities of the enzymes that are rate limiting at the different plateaux are known, the method may allow the determination, in one experiment, of the activities of up to six different enzymes in the intact bacteria. Integration of the respiration-rate measurements yields the total amount of O2 taken up. After the addition of glucose to the washed resting bacteria 37% of the amount of O2 required for the complete oxidation of the glucose was taken up. Acetate, pyruvate and succinate were all oxidized to the extent of 51%.