Figure 9. A typical transport assay carried out in the absence (·) or presence (o) of D-lactate (left) and the demonstration that D-lactate is converted stoichiometrically into pyruvate (right). The discovery that D-lactate is by far the most effective energy source for active transport of a wide variety of substrates in E. coli membrane vesicles took about 15 years, was totally fortuitous and broke open the field (Left; Control, no energy source). In addition to the fact that D-lactate is converted stoichiometrically to pyruvate (Right; Total, lactate + pyruvate), all of the accumulated substrate can be recovered from the vesicles in unmodified form which is consistent with the observations showing that addition of excess non-radioactive substrate (▲) or inhibitors such as dinitrophenol (DNP; ∆) causes rapid release of accumulated radiolabeled lactose (Left). Oxygen and an intact membrane-embedded electron transfer chain are required. In order to observe accumulation of lactose or other galactosides, the parent cells must be induced for the synthesis of lactose permease from the lacY gene, the second structural gene in the lac operon. Other transport systems such as those for many amino acids are constitutive.