Twenty-two soluble esterases have been identified in D. melanogaster by combining the techniques of native polyacrylamide gel electrophoresis and isoelectric focusing. The sensitivity of each isozyme to three types of inhibitors (organophosphates, eserine sulfate, and sulfydryl reagents) identified 10 as carboxylesterases, 6 as cholinesterases, and 3 as acetylesterases. Three isozymes could not be classified and no arylesterases were identified. The carboxyl- and cholinesterases could each be further divided into two subclasses on the basis of inhibition by organophosphates and sulfhydryl reagents, respectively. Choline- and acetylesterases have characteristic substrate preferences but both subclasses of carboxylesterases are heterogeneous in substrate utilization. Subclass 2 carboxylesterases exhibit diverse temporal expression patterns, with subclass 1 carboxylesterases generally found in larvae and subclass 1 cholinesterases and acetylesterases more characteristic of pupae and adults. Tissues showing the greatest number of isozymes are larval body wall (eight) and digestive tract (six in larvae, six in adults). Carboxylesterases are distributed across a wide range of tissues, but subclass 1 cholinesterases are generally associated with neural or neurosecretory tissues and subclass 2 cholinesterases with digestive tissues.

Förster resonance energy transfer (RET) is the nonradiative transfer of energy from a donor to an acceptor fluorophore. The Förster distance (R(0)), being the fluorophore separation corresponding to 50% of the maximum RET efficiency (E(RET)), is a critical parameter for optimization of RET biosensors. Sensitive RET-based monitoring of molecular rearrangements requires that the separation of the donor and acceptor RET pair is matched to their Förster distance. Here, for the first time, we experimentally determine the Förster distance for BRET(1), R(0) = 4.4 nm, and for BRET(2), R(0) = 7.5 nm. The latter is the largest reported value for a genetically encoded RET pair.

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