Experimental Applications

Abstract

A DNA sequence encoding the brain and muscle form of human AChE (AChE-T) was constructed in our laboratory from cloned cDNA and genomic sequences, and tentatively identified by its homology to known ChEs (Soreq et al., 1990). This putative AChE-coding sequence, bearing the 3′ alternative exon E6, was subcloned into the SP6 transcription vector from which in vitro-transcribed mRNA was prepared (see Experimental Methodologies in Chapter 2). Microinjected into mature Xenopus laevis oocytes, 5 ng in vitro-transcribed AChE mRNA directed the production of catalytically active recombinant human acetylcholinesterase (rHAChE), at levels of approx 10-fold above background oocyte levels (Table 6). Oocyte-produced rHAChE was sensitive to the AChE-specific reversible inhibitor 1,5-bis-(4-allyldimethylammoniumphenyl)-pentane-3-one dibromide (BW284C51/BW) and insensitive to inhibition by the irreversible BuChE-specific inhibitor tetraisopropyl-pyrophosphoramide (iso-OMPA), as expected for a mammalian AChE. At 10⁻⁴ M BW, endogenous oocyte AChE activity (Gundersen and Miledi, 1983) was only 50% inhibited, whereas rHAChE was essentially 100% inhibited at this concentration. A similar differential sensitivity of the amphibian and mammalian enzymes to inhibition was noted for several other anti-ChE agents (Soreq et al., 1982) and later exploited to differentiate between the two in complex mixtures.

Table 6 Catalytically Active rHAChE Expressed in Microinjected Xenopus Oocytes ^a