Experimental Applications: Human Acetylcholinesterase as a Model Nervous System Protein
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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
|
Acetylthiocholine hydrolysis, nmol/h/oocyte |
||
|---|---|---|
|
AChE mRNA-injected |
Buffer-injected |
|
|
No inhibitor |
50.4 + 5.0 |
5.2 � 0.9 |
|
+ BW284C51 |
2.3 � 0.2 |
2.5 � 0.6 |
|
+ iso-OMPA |
47.8 � 0.1 |
4.6 � 0.3 |
a In vitro-transcribed AChEmRNA encoding AChE-T was expressed in microinjected Xenopus oocytes as described in Experimental Methodologies in Chapter 2. Parallel groups were injected with modified Barth’s medium as control. Activity in total homogenates was determined in the presence and absence of 10 uM of specific AChE (BW284C51) or BuChE (iso-OMPA) inhibitors following a 40-min preincubation. Spontaneous hydrolysis of substrate was subtracted. Data represent nanomoles substrate hydrolyzed/h/ oocyte + standard evaluation of the mean (SEM) for three independent transcription reactions and microinjection experiments. Note residual endogenous, BW284C51-insensitive hydrolytic activity of approx 2.5 nmol/h/ oocyte in both groups. Adapted from Seidman et al. (1994).
