Worm Breeder's Gazette 7(2): 57

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Detergent-solubilization of the Levamisole Receptor

J.A. Lewis, R.L. Russell

Putative acetylcholine receptor mutants of C.  elegans can be 
selected as strains resistant to the cholinergic neurotoxic drug 
levamisole.  At the last C.  elegans meeting, one of us (J.A.L.) 
described a binding assay for detecting the wild type receptor 
activity and showed that same levamisole-resistant mutants were 
deficient in receptor binding activity.  The assay was based on the 
retention of an insoluble, particulate [3H]-meta-aminolevamisole 
binding activity on a glass fiber filter after incubation of the 
radioactive ligand with total worm extract and filtration.  Progress 
toward more detailed molecular studies of wild type and mutant 
receptors would require first that the presumably membrane bound 
receptor be solubilized in detergent solution without loss of binding 
activity and secondly that a suitable assay be found for detecting the 
solubilized activity.  We have now shown that the levamisole receptor 
is soluble and active in 1% solutions of the detergents Triton X-100, 
Lubrol PX, or sodium cholate.  In Triton X-100, activity can be 
detected, in order of increasing efficiency, by the bovine Y-globulin-
polyethylene glycol precipitation method of Cuatrecasas, by retention 
on DEAE filter paper, or by centrifugation through a mini-column (1ml) 
of G-25 Sephadex.  A serendipitous result of detergent solubilization 
in 1% Triton X-100 is that the affinity of the solubilized receptor 
for [3H] MAL is increased roughly 100 fold due to a corresponding 
increase in the rate of association between ligand and receptor.  The 
equilibrium dissociation constant for solubilized receptor is thus 
~10+E-10 M as compared to ~10+E-8 M for the 'native' receptor found in 
untreated extracts.  A much lower amount of radioactive ligand is thus 
required to assay solubilized receptor, resulting in more than a four 
fold improvement in the signal-to-noise ratio of the assay.  Whereas 
the filter assay of insoluble receptor requires assays in 
quadruplicate for accuracy, single determinations of solubilized 
receptor activity by the Sephadex assay will suffice for most purposes.
These vastly improved assay capabilities should allow fine 
distinctions to be made between wild type and mutant receptor 
properties and make purifying the receptor much easier as well.
Previously, we had shown by assay of total, insoluble activity that 
mutants at 5 of the 7 genetic loci associated with extreme resistance 
to levamisole had some deficiency in receptor binding activity, 
ranging from about a 50% decrease (2 loci) to around a 90% decrease (3 
loci).  To confirm the authenticity of the solubilized binding 
activity, we showed that the unc-29(x29) levamisole resistant mutant, 
one of those ~90% deficient in insoluble activity, was also similarly 
deficient in solubilizable activity as detected by all three assay 
methods.  Mutants at the two resistance loci showing apparently normal 
levels of insoluble activity were tested for detergent-extractable 
activity.  These mutants, unc-63(x37) and lev-1(x21), had about a 
tenth and a third of the expected wild type detergent-soluble activity.
In the wild type, about 20% of the total activity is not detergent-
extractable and remains in the pellet.  The unc-63 and lev-1 mutants 
had wild type amounts of nonextractable activity as did the unc-29 
mutant 90% deficient in total and detergent-extractable receptor 
activities.  The nonextractable activity is thus likely to be a 
secondary (artifactual?) activity unrelated to the detergent-soluble 
receptor.  The levamisole receptors made by the unc-63 and lev-1 
mutants are probably more unstable or much less active in detergent 
than the wild type receptor.  With these results, same degree of 
receptor abnormality is now known for mutants at all 7 of the loci 
producing extreme levamisole resistance.