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Figures
- Fig. 1.
Synthesis of 4,7,10-trioxa-1,13-carboxyethyl-2-trimethylamine-tridecanediaamine (TDAC).
- Fig. 2.
RT-PCR products generated using specific primers for acetylcholine receptor genes amplified from H. monopterigium. RNA isolated from the electric organ of the coffin ray was reverse transcribed and amplified using primers specific for either the α, β, γ or δ genes of the acetylcholine receptor. Two RNA concentrations were tested with each primer pair, and RT-PCR products were run on a 0.8% agarose gel and stained with ethidium bromide. Lambda DNA cut with EcoR1 and HindIII was used to estimate the size of the DNA fragments (left-hand lane).
- Table 1.
Amino acid sequence identity between acetylcholine receptor subunits from different species
T. californica Human Species Subunit α β γ δ α β γ* ϵ* δ T. marmorata α 99 39 32 33 78 34 31 27 33 β 39 96 37 34 38 54 39 35 38 γ 32 35 97 46 32 38 54 53 47 δ 32 34 46 98 32 35 42 39 57 H. monopterigium α 98 39 32 33 79 34 30 27 33 β 38 88 38 36 38 54 40 35 39 γ 30 38 94 46 32 39 53 53 46 δ 32 34 45 95 32 35 43 39 58 N. tasmaniensis α 96 39 32 32 78 34 30 27 33 δ 32 34 46 98 32 35 42 39 57 -
Values represent the percentage identity at the amino acid sequence level between the acetylcholine receptor subunits from T. marmorata, H. monopterigium and N. tasmaniensis, compared with the equivalent proteins from T. californica and humans.
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↵* The acetylcholine receptor γ and ϵ subunits, present at the fetal and adult human neuromuscular junction, respectively, are equally homologous to the γ subunit in electric fish, representing a hybrid of the two.
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- Table 2.
Classification of the amino acid differences in the mature subunits of the acetylcholine receptor into domains and then further into secondary structure elements when compared with T. californica subunits
Amino acid differences relative to T. californica Extracellular domain Pore-forming domain Species Subunit Total amino acid changes β-strands Loops/α-helices Transmembrane α-helices Loops T. marmorata α 6 1 0 3 2 β 18 3 8 7 0 γ 12 1 4 5 2 δ 13 5 2 4 2 H. monopterigium α 7 4 0 0 3 β 51 9 17 13 12 γ 27 4 8 3 12 δ 23 7 6 8 2 N. tasmaniensis α 16 8 7 1 0 δ 13 5 2 4 2 - Fig. 3.
Purification of the acetylcholine receptor from the numb fish N. tasmaniensis. (A) The membrane fraction from the electric organ was treated with 1.5% Triton X-100, and solubilized material applied to the TDAC affinity column. Bound protein was eluted from the column using an increasing salt gradient. (B) The major contaminant (∼98 kDa) could be purified away from the acetylcholine receptor on an anion exchange column (ResourceQ), where it eluted later in the NaCl gradient. (C) The purified receptor ran close to the void volume on a Superdex200 gel permeation column (670 kDa), consistent with the receptor maintaining its dimeric form under the purification conditions used. Arrows indicate the elution positions of molecular mass markers used to calibrate the column: 670, 158, 44, 17 and 1.35 kDa. Absorbance was measured at 215 nm in A and C and at 280 nm in B.
- Fig. 4.
SDS–PAGE of the acetylcholine receptor purified from the numb fish N. tasmaniensis on the TDAC affinity column. Fractions 3, 5 and 7, correlating to the peak absorbance in Fig. 3A, were run on a 4–12% Bis-Tris NuPAGE gel and stained with Coomassie. The protein bands corresponding to the four subunits comprising the acetylcholine receptor are indicated on the right. The protein running between the α and β subunits is mostly likely the α subunit, which is known to run as a doublet.
- Fig. 5.
A rapid filtration method through DE-81 filters was used to measure the binding of 125I-α-bungarotoxin (α-Bgt) to affinity-purified acetylcholine receptors from electric rays at equilibrium. The data presented are from a typical experiment where the specific binding is plotted against increasing α-Bgt concentrations. The dissociation constants calculated from the equation for a single binding site were not significantly different in the four electric rays tested, ranging between 21 and 53 nmol l–1. Error bars represent s.e.m.
- Table 3.
Affinity constants for 125I-α-bungarotoxin binding by purified acetylcholine receptors from different species
Species Kd (nmol 1-1) T. marmorata 21±4 T. macneilli 40±6 H. monopterigium 53±15 N. tasmaniensis 28±7 -
Average results are expressed as the mean ± s.e.m. of six determinations. The Kd values were not significantly different from each other (P<0.05), calculated using a paired Student's t-test.
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- Fig. 6.
Electron microscopic images of purified acetylcholine receptors from electric rays show them to be dimers. The affinity-purified acetylcholine receptor from H. monopterigium was viewed under the electron microscope in negative stain and is shown in A. Receptor dimers are evident in this (boxes in A) and all other acetylcholine receptor preparations isolated on the TDAC affinity column: (B) T. marmorata; (C) N. tasmaniensis.
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