Tritiation of α-bungarotoxin with <i>N</i>-succinimidyl [2,3-3H]propionate. A useful reagent for labelling proteins

Dolly, J. Oliver; Nockles, E.; Lo, Mathew M. S.; Barnard, Eric A.

doi:10.1042/bj1930919

Cited by 23 publications

(13 citation statements)

References 8 publications

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“…The lack of significant phospholipase activity must be due to propionylation of a residue, presumably in the larger polypeptide, which is essential for enzymic activity. It is assumed, but not proven, that lysine groups are labelled, as demonstrated for propionylated a-bungarotoxin [32]. This assumption is supported by the demonstration of an essential residue, presumably a lysine amino group in the phospholipase A2 active site of p-bungarotoxin which can be modified by ethoxyformic anhydride [39].…”

Section: Churucter Isu T Ion Of H-p-bunguro T Oxinmentioning

confidence: 51%

“…The latter, together with thc slope obtained from the association rate plot (Fig. 5 insert), were used to calculate [42] the association rate constant k l ; its value was found to be 7.8 x lo5 M-' s-' which is similar to that reported for mono-[3 Hlpropionylated a-bungarotoxin reacting with acetylcholine receptors [32]. The dissociation constant Ka, calculated from these values of k l and k-1 (taken from this and two similar experiments), was 0.57 (+ 0.015) n M ; the latter agrees with value of 0.6 nM obtained from Scatchard analysis above.…”

Section: Churucter Isu T Ion Of H-p-bunguro T Oxinmentioning

confidence: 51%

“…The conditions of tritiation are very gentle and efficient; the extent of propionylation [32] can be controlled by varying the ratio of protein/reagent used. It yields a stable preparation that does not undergo inactivation by self-irradiation; also, the change in charge introduced allows the labelled species to be separated from native toxin (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Tritiation of 8-bungarotoxin with N-succinimidyl-[2.3-3H]prop~onate was performed by modification of the method of Dolly et al [32] described for a-bungarotoxin. The reagent (2.1 ml, 99.9 nmol) was transferred to a plastic microfuge tube; following evaporation of the solvent under a stream of N2, the toxin (0.7 mg, 33.3 nmol) was added in 0.2-0.5 ml of 0.02 M phosphate butrer, pH 7.4.…”

Section: Purrfication and Tritiation Of Fl-bungurotosinmentioning

confidence: 99%

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Preparation of Neurotoxic 3H-β-Bungarotoxin: Demonstration of Saturable Binding to Brain Synapses and Its Inhibition by Toxin I

Othman

Spokes

Dolly

2005

European Journal of Biochemistry

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Homogeneous β‐bungarotoxin, isolated from the venom of Bungarus multicinctus was radiolabelled with N‐Succinimidyl‐[2,3‐3H]propionate. Stable, di‐propionylated material was obtained which was tritiated on both subunits and had a specific radioactivity of 102 Ci/mmol. After separation from unlabelled toxin by isoelectric focussing, it was shown to exhibit significant biological activity in both the peripheral and central nervous systems but had negligible phospholipase A2 activity towards lecithin or cerebrocortical synaptosomes. The labeled neurotoxin binds specifically to a single class of non‐interacting sites of high affinity (Kd= 0.6 nM) on rat cerebral cortex synaptosomes; the content of sites is about 150 fmol/mg protein. This binding was inhibited by unlabelled β‐bungarotoxin with a potency which indicates that tritiation does not alter the affinity significantly. The association of toxin with its binding component and its dissociation were monophasic; rate constants observed were 7.8 × 105 M−1 s−1 and 5.6 × 10−4 s−1 at 37°C, respectively. β‐Bungarotoxin whose phospholipase activity had been inactivated with p‐bromophenacyl bromide inhibited to some extent the binding of tritiated toxin but with low efficacy. Taipoxin and phospholipase A2 from bee venom, but not Naja melanoleuca, inhibited the synaptosomal binding of toxin with low potencies in the presence, but not the absence, of Ca2+. Toxin I, a single‐chain protein from Dendroaspis polylepis known to potentiate transmitter release at chick neuromuscular junction, completely inhibited the binding of 3H‐β‐bungarotoxin with a Ki of 0.07 nM; this explains its ability to antagonise the neuroparalytic action of β‐bungarotoxin. Other pure presynaptic neurotoxins, α‐latrotoxin and botulinum neurotoxin failed to antagonise the observed binding; likewise tityustoxin, which is known to affect sodium channels, had no effect on 3H‐β‐bungarotoxin binding. Trypsinization of synaptosomes completely destroyed the binding activity, suggesting that the binding component is a protein; the functional role of the latter is discussed in relation to the specificity of toxin binding.

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Section: Churucter Isu T Ion Of H-p-bunguro T Oxinmentioning

confidence: 51%

Section: Churucter Isu T Ion Of H-p-bunguro T Oxinmentioning

confidence: 51%

Section: Discussionmentioning

confidence: 99%

Section: Purrfication and Tritiation Of Fl-bungurotosinmentioning

confidence: 99%

See 2 more Smart Citations

Preparation of Neurotoxic 3H-β-Bungarotoxin: Demonstration of Saturable Binding to Brain Synapses and Its Inhibition by Toxin I

Othman

Spokes

Dolly

2005

European Journal of Biochemistry

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“…Native Sap B (11) was labeled with tritiated propionic acid N-succinimidyl ester essentially as described (19). Briefly, [2,3-3 H]propionic acid N-succinimidyl ester (3.59 TBq/mmol obtained from GE Healthcare) was diluted with the unlabeled reagent to 18.5 GBq/mmol.…”

Section: Methodsmentioning

confidence: 99%

Saposin B-dependent Reconstitution of Arylsulfatase A Activity in Vitro and in Cell Culture Models of Metachromatic Leukodystrophy

Matzner

Breiden

Schwarzmann

et al. 2009

Journal of Biological Chemistry

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N‐succinimidyl propionate: Characterisation and optimum conditions for use as a tritium labelling reagent for proteins

Tang

Davis

Kitcher

1983

Labelled Comp Radiopharmac

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N-Succinimidyl propionate [ NSP] was synthesised and characterised as a pale y e l l o x waxy s o l i d . Rates of hydrolysis i n b u f f e r s range pH 5-9 were determined. Optimum conditions f o r r e a c t i o n with l y s i n e were within t h e range pH 7 t o 8. A p r o t e i n , wheat germ a g g l u t i n i n was l a b e l l e d a t pH 7.5 a t room temperature f o r two hours. Rates of r e a c t i o n of o t h e r s u b s t r a t e s under these conditions were i n v e s t i g a t e d . Key Words: P r o t e i n l a b e l l i n g , t r i t i u m , k i n e t i c s .

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Tritiation of α-bungarotoxin with N-succinimidyl [2,3-3H]propionate. A useful reagent for labelling proteins

Cited by 23 publications

References 8 publications

Preparation of Neurotoxic 3H-β-Bungarotoxin: Demonstration of Saturable Binding to Brain Synapses and Its Inhibition by Toxin I

Preparation of Neurotoxic 3H-β-Bungarotoxin: Demonstration of Saturable Binding to Brain Synapses and Its Inhibition by Toxin I

Saposin B-dependent Reconstitution of Arylsulfatase A Activity in Vitro and in Cell Culture Models of Metachromatic Leukodystrophy

N‐succinimidyl propionate: Characterisation and optimum conditions for use as a tritium labelling reagent for proteins

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