Comparison of extracellular and intracellular potency of botulinum neurotoxins.

Comparison of extracellular and intracellular potency of botulinum neurotoxins. Research Abstract Details 

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Comparison of extracellular and intracellular potency of botulinum neurotoxins. Abstract Text:

Fang Cai,Carrie B Adrion,James E Keller,

Levels of botulinum neurotoxin (BoNT) proteolytic activity were compared using a cell-free assay and living neurons to measure extracellular and intracellular enzymatic activity. Within the cell-free reaction model, BoNT serotypes A and E (BoNT/A and BoNT/E, respectively) were reversibly inhibited by chelating Zn2+ with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN). BoNT/E required relatively long incubation with TPEN to achieve total inhibition, whereas BoNT/A was inhibited immediately upon mixing. When naïve Zn2+-containing BoNTs were applied to cultured neurons, the cellular action of each BoNT was rapidly inhibited by subsequent addition of TPEN, which is membrane permeable. Excess Zn2+ added to the culture medium several hours after poisoning fully restored intracellular toxin activity. Unlike TPEN, EDTA irreversibly inhibited both BoNT/A and -E within the cell-free in vitro reaction. Excess Zn2+ did not reactivate the EDTA-treated toxins. However, application of EDTA-treated BoNT/A or -E to cultured neurons demonstrated normal toxin action in terms of both blocking neurotransmission and SNAP-25 proteolysis. Different concentrations of EDTA produced toxin preparations with incrementally reduced in vitro proteolytic activities, which, when applied to living neurons showed undiminished cellular potency. This suggests that EDTA renders the BoNT proteolytic domain conformationally inactive when tested with the cell-free reaction, but this change is corrected during entry into neurons. The effect of EDTA is unrelated to Zn2+ because TPEN could be applied to living cells before or after poisoning to produce rapid and reversible inhibition of both BoNTs. Therefore, bound Zn2+ is not required for toxin entry into neurons, and removal of Zn2+ from cytosolic BoNTs does not irreversibly alter toxin structure or function. We conclude that EDTA directly alters both BoNTs in a manner that is independent of Zn2+.

Comparison of extracellular and intracellular potency of botulinum neurotoxins. Publishing Authors By Initials

F Cai,CB Adrion,JE Keller,

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Comparison of extracellular and intracellular potency of botulinum neurotoxins. Journal Published:

PUBLICATION TYPE: Research Support, N.I.H., Extr

Journal: Infection and immunity

VOLUME: 74

Page Numbers: 5617-24

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ISSN: 0019-9567

DAY: 3

MONTH: Oct

YEAR: 2006

Comparison of extracellular and intracellular potency of botulinum neurotoxins. Information

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LANGUAGE: eng

NlmUniqueID: 246127

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Grant and Affiliation Information for Comparison of extracellular and intracellular potency of botulinum neurotoxins.

AFFILIATION: Laboratory of Bacterial Toxins, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA.

Country: United States

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MEDLINETA: Infect Immun

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