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ADP-ribosylation of a Mr 21,000 membrane protein by type D botulinum toxin
Abstract:
When crude membrane fraction from bovine adrenal gland was incubated with type D botulinum toxin in the presence of NAD, a membrane protein with a molecular weight of 21,000 was specifically ADP-ribosylated. This ADP-ribosylation occurred dependent on the dose of the toxin and was abolished by prior boiling ADP-ribose transfer to the membrane protein was significantly suppressed when agmatine and L-arginine methyl ester were included in the reaction mixture. Dithiothreitol stimulated this ADP-ribosylation about 3-fold. Incubation of membrane fractions from mouse brain and pancreas with this toxin also resulted in ADP-ribosylation of a protein of the same molecular weight. These results suggested that type D botulinum toxin catalyzed transfer of an ADP-ribose moiety of NAD to the specific membrane protein common to secretory cells.
Insights
Type D botulinum toxin specifically ADP-ribosylates a 21 kDa membrane protein in bovine adrenal gland and mouse secretory cells. This process, dependent on NAD, is modulated by specific inhibitors and activators, suggesting a role in secretory cell function.
Area of Science:
- Biochemistry
- Molecular Biology
- Neuroscience
Background:
- Botulinum toxins are potent neuroparalytic agents.
- Understanding the molecular targets of botulinum toxins is crucial for elucidating their mechanisms of action.
- Secretory cells possess unique membrane proteins involved in cellular processes.
Purpose of the Study:
- To identify the specific molecular target of type D botulinum toxin in cellular membranes.
- To characterize the ADP-ribosylation activity of type D botulinum toxin on membrane proteins.
- To investigate the prevalence of this target protein across different secretory cell types.
Main Methods:
- Incubation of crude membrane fractions with type D botulinum toxin and NAD.
- Analysis of protein ADP-ribosylation using molecular weight determination.
- Dose-dependency studies and assessment of inhibitor/activator effects (agmatine, L-arginine methyl ester, dithiothreitol).
- Comparative analysis using membrane fractions from bovine adrenal gland, mouse brain, and pancreas.
Main Results:
- Type D botulinum toxin specifically ADP-ribosylated a 21 kDa membrane protein.
- ADP-ribosylation was toxin dose-dependent and abolished by heat denaturation.
- Agmatine and L-arginine methyl ester suppressed the reaction, while dithiothreitol enhanced it.
- Identical ADP-ribosylation was observed in mouse brain and pancreatic membrane fractions.
Conclusions:
- Type D botulinum toxin catalyzes the transfer of ADP-ribose from NAD to a specific 21 kDa membrane protein.
- This target protein appears to be common in secretory cells, including those in the adrenal gland, brain, and pancreas.
- The findings suggest a potential role for this toxin-mediated modification in the function of secretory cells.