A radioassay for synaptic core complex assembly: screening of herbal extracts for effectors.
Synaptic core complex formation between syntaxin and synaptosome-associated protein of 25 kDa (SNAP25) on the plasma membrane and synaptobrevin on the vesicle membrane is responsible for membrane fusion and neurotransmitter release. A radiolabeled protein binding assay for synaptic core complex formation was developed. The components of this assay included recombinant glutathione S-transferase (GST)-syntaxin immobilized on glutathione agarose beads, SNAP25 and (125)I-labeled synaptobrevin. Reactions were performed in tubes containing filter inserts to facilitate removal of unbound protein. The radiolabeled protein bound was then quantified by gamma counter. A K(d) of 1.6 microM was determined for the GST-syntaxin/SNAP25/synaptobrevin complex, and a K(d) of 12 microM was determined for the GST-syntaxin/synaptobrevin complex. The assay was used to screen 14 herbal extracts for effectors of core complex formation. Herbs traditionally used to treat neurological conditions such as depression, anxiety, and stress were chosen. A Hypericum perforatum extract was found to have a nonspecific effect via protein complexation, whereas an Albizzia julibrissin extract was found to reduce the level of core complex formation. The assay was used to further investigate the effect of the A. julibrissin extract. The discovery of an inhibitor of core complex formation demonstrates the efficacy of the assay in screening natural products for substances that affect core complex formation.
Riley
Lisa G
LG
School of Molecular and Microbial Biosciences, University of Sydney, Sydney, NSW 2006, Australia.
Roufogalis
Basil D
BD
Li
George Q
GQ
Weiss
Anthony S
AS
D000818:Animals; D004353:Drug Evaluation, Preclinical; D006801:Humans; D007457:Iodine Radioisotopes; D008970:Molecular Weight; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D010936:Plant Extracts / Q000737:chemistry* / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D050683:R-SNARE Proteins / Q000737:chemistry / Q000378:metabolism*; D011869:Radioligand Assay / Q000379:methods*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry / Q000378:metabolism*
D007457:Iodine Radioisotopes; D046912:Multiprotein Complexes; D010936:Plant Extracts; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; D050825:Synaptosomal-Associated Protein 25