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40. Self-cleavage of mistletoe lectin I into the A and B subunits by thiol-disulfide exchange reaction.

Mistletoe plant has been shown to contain three groups of lectins of therapeutical interest belonging to class II of ribosome-inactivating proteins (RIP II). The lectins ML I, ML II and ML III share the typical A-S-S-B structure of RIP II and recognize galactoside/N-acetyl-galactosaminide containing glycoconjugates. The subunits are connected via an interchenar disulfide bond which can be easily broken in the absence of reducing agents as shown by SDSPAGE electrophoresis. The self-cleavage of interchenar disulfide bond is completely inhibited by thiol-oxidizing or -blocking reagents like iodacetamide. The cleavage of the hololectin into the A and B subunits is the result of an intraprotein thiol-disulfide exchange reaction by which a disulfide bond is formed and rearranged in a protein. A thiol group (as thiolate) located close to the interchenar disulfide bond attacks its own disulfide bond. The content of free thiol groups in native ML I of different origin amounts up to 0.4 per monomer molecule. Using fluorescence labelling techniques and thiol-specific reagents the critical free thiol group was localized in the B chain which contains four disulfide bonds. The labelled ML I isoforms were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Obviously one disulfide bridge in the B chain can be open to a small extent. Effects and agents driving and facilitating the thiol/disulfide exchange reaction are higher temperature, denaturating agents like SDS, chaotropic ions, and basic conditions. Therefore, we examined the effect of these agents at pH 7-9 and at moderately enhanced temperatures on purified ML I isoforms. There remains the question whether disulfide bond isomerization is going on in ML I but not in ML II and ML III. Thiol-disulfide isomerization in ML I may affect the actual concentration in extracts and preparations and even the function of this hololectin. Possible implications for the formulation and handling of mistletoe extracts and preparations were discussed.

Keywords: Mistletoe lectin I; Thiol-disulfide exchange; Subunits; Electrophoresis; SDS; Biological effects

U. Pfuller*, Karola Pfuller

Institute of Phytochemistry, Private University Witten/Herdecke gGmbH, Stockumer Strasse 10, 58453 Witten, Germany

*Corresponding author. Tel.: +49 2302 669328; fax: +49 2302 669343.

E-mail address: uwep@uni-wh.de (U. Pfuller).
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Author:Pfuller, U.; Pfuller, Karola
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:4EUGE
Date:Oct 1, 2007
Words:356
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