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: email@example.com (U. Pfuller).