Abstract
A toxic protein, viscumin, was isolated from extracts of mistletoe by affinity chromatography on acid-treated Sepharose 4B. Viscumin was selectively bound to the column and could be eluted with lactose. It migrated in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate corresponding to Mr = 60,000. In addition, two bands migrating corresponding to Mr = 29,000 and 32,000 were found. After treatment with 2-mercaptoethanol, only 2 bands (Mr = 29,000 and 34,000) were found. Apparently, viscumin consists of two chains which, in some of the molecules, are disulfide-linked. Protection experiments with antiserum against viscumin indicated that the major part of the cytotoxic activity in mistletoe extracts is due to viscumin. Gel filtration experiments on Sephacryl 200 indicated that, at low concentrations, viscumin occurs as a monomer and at higher concentrations as a dimer. Viscumin was found to inhibit protein synthesis in cell-free systems. When the two constituent peptide chains of viscumin were eluted from polyacrylamide gels and tested for ability to inhibit cell-free protein synthesis, this property was found to be associated with the fastest migrating chain, here denoted the A chain. The heavier chain was denoted the B chain. The A chain was found to inhibit protein synthesis by inactivating the ribosomes catalytically. Reconstitution experiments with isolated ribosomal subunits from untreated and A chain-treated ribosomes showed that the 60 S ribosomal subunit was selectively inactivated.
Highlights
Of viscumin were eluted from polyacrylamidgeels and Here we demonstrate that mistletoe extract contains tested for ability to inhibit cell-free proteinsynthesis, a cytotoxin with similar structure and mechanisomf action as this property was foundto be associated with thefast- abrin, ricin, and modeccin
Purification of Viscumin-The green parts of mistletoe were extracted as described under"ExperimentalProcetions
The incubation was terminated by adding 1ml of100mM KOH and thterichloroacetic acid-precipitable radioactivity was measured as previously described viscumin isolated here
Summary
0.2 ml was mixed with Freund's complete adjuvant and injected subcutaneously intoa rabbit. The crude extract was first passed through acolumn of DEAE-cellulose to which several proteins,including the most indicated amounts of toxin were added and the cells were incubated toxic one, denoted viscumin, were bound (Table I). Protein synthesis was measured in 23-pI samples containing 0.1 pCi of ['4C]leucine (342 mCi/mol), with and without toxin, as described in legends to figures. The incubation was terminated by adding 1ml of100mM KOH and thterichloroacetic acid-precipitable radioactivity was measured as previously described viscumin isolated here (see Ref. 9). The reason for this discrepancy is not clear.
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