Gelonin, a new inhibitor of protein synthesis, nontoxic to intact cells. Isolation, characterization, and preparation of cytotoxic complexes with concanavalin A.

The protein here named gelonin was extracted from the seeds of Gelonium multiflorum by a buffered phosphate solution and purified in a single step by chromatography on a carboxymethyl cellulose column. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and gel filtration on Sephacryl 300 superfine showed that gelonin consists of one polypeptide chain with a molecular weight of about 28,000 to 30,000. Gelonin bound to a column containing immobilized concanavalin A and could be eluted with amethylmannoside. Pretreatment of gelonin with jackbean mannosidase prevented this binding, indicating that it is a glycoprotein containing terminal mannose residues. The protein proved t o be extremely stable, as measured by its biological activity, toward treatment with sodium dodecyl sulfate, urea, acid, base, and heat. Gelonin strongly inhibited protein synthesis in a reticulocyte lysate. It inactivated the 60 S ribosomal subunit with no effect on the 40 S ribosomal subunit. A t a concentration of 100 pg/ml, gelonin only slightly inhibited protein synthesis in intact HeLa cells and it gave no microscopically visible cytopathogenic effect. When the inhibitor was linked by a disulfide bridge to concanavalin A, the complex gave 50% inhibition of cellular protein synthesis at a concentration of about 1 pg/ml, corresponding to about 0.2 pg/ml of gelonin. The results show that gelonin is a single chain protein which acts in a cell-free system like the A chains of abrin, ricin, and modeccin and suggest that it lacks the ability to bind to the cell surface and to enter intact cells. When coupled through a disulfide bond to a protein capable of binding to cells, it is rendered toxic to intact cells.