Arming alpha 2-macroglobulin with ricin A chain forms a cytotoxic conjugate that inhibits protein synthesis and kills human fibroblasts.
A conjugate containing alpha 2-macroglobulin and highly purified ricin A chain was made using N-succinimidyl-3-(2-pyridyldithio)propionate. Radioimmunoassay indicated that it contained 1.2 mol A chain per mol alpha 2-macroglobulin. The conjugate inhibited polyuridylic-acid directed translation by rat liver ribosomes and protein synthesis in human fibroblasts. There was a 90 min lag period before the beginning of inhibition in fibroblasts, but complete inhibition could be achieved. By measuring protein synthesis as a function of protein concentration, it was demonstrated that 8.25 X 10(-9) M conjugate was required to inhibit 50% of protein synthesis in 6 h. To achieve the same level of inhibition, 165-times more (1.3 X 10(-6) M) unconjugated A chain was required, and 180-times less ricin (4.6 X 10(-11) M). Ricin was more than 28 000 times more inhibitory than A chain alone. The presence of alpha 2-macroglobulin did not increase the cytotoxicity of unconjugated A chain, and it even protected the cells to a slight extent. The inhibitory action of the conjugate was blocked by antibodies specific for alpha 2-macroglobulin or ricin, and it was not prevented by galactose or antibodies specific for ricin B chain. Electron microscopy of the conjugate indirectly labelled with ferritin demonstrated that it was internalized by receptor mediated endocytosis through coated pits. These data indicate that the A chain portion of the conjugate survives the conditions in the lysosomes to the extent that it retains its ability to inactivate cytoplasmic ribosomes.
D000818:Animals; D002470:Cell Survival / Q000187:drug effects*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D003603:Cytotoxins / Q000008:administration & dosage*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D051381:Rats; D012276:Ricin / Q000008:administration & dosage* / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000008:administration & dosage* / Q000378:metabolism
D003603:Cytotoxins; D000511:alpha-Macroglobulins; D012276:Ricin