Ricin is a glycoprotein which is present in the seeds ofRicinus communis and is extremely toxic to animals and man (1, 2). After intravenous administration the LDs0 dose in mice is about 65 ng. The toxin inhibits the protein synthesis by inactivating the ribosomes. Ricin, which has a mol wt of 65,000 consists of two polypeptide chains joined together by an SS bond. The A chain or "effectomer" is an enzyme capable of inactivating specifically the 60S ribosomal subunit (2-8). The B chain or "haptomer" is responsible for the binding of the toxin to receptors on the cell surface, which is a prerequisite for toxic effect on intact cells. The binding is inhibited by lactose, and there is now good evidence that the B chain of the toxin interacts with membrane glycoproteins containing nonreducing terminal galactose residues (1, 9-13).The B chain of the toxin binds to animal cells in general including erythrocytes. Thus, to make the toxic effect selective against certain kinds of cells such as tumor cells, a number of points related to the introduction of the toxin into cells have to be clarified. In this study we have asked the following questions: (a) Can the toxin be introduced into cells through other cell membrane receptors than via the B-chain receptor? (b) Does internalization through endocytosis (most likely including exposure of the toxin to lysosomal enzymes) neutralize the toxic effect?In attempts to throw light on these questions we have studied the effect of [ricin.antiricin B] complexes on mouse macrophages and rat Kupffer cells. The results indicate that such complexes, subsequent to binding to Fc receptors of cells, are indeed internalized and lead to inhibition of protein synthesis and cell death.
Materials and MethodsIsolation ofRicin. Ricin was extracted from the seeds ofRicinus communis (a batch of Castor beans obtained from Deutsche Rizinus Oelfabrik, Boley & Co, Krefeld-Urdingen, West Germany) and purified by chromatography using and Sepharose 4B columns (I,9,10). To prepare its constituent polypeptide chains, ricin was treated with 5% 2-mercaptoethanol in the presence of 0.5 M D-galactose, and the A and the B chain were separated on DE-52 and CM-52 columns as described elsewhere (1, 9, 10). Dilution of the toxin used in these experiments was always done in cell culture medium (containing 0.02% fetal calf serum).Preparation of Antitoxin. Antiserum against ricin was raised in rabbits. Ricin was first treated for 3 days with 5% formaldehyde at room temperature in 0.05 M sodium phosphate buffer
The time course of the conversions of chemical components in herring extracts during anaerobic growth of Proteus sp., str. NTHC 153, Aeromonas sp., str. NTHC 154, and Enterobacter sp., str. NTHC 151 (Strøm & Larsen 1979) has been studied. When the Proteus sp. or the Aeromonas sp. were inoculated into the herring extracts and incubated at 15°C under anaerobic conditions, the sugar components (i.e. mainly ribose, free and bound) were the first substrates utilized. These compounds were converted to acetate and CO2 by the use of trimethylamine oxide (TMAO) as an external hydrogen acceptor. Growth of bacteria ceased when all TMAO was reduced to trimethylamine (TMA). By adding an extra amount of TMAO to the herring extracts an increased growth of the Proteus sp. and the Aeromonas sp. ensued. The increased growth occurred concomitantly with a further conversion of TMAO to TMA and of lactate to acetate and CO2. The Enterobacter sp., which did not utilize lactate, did not give an increased growth in herring extracts enriched with TMAO.
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