Modeccin, the toxin of Adenia digitata. Purification, toxicity and inhibition of protein synthesis in vitro.

1. Modeccin, the toxin of Adenia digitata (Modecca digitata), was purified from the roots of this plant by affinity chromatography on Sepharose 4B. 2. This toxin is a protein with mol.wt. 57000, which on treatment with 2-mercaptoethanol can be dissociated into two subunits of mol.wts. 25000 and 32000. 3. Modeccin inhibits protein synthesis in vitro in a lysate of rabbit reticulocytes and in Ehrlich ascites cells; the effect on cells is decreased in the presence of lactose. 4. Dissociation of modeccin into subunits decreases the toxicity to animals and the inhibition of protein synthesis in cells, but enhances the inhibition of protein synthesis in the lysate system.     

Properties and action mechanism of the toxic lectin modeccin: Interaction with cell lines resistant to modeccin,abrin, and ricin

The toxic lectin modeccin, which inhibits protein synthesis in eukaryotic cells, is cleaved upon treatment with 2-mercaptoethanol into two peptide chains which move in polyacrylamide gels at rates corresponding to molecular weights 28,000 and 38,000. After reduction, the toxin loses its effect on cells, while its ability to inhibit cell-free protein synthesis increases. Like abrin and ricin it inhibits protein synthesis by inactivating the 60S ribosomal subunits. Modeccin binds to surface receptors containing terminal galactose residues. Competition experiments with various glycoproteins indicate that the modeccin receptors are different from the abrin receptors. In addition, they were present on HeLa cells in much smaller numbers. Moreover, mutant lines resistant to abrin and ricin were not resistant to modeccin and vice-versa. The toxin resistance of various mutant cell lines could not be accounted for by a reduced number of binding sites on cells. The data are consistent with the view that the cells possesss different populations of binding sites with differences in ability to facilitate the uptake of the toxins and that in the resistant lines the most active receptors have been reduced or eliminated.     

Purification and immunological characterization of HPLC-purified pertussis toxin subunits.

Pertussis toxin (PT), an oligomeric exotoxin of Bordetella pertussis containing five dissimilar subunits, is considered to be an essential immunogen in acellular and component pertussis vaccines against whooping cough. A rapid single-step procedure for isolating PT subunits was developed using reverse-phase high-performance liquid chromatography. Recoveries of individual subunits were 75% (S1), 70% (S2), greater than 90% (S3), greater than 90% (S4), and 50% (S5), as judged by SDS-PAGE and amino acid analysis. Lyophilized subunits were solubilized in urea followed by step-wise dialysis to remove the urea. All subunits were inactive in histamine sensitization, lymphocytosis, and hemagglutination assays. However, purified S1 retained residual NAD-glycohydrolase and ADP-ribosyltransferase activity. A partially active holotoxin could be generated by mixing the five individual subunits. All subunits were immunogenic in rabbits and mice. Monospecific antisera raised in both animal species were able to neutralize the PT-mediated clustering of Chinese hamster ovary cells, but active immunization of mice with single subunits failed to protect them in the intracerebral challenge assay. These subunit preparations therefore retained neutralizing determinants, but did not contain protective epitopes.     

Evidence that diphtheria toxin and modeccin enter the cytosol from different vesicular compartments

Inhibition of protein synthesis in Vero cells was measured at different periods of time after treatment with diphtheria toxin and the related plant toxin modeccin. Diphtheria toxin acted much more rapidly than modeccin. Cells were protected against both toxins with antiserum as well as with agents like NH4Cl, procaine, and the ionophores monensin, FCCP, and CCCP, which increase the pH of intracellular vesicles. Antiserum, which is supposed to inactivate toxin only at the cell surface, protected only when it was added within a short period of time after modeccin. Compounds that increase the pH of intracellular vesicles, protected even when added after 2 h, indicating that modeccin remains inside vesicles for a considerable period of time before it enters the cytosol. After addition of diphtheria toxin to the cells, compounds that increase the pH of intracellular vesicles protected only approximately to the same extent as antitoxin. This indicates that after endocytosis diphtheria toxin rapidly enters the cytosol. At 20 degrees C, the cells were more strongly protected against modeccin than against diphtheria toxin. The residual toxic effect of diphtheria toxin at 20 degrees C could be blocked with NH4Cl whereas this was not the case with modeccin. This indicates that at 20 degrees C the uptake of diphtheria toxin occurs by the normal route, whereas the uptake of modeccin occurs by a less efficient route than that dominating at 37 degrees C. The results indicate that after endocytosis diphtheria toxin rapidly enters the cytosol from early endosomes with low pH (receptosomes). Modeccin enters the cytosol much more slowly, possibly after fusion of the endocytic vesicles with another compartment.     

Preparation and properties of a hybrid toxin of modeccin A-chain and ricin B-chain

Hybrid molecules were prepared from the A- and B-chains of the two toxic lectins ricin and modeccin by dialyzing mixtures of isolated chains to allow a disulfide bridge to be formed between them. Whereas the hybrid consisting of ricin A-chain and modeccin B-chain was non-toxic, the converse hybrid, modeccin A-chain/ricin B-chain, was even more toxic to Vero cells than were the parent toxins, native ricin and modeccin. A number of drugs (NH₄Cl, monensin, trifluoperazine, verapamil, ionophore A23187) which protect cells against modeccin, but not against ricin, protected to some extent against the toxic hybrid, but less so than against native modeccin. The possibility is discussed that the modeccin A-chain of the hybrid may enter the cytosol by two routes, one which is highly efficient and identical to that used by native modeccin and another less efficient one which cannot be used by native modeccin.     

Purification and properties of five different forms of human procarboxypeptidases

Three different procarboxypeptidases A and two different procarboxypeptidases B have been isolated for the first time, in a pure and native state, from human pancreatic extracts. These proteins were purified in one or two quick steps by anion-exchange HPLC. All these forms have been biochemically characterized. Two of the procarboxypeptidases A, the A1 and A2 forms, are obtained in a monomeric state while the other, the A3 form, is obtained as a binary complex of a procarboxypeptidase A with a proproteinase E. This complex is stable in aqueous buffers at various ionic strengths and develops carboxypeptidase A and proteinase E activities in the presence of trypsin. The A1 and A2 forms show clear differences in electrophoretic mobility in SDS/polyacrylamide gels, isoelectric point, proteolytic activation process with trypsin and susceptibility to thermal denaturation. In contrast, these properties are similar in the A1 and A3 (binary complex) forms. On the other hand, with respect to the properties listed above, the B1 and B2 forms differ from each other mainly in isoelectric point. An overall comparison of the above properties reveals the unusual character of the A2 form, midway between the other A and B forms. N-terminal extended sequence analysis carried out on these proenzymes confirm that they constitute different isologous forms.     

鳞毛蕨凝集素的分离纯化与性质

阔鳞鳞毛蕨[Dryopteris championi(Benth.)C.CHr.]的叶片组织经硫酸铵沉淀、活性炭柱及DEAE-SepharoseFF离子交换柱等步骤纯化得到鳞毛蕨凝集素(Dryopteris championi lectin)。纯化的鳞毛蕨凝集素(DCL)在聚丙烯酰胺凝胶电泳上显示1条蛋白质着色带。其中性糖含量高,氨基酸组成中队(苯丙氨酸)含量最高,His(组氨酸)含量最低。对不同动物红细胞及人的不同血型红细胞的凝集有专一性。其凝血活性能被果糖、半乳糖和N-乙酰半乳糖胺所抑制,对温度变化较不敏感,Mn^2 和Mg2 在一定浓度范围能激活其为EDTA-Na2所抑制的活性。     

Human placenta beta-galactoside-binding lectin. Purification and some properties

A beta-galactoside-binding lectin was extracted from human placenta homogenate with lactose solution and purified to apparent homogeneity by affinity chromatography on asialofetuin-Sepharose. The apparent subunit molecular weight of the lectin was 13,800 and its isoelectric point was about 5. Several saccharides containing D-galactose inhibited the hemagglutinating activity. The lectin resembles other vertebrate beta-galactoside-binding lectins in various biochemical characteristics.     

Separation and characterization of four different amylases of Entamoeba histolytica. I. Purification and properties

Cell homogenate of Entamoeba histolytica trophozoites was investigated for amylolytic activity against various biogenic and synthetic substrates. After gel filtration of the cell homogenate on Sephadex G-150, six partly separated amylases (I to VI) differing in their substrate specificities were detected using maltose, amylose, amylopectin, 4-nitrophenyl alpha-glucoside and 4-nitrophenyl alpha-maltotetraoside. All enzymes are able to degrade amylose, amylopectin, glycogen and biogenic malto-oligosaccharides. Since amylase I and II, which accepted maltose as substrate, were found in fresh (cell-free) medium containing calf serum, the possibility cannot be excluded that these enzymes originate from the medium and therefore are not associated with E. histolytica trophozoites. Amylases III to VI, which were not found in fresh medium, were further purified by isoelectric focusing and chromatographic procedures using DEAE, CM ion exchange materials and Con A Sepharose 4B. pH, temperature optima and relative molecular masses were determined.     

Effects of retinoids and phorbol esters on the sensitivity of different cell lines to the polypeptide toxins modeccin, abrin, ricin and diphtheria toxin.

The effects of retinoic acid and 12-O-tetradecanoylphorbol 13-acetate on the sensitivities of a number of cell lines to the toxins modeccin, abrin, ricin and diphtheria toxin were studied. Retinoic acid and some other retinoids were found to protect a number of the cell lines against the toxins. HeLa cells that were protected bound much more retinoic acid than L-cells that were not protected. The tumour promoter 12-O-tetradecanoylphorbol 13-acetate was found to increase the sensitivity of cells to abrin, ricin and modeccin in the absence as well as in the presence of retinoic acid. Neither retinoic acid nor 12-O-tetradecanoylphorbol 13-acetate affected the extent of binding and pinocytotic uptake of toxins by the cells. Apparently retinoic acid and 12-O-tetradecanoylphorbol 13-acetate interfere with the entry of the toxins through the cell membrane.     

Induction of cytokines by toxins that have an identical RNA N-glycosidase activity: Shiga toxin, ricin, and modeccin

Shiga toxin (Stx) has an A1-B5 subunit structure, and the A subunit is an RNA N-glycosidase that inhibits cellular protein synthesis. We previously reported that in Caco-2 cells Stx induced cytokines and that the RNA N-glycosidase activity was essential for the cytokine induction. It is known that the binding of the Stx-B subunit to its receptor glycolipid, Gb3, mediates an A subunit-independent signal in some types of cells, but the involvement of this signal in the cytokine induction is unclear. In this study, we investigated whether RNA N-glycosidase itself induces cytokines. IL-8 production was enhanced by Stx, ricin, and modeccin, three toxins that inhibit protein synthesis through an identical RNA N-glycosidase activity, but not by two other types of protein synthesis inhibitors, diphtheria toxin and cycloheximide. The RNA N-glycosidase-type toxins showed a similar induction pattern of cytokine mRNAs. Brefeldin A, a Golgi apparatus inhibitor, completely suppressed the cytokine induction by the toxins. Analysis by using inhibitors of toxin binding and also Stx-B subunit showed that the cytokine-inducing activity was independent of Gb3-mediated signaling. These results indicate that RNA N-glycosidase itself induces the cytokine production and that intracellular transport of toxins through the Golgi apparatus is essential for the activity.     

Purification and properties of a beta-galactoside-binding lectin from neonatal marmoset.

A beta-galactoside-binding lectin was extracted from whole neonatal marmoset homogenate with lactose solution and purified to homogeneity by ion-exchange chromatography on Q Sepharose Fast Flow and by affinity adsorption to trypsinized and glutaraldehyde-fixed ghosts of rabbit erythrocytes. The lectin has a dimeric structure composed of two 15K subunits. Its amino acid composition and partial amino acid sequences were quite similar to those of beta-galactoside-binding lectins from human placenta and lung.     

Purification and properties of a lectin from the seeds of Croton tiglium with hemolytic activity toward rabbit red cells

The pH dependence of the chemical shift of the inorganic phosphate (P_i) inside mitochondria observable by ³¹P nmr has been examined and used for the measurement of the internal pH. The pH gradient and the P_i concentration gradient were in the simple relation expected for the neutral exchange process of H₂PO₄^? and OH^?. This P_i distribution across the mitochondrial membrane was not influenced by the cross-membrane electrical potential. Both the P_i, distribution and the pH titration curve of the internal P_i indicate that the activity of the internal P_i can be well represented by the concentration of P_i measured by ³¹P nmr peak intensity. The present results give a sound base for applying ³¹P nmr to study bioenergetics and cell metabolism.     

Purification and properties of two forms of rat alpha-lactalbumin.

Two species of alpha-lactalbumin, alpha-lactalbumin1 and alpha-lactalbumin2, were separated from rat milk and purified to homogeneity by gel filtration, followed by the DEAE-cellulose chromatography. alpha-Lactalbumin1 is a bigger molecule in contrast to other known alpha-lactalbumins, and has a molecular weight of 21,500 as determined by sedimentation equilibrium and sodium dodecyl sulfate-polyacrylamide gel analysis. alpha-Lactalbumin2 has a molecular weight of 16,000 measured by sedimentation analysis. alpha-Lactalbumin2, however, exhibits abnormally high molecular weight of 22,500 on sodium dodecyl sulfate polyacrylamide gels. Both alpha-lactalbumins are active in lactose synthase assay and are glycoproteins containing 7 to 9% carbohydrate. Antiserum raised against alpha-lactalbumin1 cannot discriminate between the two species in a radioimmunoassay.