Resistance of Tetrahymena to ricin, a toxic enzyme from Ricinus communis.

The protozoan $\text{Tetrahymena}\text{pyriformis}$ was found to be resistant to the toxic action of ricin $\text{in}\text{vivo}$. Isolated $\text{Tetrahymena}$ ribosomes were strongly resistant to the A subunit of ricin when tested in a cell free protein synthesis system under different conditions and also lacked the ability to bind A chain stoichometrically. This suggests that $\text{Tetrahymena}$ is resistant $\text{in}\text{vivo}$ because it contains a ribosome which is not susceptible to the toxic action of ricin.     

Cloning and characterization of the calreticulin gene from Ricinus communis L.

A full-length cDNA encoding a calreticulin-like protein was isolated by immune-screening a germinating castor bean endosperm cDNA library with antisera raised to the total lumenal fraction of purified plant endoplasmic reticulum. The calcium-binding properties of the recombinant protein were characterized and shown to be essentially identical to those reported for the mammalian calreticulin. Calcium overlays and immune blot analysis confirmed the endoplasmic lumenal identity of this reticuloplasmin. Probing protein blots of endoplasmic reticulum subfractions with radio-iodinated calreticulin showed specific associations with various polypeptides including one identified as the abundant reticuloplasmin protein disulfide isomerase.Characterization of the corresponding genomic clones revealed that calreticulin is encoded by a single gene of 3 kb in castor. The full genomic sequence reveals the presence of 12 introns, 12 translated exons, and one exon containing the last three amino acids of the translated sequence and the 3-untranslated region of the gene. Northern blot analysis of RNA isolated from various organ tissues showed a basal constitutive level of expression throughout the plant, but more abundant mRNA being detected in tissues active in secretion. This was confirmed by analysis of transgenic tobacco plants containing 1.8 kb of 5-untranslated genomic sequence fused to the -glucuronidase reporter gene (GUS) showed a more localized pattern of expression. Activity being localized to the vasculature (phloem, root hairs and root tip) in vegetative tissue, and being strongly expressed in the floral organs including the developing and germinating seed.     

Characterization of a cDNA encoding ricin E,a hybrid ricin-Ricinus communis agglutinin gene from the castor plant Ricinus communis

Two classes of ricin cDNA clones have been identified and sequenced. The cDNA clone pBL-1 closely matches in nucleotide sequence the ricin genomic clone pAKG previously described by Halling et al., 1985 (Nucl. Acids Res. 13:8019). A second group of cDNA clones, represented by pBL-3, encode a hybrid protein (ricin E), having a ricin-like A chain and N-terminal half of the B chain and an RCA (Ricinus communis agglutinin)-like C-terminal half of the B chain.     

Crystalline ricin D, a toxic anti-tumor lectin from seeds of Ricinus communis

A toxic lectin, ricin D, present in the seeds of Ricinus communis has been purified and crystallized in a form suitable for high resolution crystallographic structure studies. This protein is different from a previously found form of ricin (also present in the same seeds), the only ricin for which a preliminary x-ray investigation has been reported so far. Ricin D crystallizes from an aqueous solution in an orthorhombic unit cell of symmetry P2(1)2(1)2(1) and a = 79.0, b = 114.7, and c = 72.8 A. The asymmetric unit contains one molecule with an average molecular weight of 62,400. The crystal is fairly stable to x-radiation and has a water content of approximately 54% by volume. It appears to comprise two closely related species of proteins, the major species corresponding to recin D and the other presumably corresponding to a deamidation product of ricin D. The two species have nearly identical molecular size and amino acid compositions, but different charges.     

The primary sequence of Ricinus communis agglutinin. Comparison with ricin

A mixture of synthetic oligonucleotides representing all possible sequences of a peptide present in the ricin B chain has been used to screen a cDNA library constructed using ripening castor bean seed poly(A+) RNA. The eight largest recombinant plasmids selected, by hybridization, a single mRNA species whose translational product was identified as preprolectin by immunoprecipitation. Restriction enzyme analysis of these clones demonstrated that two classes were present representing sequences complementary to two distinct but closely related preprolectin mRNA species. The nucleotide sequence of the cloned cDNA from one of these classes encodes preproricin and has been presented elsewhere (Lamb, F. I., Roberts, L. M., and Lord, J. M., (1985) Eur. J. Biochem. 148, 265-270). The nucleotide sequence of the second class is presented here and shown to represent prepro-Ricinus communis agglutinin. The entire coding sequence was deduced from two overlapping cDNA clones having inserts of 1668 and 1151 base pairs. The coding region defines a preproprotein with a 24-amino acid N-terminal signal sequence preceding the A chain (266 amino acids) which is joined to the B chain (262 amino acids) by a 12-amino acid linking peptide. The protein was confirmed as R. communis agglutinin since the deduced B chain N-terminal sequence corresponds exactly with that determined for purified R. communis agglutinin B chain over a region where several residue differences occur in the ricin B chain. The nucleotide and deduced amino acid sequences of the R. communis agglutinin precursor are compared with those of the ricin precursor.     

Purification and characterization of Ricinus communis invertase

An invertase from Ricinus communis leaves was purified 4,400-fold. The preparation was homogeneous by criteria of gel electrophoresis, gel permeation, adsorption, and ionic exchange chromatography. One optimum pH at 3.5 was observed with crude invertase; however, purified preparations showed two optima, at pH 3.5 and 5.5. Addition of bovine serum albumin restored one maximum at pH 3.5 and elicited a 30% activation of the invertase. The effect was caused by many other proteins and by heparin, dextran sulfate, and polyvinylpyrrolidone. Fructose, fructose 1,6-diphosphate, maleic, trans-aconitic, malic, and ascorbic acids were simple competitive inhibitors of the purified enzyme. Glucose was a noncompetitive inhibitor. The activation by proteins suppressed these inhibitory effects. The minimum concentration of activator necessary to reach the maximal activation or "point of optimal activation" was always reached at a concentration of 1 X 10(-6) M, independently of the nature of the activator, when 8.6 X 10(-12) mol of enzyme were used. Apparent molecular weight determinations of the enzyme in the presence and absence of activator and molecular weight determinations based on determinations of the point of optimal activation suggested that the purified enzyme is a heptamer (Mr of 77,900, Stokes radius 32 A, frictional ration f/fo 1.1, partial specific volume 0.749 ml/g) and that the activated form is a trimer consisting of two enzyme subunits and one activator molecule. The activation was lost by dilution of the trimer. The enzyme subunit, as isolated by gel filtration in the presence of sodium dodecyl sulfate (Mr 11,000) was inactive but quickly regained activity upon removal of sodium dodecyl sulfate.     

Crystallographic characterization of a principal non-toxic lectin from seeds of Ricinus communis

Large hexagonal crystals of ricinus lectin, present as a major component in the seeds of Ricinus communis, have been obtained at 4 °C in the presence of polyethylene glycol 6000 by vapor diffusion against media containing acetic acid. The crystals are of space group P622, with hexagonal unit cell parameters $\text{a = b = 166}\text{A}\text{?}\text{and}\text{c = 341}\text{A}\text{?}$. The asymmetric unit contains two molecules of molecular weight 125,000. The crystals are extremely unstable, both to environmental changes and to X-irradiation, and have a solvent content of approximately 55% by volume.     

Precursors of ricin and Ricinus communis agglutinin. Glycosylation and processing during synthesis and intracellular transport

During synthesis in vivo the castor bean lectin precursors initially appear in the endoplasmic reticulum as a group of core glycosylated polypeptides of relative molecular mass 64 000-68 000. Pretreatment of intact castor bean endosperm tissue with tunicamycin partially inhibits the cotranslational core glycosylation step and results in the accumulation of a single sized unglycosylated precursor polypeptide of relative molecular mass 59 000. The glycosylated precursors in the endoplasmic reticulum were enzymically converted to the 59 000-Mr form by incubation with endoglucosaminidase H. Intracellular transport of the glycosylated lectin precursors from the endoplasmic reticulum to a denser vesicle fraction was accompanied by modifications to the oligosaccharide moieties which conferred resistance to the action of endoglucosaminidase H. The post-translational addition of fucose to the carbohydrate chain was identified as one of the oligosaccharide modification steps. Fucose addition was catalysed by a glycosyltransferase associated with a smooth-surfaced membrane fraction which was distinct from the endoplasmic reticulum and which was tentatively identified as the Golgi apparatus. Glycosylation was not essential for intracellular transport of the lectin precursors: unglycosylated precursor synthesized in the presence of tunicamycin gave rise to unglycosylated lectin subunits in the protein bodies.     

Variants of hamster fibroblasts resistant to Ricinus communis toxin (ricin).

1. Variant baby-hamster kidney (BHK) cell lines were isolated that grow in the presence of high concentrations of ricin, the toxic lectin of castor beans (Ricinus communis). The variant lines were independently derived from several cultures of normal BHK cells which had been exposed to the mutagen, methyl-N-nitro-N-nitrosoguanidine, before selection by ricin. 2. The cell lines maintain a high degree of resistance to ricin after growth in lectin-free medium for prolonged periods and therefore exhibit stable phenotypes that are different from normal BHK cells. 3. A preliminary classification of the phenotypes was made. Several cell lines bind normal amounts of 125I-labelled ricin, whereas other bind the lectin poorly. 4. A loss of surface receptors for two other lectins, R. communis RCA and Axinella polyploides, which have specificities similar to ricin, was also found in some but not all of the cell lines showing decreased surface concentrations of ricin receptors. 5. The binding to the ricin-resistant cells of lectins of different sugar specificity, namely Lens culinaris lectin and concanavalin A, was similar to, or higher than, to normal BHK cells. 6. Several of the ricin-resistant cell lines were shown to be cross-resistant to the weak cytotoxicity of Phaseolus vulgaris lectin. By contrast, some cell lines were more sensitive to concanavalin A than were normal BHK cells.     

Structure and analysis of phospholipase D gene from Ricinus communis L

Phospholipase D (PLD; EC 3.1.4.4) has been proposed to play a pivotal role in various cellular processes, but molecular understanding of this enzyme is rather limited. This report describes the nucleotide sequence, structure, and genomic organization of a PLD gene from castor bean (Ricinus communis L. cv. Hale). The PLD gene was isolated from a castor bean genomic library using the PLD cDNA as a hybridization probe. Sequence comparison with the PLD cDNA revealed that the PLD gene consisted of four exons and three introns, one of which interrupts the 5-untranslated region. Southern blot analysis indicated that the cloned PLD gene was present as a single-copy gene, and yet there were other PLD or PLD-related sequences in the castor bean genome.     

Acetyl-coenzyme A carboxylase from the developing endosperm of Ricinus communis. I. Isolation and characterization

Acetyl-coenzyme A carboxylase has been purified from the plastids of developing castor oil seeds. High concentrations of the enzyme are required for stability as well as the presence of dithiothreitol, glycerol, bicarbonate, Triton X-100, and polyvinyl-pyrrolidone. It has a molecular weight of approximately 528,000 and appears to be membrane associated. Acetyl-CoA carboxylase is active over a wide pH range with an optimum at 8.0. Arrhenius plots are biphasic. The enzyme displays normal Michaelis-Menten kinetics with limiting Michaelis constants of KATP, 0.1 mM; KHCO-3, 3.0 mM; and Kacetyl-CoA, 0.05 mM. Monovalent cations, such as K+ and Cs+, exert a small activating effect on the enzyme while a divalent cation, Mn2+ or Mg2+, is essential for activity. The enzyme does not appear to be highly regulated by cellular metabolites.     

Enolase isozymes from Ricinus communis: Partial purification and characterization of the isozymes

The plastid and cytosolic isozymes of enolase from developing endosperm of castor oil seeds, Ricinus communis L. cv. Baker 296, were separated and partially purified. Each purified isozyme had a specific activity of approximately 200 μmol min^?1 mg protein. The isozymes have similar pH optima for the forward reaction, but different optima for the reverse reaction. The divalent metal specificity is the same for both isozymes. In addition to differences in charge, the isozymes can be distinguished by their different kinetic constants, thermostability and sensitivity to fluoride inhibition. Antibodies against yeast enolase isozyme I cross-react with Ricinus plastid enolase but not with the cytosolic isozyme.