Analysis of glycoconjugate saccharides in organelles isolated from castor bean endosperm

The presence of lipid- and protein-bound sugars in the major organelle fractions isolated from germinating castor bean (Ricinus communis L.) endosperm has been established. Microsomes, glyoxysomes and mitochondria were subfractionated into a membrane fraction and a fraction containing peripheral membrane and soluble matrix proteins. The membranes were further subfractionated into monosaccharide lipid, oligosaccharide lipid and lipid-free protein components. The constituent sugars present in the prepared fractions were released and identified by gas-liquid chromatography. While all derived protein fractions contained the N-acetylglucosamine and mannose typically found in the inner core region attached to asparagine residues in many glycoproteins, some differences were noted in the organellar distribution of peripheral sugars such as fucose, arabinose, and xylose.     

A single affinity column step method for the purification of ricin toxin from castor beans (Ricinus communis)

A rapid method for purifying ricin toxin from castor beans is presented which uses a single affinity column step to obtain pure toxin from a crude extract of castor beans. A galactosyl-Sepharose affinity matrix was used to bind ricin toxin and its associated agglutinin, which both bind specifically to galactose, from a crude extract. The selective elution of ricin toxin and agglutinin was then achieved by eluting the affinity column with a galactose gradient, which sequentially elutes the two proteins due to a difference in binding avidity to the matrix.     

The complete amino acid sequence of the B-chain of ricin E isolated from small-grain castor bean seeds. Ricin E is a gene recombination product of ricin D and ricinus communis agglutinin

The complete amino acid sequence of the B-chain of ricin E has been determined. The reduced and carboxymethylated B-chain was digested with trypsin, followed by separation and purification of the resulting peptides using reverse-phase HPLC. The amino acid sequence of each tryptic peptide was determined employing the DABITC/PITC double-coupling method. The B-chain of ricin E proved to consist of 262 amino acid residues. By comparing the amino acid sequence of the B-chain of ricin E with those of ricin D and of Ricinus communis agglutinin, it was found that the B-chain of ricin E was composed of the N-terminal half of ricin D and the C-terminal half of R. communis agglutinin. This result suggested that the gene recombination probably occurred at the center region of two B-chain genes of ricin D and R. communis agglutinin.     

Cloning and characterization of the acid lipase from castor beans

Castor bean endosperm contains a well known acid lipase activity that is associated with the oil body membrane. In order to identify this enzyme, proteomic analysis was performed on purified oil bodies. A approximately 60-kDa protein was identified (RcOBL1), which shares homology with a lipase from the filamentous fungus Rhizomucor miehei. RcOBL1 contains features that are characteristic of an alpha/beta-hydrolase, such as a putative catalytic triad (SDH) and a conserved pentapeptide (GXSXG) surrounding the nucleophilic serine residue. RcOBL1 was expressed heterologously in Escherichia coli and shown to hydrolyze triolein at an acid pH (optima approximately 4.5). RcOBL1 can hydrolyze a range of triacylglycerols but is not active on phospholipids. The activity is sensitive to the serine reagent diethyl p-nitrophenyl phosphate, indicating that RcOBL1 is a serine esterase. Antibodies raised against RcOBL1 were used to show that the protein is restricted to the endosperm where it is associated with the surface of oil bodies. This is the first evidence for the molecular identity of an oil body-associated lipase from plants. Sequence comparisons reveal that families of OBL1-like proteins are present in many species, and it is likely that they play an important role in regulating lipolysis.     

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.     

OTA-producing fungi isolated from stored cocoa beans

Aims:  The aim of this study was to identify fungal populations in unroasted cocoa beans stored in Spain in order to evaluate the ochratoxin A (OTA)-production ability of certain Aspergillus isolates.
Methods and Results:  Twenty batches of cocoa beans from different origins and with different OTA content were selected for this study. Three Aspergillus carbonarius and 13 Aspergillus niger aggregate strains isolated from these cocoa bean samples were selected to evaluate their OTA synthesis ability, being the only A. carbonarius isolates which are OTA producers [−1 culture medium; LOD = 6  μ g kg⁻¹ culture medium].
Conclusions:  No correspondence was found between the OTA levels in cocoa beans and the presence of OTA-producing fungi. Nonetheless, some samples contained A. carbonarius with a high OTA-producing ability and, consequently, specific fungal controls should be set up during storage to avoid this toxin.
Significance and Impact of the Study:  Toxigenic fungi in cocoa beans are not well understood. This study attempted to identify these fungi and evaluate their OTA-producing ability.     

Effects of leupeptin on proteinase and germination of castor beans

Leupeptin, a tripeptide inhibitor of some proteinases, was shown previously to maintain the stability of several enzymes (isocitrate lyase, fumarase, and catalase) in crude extracts of castor bean endosperm. This reagent is now shown to inhibit the breakdown of water-soluble and crystalloidstorage proteins of the protein bodies isolated from castor beans by the SH-proteinase and it also inhibits the endopeptidase from mung beans. When suitably introduced into the endosperm of dry castor beans it strongly inhibits germination and seedling development. Application of leupeptin to endosperm halves removed from the seed prevents the normal development of enzymes concerned with gluconeogenesis from fat and drastically curtails sugar production. The results suggest that the SH-proteinase is intimately involved in the mobilization of storage proteins.     

Binding of vitellogenin to membranes isolated from mosquito ovaries

The presence of specific receptors for vitellogenin (Vg) in ovary membranes of the mosquito, Aedes aegypti, was demonstrated by an in vitro binding assay. The binding reaction, which is dependent on pH and Ca2+, uses 4 micrograms membrane protein, 35S-Vg labeled metabolically by fat body culture in vitro, and unlabeled vitellin (Vn) for competition. At pH 7.0 and in the presence of 5 mM Ca2+, the binding of Vg to its receptor reaches equilibrium within 60-90 min at both 4 and 25 degrees C. The binding is specific to membranes prepared only from ovaries. While mosquito Vg and Vn bind with equal affinity to Vg receptors on ovary membranes, neither locust Vg nor mouse IgG has any measurable affinity towards these sites. Nonlinear least square analysis of the saturation isotherms is consistent with the presence of a single class of Vg receptors on ovary membranes with a dissociation constant (Kd) of 0.18 microM.     

Mycotoxin-producing potential of fungi isolated from red kidney beans

The predominant fungi present in samples of reject and retail red kidney beans were Aspergillus glaucus, Penicillium spp. and Alternaria spp. Together with A. ochraceus, A. flavus, Fusarium spp., and Trichoderma, these isolates from the reject beans were screened for numerous mycotoxins by TLC. The most consistently produced mycotoxins were penicillic acid (from A. ochraceus and Penicillium spp.) and Alternaria toxins (tenuazonic acid and alternariol). A. glaucus strains were tested for cytotoxicity in three tissue culture cell lines with positive results.     

Isoenzymes of sugar phosphate metabolism in endosperm of germinating castor beans

Two isoenzymes each of phosphoglucomutase, hexose phosphate isomerase, aldolase, fructose diphosphatase, phosphofructokinase, and 6-phosphogluconate dehydrogenase have been separated by DEAE-cellulose column chromatography of extracts from endosperm of germinating castor beans (Ricinus communis cv. Hale). One of each of the enzymes is localized in the cytosol and the other is confined to plastids. Developmental studies of these isoenzymes were carried out to clarify their roles in the endosperm. In extracts from ungerminated seeds the activities of marker enzymes of mitochondria (fumarase), plastids (ribulose bisphosphate carboxylase), and glyoxysomes (catalase) were low, but phosphoglucomutase, hexose phosphate isomerase, aldolase, and 6-phosphogluconate dehydrogenase were present in relatively high activity. The total amounts of these enzymes increased 3- to 4-fold during the first 5 days of growth. The activities of isoenzymes in the plastids rose in parallel with that of ribulose bisphosphate carboxylase to reach a maximum at day 4, and like the carboxylase they declined sharply thereafter. The activities of the cytosolic isoenzymes peaked at day 5. These changes are consistent with the roles previously proposed for the sequences present in plastid and cytosol.     

Isolation and characterization of the protein body membrane of castor beans

Intact protein bodies were isolated from dry castor bean seeds (Ricinus communis L.) after homogenization in nonaqueous medium. After repeated washing with glycerol to remove trapped lipid globules, the soluble matrix proteins were removed by the addition of aqueous buffer. The membrane remained attached to the insoluble protein crystalloids and was subsequently released by sonication. Purification of the membrane vesicles in a sucrose gradient produced a single band at a density of 1.21 grams per cubic centimeter. Treatment with 6 molar urea, 1 molar KCl, or 0.25 molar galactose had no effect on the equilibrium density of the membrane. Electron microscopy revealed a highly pure and uniform collection of membrane vesicles. No enzyme activity was specifically associated with the membrane. Sodium dodecyl sulfate gel electrophoresis of the protein body fractions showed that the membrane contained unique proteins, two of which were glycosylated. The membrane contained 153 nanomoles of phospholipid per milligram of protein. The composition of the phosphoglycerides was 51% ethanolamine, 41% choline, 8% inositol, and a trace of serine.