Lectins as membrane components of mitochondria from Ricinus communis.
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1. Mitochondria were isolated from developing endosperm of Ricinus communis and were fractionated into outer membrane and inner membrane. The relative purity of the two membrane fractions was determined by marker enzymes. The fractions were also examined by negative-stain electron microscopy. 2. Membrane fractions were sequentially extracted in the following way. (a) Suspension in 0.5M-potassium phosphate, pH7.1; (b)suspension in 0.1M-EDTA (disodium salt)/0.05M-potassium phosphate, pH7.1; (c) sonication in 0.05M-potassium phosphate, pH7.1;(d)sonication in aq. Triton X-100 (0.1%). The membranes were pelleted by centrifugation at 100 000g for 15 min, between each step. Agglutination activity in the extracts was investigated by using trypsin-treated rabbit erythrocytes. 3. The addition of lactose to inner mitochondrial membrane resulted in the solubilization of part of the lectin activity, indicating that the protein was attached to the membrane via its carbohydrate-binding site. Pretreatment of the membranes with lactose before tha usual extraction procedure showed that lactose could extract lectins that normally required more harsh treatment of the membrane for solubilization. 4. Lectins extracted from inner membranes were purified by affinity chromatography on agarose gel. Polyacrylamide-gel electrophoresis of purified samples in sodium dodecyl sulphate indicated that at least part of the lectin present in inner mitochondrial membrane was identical with the R. communis agglutinin of mol.wt. 120 000. (+info)
Expression of Ricinus communis receptors on epithelial cells in oral carcinomas and oral wounds.
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The histological distribution of receptors for Ricinus communis Fraction 1 (RCA1) in oral carcinomas and in oral epithelial cells during wound healing has been studied by use of fluorescein-tagged RCA1. Biopsies from 15 human oral carcinomas and adjacent normal mucosa showed RCA1 receptors at the cell membranes in the basal and spinous layer of the normal epithelium, whereas receptors could not be demonstrated in invading islands of the tumors. In healing oral wounds from eight humans and three monkeys, RCA1 receptors were demonstrated both in normal epithelium adjacent to the wounds and in the epithelial outgrowth from the wound margin. Titrations, however, showed that the epithelial outgrowth reacted more weakly than did the normal adjacent epithelium. These results support previous in vitro studies showing changes in carbohydrate composition of moving normal cells and of malignant cells, a finding that may be of interest in relation to formation of metastases. (+info)
Nitrate reductases from leaves of Ricinus (Ricinus communis L.) and spinach (Spinacia oleracea L.) have different regulatory properties.
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The activity of nitrate reductase (+Mg(2+), NR(act)) in illuminated leaves from spinach, barley and pea was 50-80% of the maximum activity (+EDTA, NR(max)). However, NR from leaves of Ricinus communis L. had a 10-fold lower NR(act), while NR(max) was similar to that in spinach leaves. The low NR(act) of Ricinus was independent of day-time and nitrate nutrition, and varied only slightly with leaf age. Possible factors in Ricinus extracts inhibiting NR were not found. NR(act) from Ricinus, unlike the spinach enzyme, was very low at pH 7.6, but much higher at more acidic pH with a distinct maximum at pH 6.5. NR(max) had a broad pH response profile that was similar for the spinach and the Ricinus enzyme. Accordingly, the Mg(2+)-sensitivity of NR from Ricinus was strongly pH-dependent (increasing sensitivity with increasing pH), and as a result, the apparent activation state of NR from a Ricinus extract varied dramatically with pH and Mg(2+)concentration. Following a light-dark transition, NR(act) from Ricinus decreased within 1 h by 40%, but this decrease was paralleled by NR(max). In contrast to the spinach enzyme, Ricinus-NR was hardly inactivated by incubating leaf extracts with ATP plus okadaic acid. A competition analysis with antibodies against the potential 14-3-3 binding site around ser 543 of the spinach enzyme revealed that Ricinus-NR contains the same site. Removal of 14-3-3 proteins from Ricinus-NR by anion exchange chromatography, activated spinach-NR but caused little if any activation of Ricinus-NR. It is suggested that Mg(2+)-inhibition of Ricinus-NR does not require 14-3-3 proteins. The rather slow changes in Ricinus-NR activity upon a light/dark transient may be mainly due to NR synthesis or degradation. (+info)
Ultrastructural localization of lectin-binding sites on the zonae pellucidae and plasma membranes of mammalian eggs.
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Receptors for Ricinus communis agglutinin I (RCAI), concanavalin A (Con A), and wheat germ agglutinin (WGA) were localized on the zonae pellucidae and plasma membranes of hamster, mouse, and rat eggs with ferritin-lectin conjugates. Intact eggs labeled with the ferritin conjugates showed dense concentrations of RCAI and WGA receptors in the outermost regions of their zonae pellucidae and sparse distributions of Con A receptors throughout the zonae. Ferritin-lectin labeling was specific, since inhibitory saccharides effectively blocked labeling. The asymmetric density of RCAI receptors across the zona was confirmed by ferritin-RCAI and fluorescein-RCAI labeling of mechanically isolated zonae pellucidae, indicating that the RCAI-binding sites are more densely distributed in the exterior zona regions. Plasma membranes of rodent eggs contained RCAI, WGA, and Con A receptors. These receptors were found to be more or less randomly distributed on surfaces of aldehyde-fixed eggs or on eggs labeled near 0 degrees C. However, eggs incubated at 25 degrees C showed aggregated WGA- and Con A-binding site distributions on their plasma membranes. This indicates that lectin-induced receptor redistribution occurs at this temperature. The possibility that plasma membrane receptor mobility is a requirement for sperm-egg fusion is discussed. (+info)
Leaf development in Ricinus communis during drought stress: dynamics of growth processes, of cellular structure and of sink-source transition.
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Dicot leaf growth is characterized by partly transient tip-to-base gradients of growth processes, structure and function. These gradients develop dynamically and interact with dynamically developing stress conditions like drought. In Ricinus communis plants growing under well-watered and drought conditions growth rates peaked during the late night and minimal values occurred in the late afternoon. During this diurnal course the leaf base always showed much higher rates than the leaf tip. The amplitude of this diurnal course decreased when leaves approached maturity and during drought stress without any significant alteration of the diurnal pattern and it increased during the first days after rewatering. Unique relationships between leaf size and cytological structure were observed. This provided the framework for the analysis of changes in assimilation, transpiration and dark respiration, chlorophyll, protein, carbohydrate, and amino acid concentrations, and of activities of sink-source-related enzymes at the leaf tip and base during leaf development in well-watered and drought-stressed plants. Gas exchange was dominated by physiological rather than by anatomical properties (stomatal density). Tip-to-base gradients in carbohydrate concentrations per dry weight and sink-source-related enzymes were absent, whereas significant gradients were found in amino acid concentrations per dry weight. During drought stress, growing leaves developed source function at smaller leaf size, before specific physiological adaptations to drought occurred. The relevance of the developmental status of individual leaves for the drought-stress response and of the structural changes for the biochemical composition changes is discussed. (+info)
Characterization of group N streptococcus lipoteichoic acid.
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Lipoteichoic acid was extracted from the group N organism Streptococcus lactis ATCC 9936 with hot aqueous phenol and purified by gel chromatography followed by affinity chromatography using Ricinus communis lectin as the specific absorbent. The teichoic acid moiety of the lipoteichoic acid was calculated to contain 16 to 17 glycerol phosphate units, approximately half of which were substituted with alpha-D-galactosyl residues; the glycolipid moiety contained O-alpha-D-glucosyl-1 yields 2-O-alpha-D-glucosyl-1 yields 1-glycerol. The finding of 2-O-alpha-D-galactosyl glycerol in the lipid fraction of hydrofluoric acid hydrolysates suggests that fatty acids also occur as substituents on the main chain of the lipoteichoic acid. The reactivity of the lipoteichoic acid with R. communis lectin was studied by the quantitative precipitin method and compared with the reactivity of Lactobacillus fermenti lipoteichoic acid, which has a lower degree of alpha-D-galactosyl substitution. Group N antiserum reacted strongly with the S. lactis lipoteichoic acid and cross-reacted with L. fermenti lipoteichoic acid. From inhibition studies it is concluded that the antibodies are specific for alpha-D-galactosyl substituents. In addition to lipoteichoic acid, a fraction was obtained by gel chromatography which contained galactose and reacted with group N antiserum but could be distinguished from the lipoteichoic acid by immunoelectrophoresis. (+info)
The ricinosomes of senescing plant tissue bud from the endoplasmic reticulum.
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The ricinosome (synonym, precursor protease vesicle) is a novel organelle, found so far exclusively in plant cells. Electron microscopic studies suggest that it buds off from the endoplasmic reticulum in senescing tissues. Biochemical support for this unusual origin now comes from the composition of the purified organelle, which contains large amounts of a 45-kDa cysteine endoprotease precursor with a C-terminal KDEL motif and the endoplasmic reticulum lumen residents BiP (binding protein) and protein disulfide isomerase. Western blot analysis, peptide sequencing, and mass spectrometry demonstrate retention of KDEL in the protease proform. Acidification of isolated ricinosomes causes castor bean cysteine endopeptidase activation, with cleavage of the N-terminal propeptide and the C-terminal KDEL motif. We propose that ricinosomes accumulate during senescence by programmed cell death and are activated by release of protons from acidic vacuoles. (+info)
Lipolytic activity of ricin from Ricinus sanguineus and Ricinus communis on neutral lipids.
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The present study was carried out with a view of determining ricin lipolytic activity on neutral lipids in emulsion and in a membrane-like model. Using 2,3-dimercapto-1-propanol tributyrate (BAL-TC(4)) as substrate, the lipolytic activity of ricin was found to be proportional to ricin and substrate concentrations, with an apparent K(m) (K(m,app)) of 2.4 mM, a k(cat) of 200 min(-1) and a specific activity of 1.0 unit/mg of protein. This work was extended to p-nitrophenyl (pNP) fatty acid esters containing two to twelve carbon atoms. Maximum lipolytic activity was registered on pNP decanoate (pNPC(10)), with a K(m,app) of 3.5 mM, a k(cat) of 173 min(-1) and a specific activity of 3.5 units/mg of protein. Ricin lipolytic activity is pH and galactose dependent, with a maximum at pH 7.0 in the presence of 0.2 M galactose. Using the monolayer technique with dicaprin as substrate, ricin showed a lipolytic activity proportional to the ricin concentration at 20 mN/m, which is dependent on the surface pressure of the lipid monolayer and is detectable up to 30 mN/m, a surface pressure that is of the same order of magnitude as that of natural cell membranes. The methods based on pNPC(10) and BAL-TC(4) hydrolysis are simple and reproducible; thus they can be used for routine studies of ricin lipolytic activity. Ricin from Ricinus communis and R. sanguineus were treated with diethyl p-nitrophenylphosphate, an irreversible serine esterase inhibitor, and their lipolytic activities on BAL-TC(4) and pNPC(10), and cytotoxic activity, were concurrently recorded. A reduction in lipolytic activity was accompanied by a decrease in cytotoxicity on Caco2 cells. These data support the idea that the lipolytic activity associated with ricin is relevant to a lipase whose activity is pH and galactose dependent, sensitive to diethyl p-nitrophenylphosphate, and that a lipolytic step may be involved in the process of cell poisoning by ricin. Both colorimetric tests used in this study are sensitive enough to be helpful in the detection of possible lipolytic activities associated with other cytotoxins or lectins. (+info)