178 research outputs found
A- and B-Subunit Variant Distribution in the Holoprotein Variants of Protein Toxin Abrin: Variants of Abrins I and III Have Constant Toxic A Subunits and Variant Lectin B Subunits
No full textThe cytotoxic lectin abrin shows more than 30 variant forms (R. Hegde, T. K, Maiti, and S, K. Podder, 1991, Anal. Biochem. 194, 101-109). The lectin B subunit as cause for variance in abrins I and III was detected by a combination of one- and two-dimensional electrophoresis and Western blotting. Intriguingly, in abrin I but not in abrin III, association of a single A subunit with the variant B subunits shifts the holoprotein pI toward the alkaline side indicating that the subunit association involves neutralization of few negative charges. The B-subunit variants of abrins I and III overlap in their pI, and the A-subunit association gives the holoproteins a distinctness on isoelectric focusing gel. The results were also confirmed by analyzing the pH titration curves, These differences in the subunit association pattern between abrins I and III are in corroboration with the previously observed differences in the kinetics of protein synthesis inactivation and accessibility of the disulfide bridge to reducing agents in the presence or absence of putative receptor (R. Hegde, A. Karande, and S. K, Podder, 1993 Eur. J. Biochem. 215, 411-419). Further, the genetic origin of variance was confirmed by peptide mapping of the individual subunit variants, Considering a theoretical value of 0.1 to 0.2 pI/charge, a 15-17 charge difference could be predicted between the variants of two extreme pIs, The fact that the A subunits are not shared between the groups was taken to interpret that the protein synthesized as prepro form is processed posttranslationally and the processing takes place only after the disulfide bond formation between A and B subunits, The N-terminal 16 amino acids of A subunits of abrins I and III showed 26% dissimilarity. The A subunits of abrins I and III did not react with concanavalin A, indicating that the heterogeneity in the molecular weight is because of differential processing but not because of glycosylation
Studies on the variants of the protein toxins ricin and abrin
No full texthis study elucidates some structural and biological features of galactose-binding variants of the cytotoxic proteins ricin and abrin. An isolation procedure is reported for ricin variants from Ricinus communis seeds by using lactamyl-Sepharose affinity matrix, similar to that reported previously for variants of abrin from Abrus precatorius seeds [Hegde, R., Maiti, T. K. & Podder, S. K. (1991) Anal. Biochem. 194, 101–109]. Ricin variants, subfractionated on carboxymethyl-Sepharose CL-6B ion-exchange chromatography, were characterized further by SDS/PAGE, IEF and a binding assay. Based on the immunological cross-reactivity of antibody raised against a single variant of each of ricin and abrin, it was established that all the variants of the corresponding type are immunologically indistinguishable. Analysis of protein titration curves on an immobilized pH gradient indicated that variants of abrin I differ from other abrin variants, mainly in their acidic groups and that variance in ricin is a cause of charge substitution. Detection of subunit variants of proteins by two-dimensional gel electrophoresis showed that there are twice as many subunit variants as there are variants of holoproteins, suggesting that each variant has a set of subunit variants, which, although homologous, are not identical to the subunits of any other variant with respect to pI. Seeds obtained from polymorphic species of R. communis showed no difference in the profile of toxin variants, as analyzed by isoelectric focussing. Toxin variants obtained from red and white varieties of A. precatorius, however, showed some difference in the number of variants as well as in their relative intensities. Furthermore, variants analyzed from several single seeds of A. precatorius red type revealed a controlled distribution of lectin variants in three specific groups, indicating an involvement of at least three genes in the production of Abrus lectins. The complete absence or presence of variants in each group suggested a post-translational differential proteolytic processing, a secondary event in the production of abrin variants
Evolution of tetrameric lectin Ricinus communis agglutinin from two variant groups of ricin toxin dimers
No full textSeeds of Ricinus communis contain two types of lectins; the toxin ricin (approximate to 60 kDa) and R. communis agglutinin (approximate to 120 kDa). The toxin is a heterodimer composed of a toxic A subunit and a lectin B subunit, while R. communis agglutinin is a tetramer, constituted of two ricin-like dimers held together by noncovalent forces. The lactamyl Sepharose affinity-purified ricin consists of two major groups of variants designated ricin II and III [Hegde, R. & Podder, S. K. (1992) Eur: J. Biochem. 204, 155-164]. The purified A subunits of all the variants of ricins and R. communis agglutinin show heterogeneity in the molecular mass as shown for ricin before [Fulton, J. R., Blakey, C. D., Knowles, P. P., Uhr, J. W., Thorpe, P. E. & Vitetta, E. S. (1986) J. Biol. Chem. 261, 5314-5319]. Since the isoelectric points of the R. communis agglutinin variants fall between the isoelectric points of ricin II and III, we investigated the possibility that this lectin is made up of ricin II and III. The isoelectric points of the purified B subunits of R. communis agglutinin matched well with those of ricin II and III on urea-polyacrylamide isoelectric focussing gel. Further, two-dimensional electrophoretic analysis of the ricin constituants of R. communis agglutinin in the presence of 9 M urea, showed two protein bands, differing by nearly pH 2 in their isoelectric points, the more alkaline one corresponding to that of ricin III analyzed under the same conditions, while the other, although a higher molecular mass variant, corresponding well with ricin II in its isoelectric point. Based on these results and those obtained from adenine binding to A chains of both ricin and R. communis agglutinin, we provide a plausible evolutionary relationship between R. communis agglutinin and two groups of ricin variants; ricin II and III. The model predicts that one half of R. communis agglutinin is derived from ricin I and II, and the other half from ricin III. The results presented, contrary to the existing notion, unequivocally show that the two halves of R. communis agglutinin are not identical protein units, but differ both in surface charge and molecular mass
Elucidation of the mechanism of interaction of sheep spleen galectin-1 with splenocytes and its role in cell-matrix adhesion
No full textThe binding of a 14 kDa beta-galactoside animal lectin to splenocytes has been studied in detail. The binding data show that there are two classes of binding sites on the cells for the lectin: a high-affinity site with a K-a ranging from 1.1 x 10(6) to 5.1 x 10(5) M-1 and a low affinity binding site with a K-a ranging from 7.7 x 10(4) to 3.4 x 10(4) M-1 The number of receptors per cell for the high- and low-affinity sites is 9 +/- 3 x 10(6) and 2.5 +/- 0.5 x 10(6) respectively. The temperature dependence of the K value yielded the thermodynamic parameters. The energetics of this interaction shows that, although this interaction is essentially enthalpically driven (Delta H - 21 kJ lambda mol(-1)) for the high-affinity sites, there is a very favorable entropy contribution to the free energy of this interaction (-T Delta S - 17.5 Jmol(-1)), suggesting that hydrophobic interaction may also be playing a role in this interaction. Lactose brought about a 20% inhibition of this interaction, whereas the glycoprotein asialofetuin brought about a 75 % inhibition, suggesting that complex carbohydrate structures are involved in the binding of galectin-1 to splenocytes, Galectin-1 also mediated the binding and adhesion of splenocytes to the extracellular matrix glycoprotein laminin, suggesting a role for it in cell-matrix interactions. Copyright (C) 2000 John Wiley & Sons, Ltd
Complex carbohydrate–lectin interaction at the interface: a model for cellular adhesion. II. Reactivity of both the oligosaccharide chain and sugar‐binding domain of a glycoprotein lectin
No full textComplex carbohydrate-lectin interaction at the interface: a model for cellular adhesion. II. Reactivity of both the oligosaccharide chain and sugar-binding domain of a glycoprotein lectin
No full textWe describe studies of a new model cell adhesion system involving liposomes bearing lectins and the glycosphingolipid, asialomonosialoganglioside (asialoGM1). The model provides a simple analysis of experimental data to elucidate the mechanism of heterophilic cell-cell adhesion mediated by multiple protein-carbohydrate interactions. Phospholipid vesicles bearing the fatty acid conjugate of a glycoprotein lectin from Ricinus communis (RCAI vesicle) are shown to react with vesicles bearing the fatty acid conjugate of Concanavalin A (Con A) and asialoGM1 (Con A vesicle). The kinetics of aggregation and monosaccharide-induced disaggregation of the two types of vesicles were followed by monitoring the time-dependent change in turbidity. Depending on the surface density of the asialoGM1, 40-60% of the resulting precipitin complex was dissociable only in the presence of both RCAI-specific galactose and Con A-specific ?-methyl-D-mannoside. Results indicate simultaneous participation of both the saccharide-binding domain and carbohydrage sequence of RCAI, a model cell adhesion molecule, to stabilize the encounter complex by two types of interactions. These findings support the possibility of stable cell-cell adhesion in vivo occurring via interactions between cell adhesion molecules on apposing cell-surface membranes
Difference spectroscopic studies on binding of Cibacron blue F3GA to ribosome inactivating proteins: effect of beta-mercaptoethanol on the interaction with ricin
No full textThe interaction of Cibacron blue F3GA with ribosome inactivating proteins, ricin, ricin A-chain and momordin has been investigated using difference absorption spectroscopy. Ricin was found to bind the dye with a 20- and 2-fold lower affinity than ricin A-chain and momordin, respectively. A time dependent increase in the amplitude of Cibacron blue difference spectrum in the presence of ricin was observed on addition of beta-mercaptoethanol. Analysis of the kinetic profile of this increase showed a biphasic phenomenon and the observed rates were found to be independent of the concentration of beta-mercaptoethanol. Kinetics of reduction of the intersubunit disulphide bond in ricin by beta-mercaptoethanol showed that reduction pet se is a second order reaction. Therefore, the observed changes in the difference spectra of Cibacron blue probably indicate a slow change in the conformation of ricin, triggered by reduction of the intersubunit disulphide bond
An Efficient Parallel Block Processing Approach for K -Means Algorithm for High Resolution Orthoimagery Satellite Images
No full textAbstractA parallel block processing for remote sensed images for classification problem is presented in this paper. Due to increase in computational time for processing the remote sensing images for pixel dimension more than 1000 × 1000. Block processing approach is applied for an image in parallel by distributing the task among the cores. K -means is one of the widely used clustering method for analyzing features in images. Hence it is considered for the parallel block processing approach. The parallel Block Processing approach was implemented using Matlab 2014a programming environment. The experiment is carried out on data sets comprising of 200 samples of high resolution orthoimagery satellite images. The result obtained from parallel block processing approach lead to efficient usage of hardware resources, depletion in time compared to sequential K -means algorithm. Results are acceptable and this approach can be applied for image processing operations
On the specificity of carbohydrate-lectin recognition: the interaction of a lectin from Ricinus communis beans with simple saccharides and concanavalin A
No full textA galactose-specific protein (RC1) isolated from Ricinus communis beans was found to give a precipitin reaction with concanavalin A. Its carbohydrate content amounted to 8-9% of the total protein and was found to be rich in mannose. The interaction of RC1 with galactose and lactose was measured in 0.05 M phosphate buffer containing 0.2 M NaCl (pH 6.8) by the method of conventional equilibrium dialysis. From the analysis of the binding data according to Scatchard method the association constant (Ka) at 5°C was calculated as 3.8 mM−1 and 1.2 mM−1 for lactose and galactose, respectively. In both cases the number of binding sites per molecule of RC1 with molecular weight of 120000 was found to be 2. From the temperature-dependent Ka values for the binding of lactose, the values of -5.7 kcal/mol and -4.3 cal×mol−1×K−1 were calculated for ΔH and ΔS, respectively. The addition of concanavalin A to RC1 or vice versa led to the formation of the insoluble complex RC1·ConA4 containing one molecule of RC1 and one molecule of tetrameric concanavalin A (ConA4) which could be dissociated upon addition of concanavalin A-specific sugars. The complex formation results in a time-dependent appearance of turbidity in the time range from 10s to 10 min. From the measurement of the time-dependent appearance and disappearance of the turbidity the formation (kf) and dissociation (kd) rate constants were calculated as 3 mM−1×s−1 and 0.07 ks−1 respectively. The ratio kf/kd (43μM −1), that corresponds to the association constant of complex RC1·ConA4, is higher than that of mannoside·ConA4 and thereby suggests that protein-protein interaction contributes significantly in stabilising glycoprotein·lectin complexes. The relevance of this finding to the understanding of the chemical specificities that are involved in a model cell-lectin interaction is discussed
On the Specificity of Carbohydrate-Lectin Recognition The Interaction of a Lectin from Ricinus communis Beans with Simple Saccharides and Concanavalin A
No full textA galactose-specific protein (RC1) isolated from Ricinus communis beans was found to give a precipitin reaction with concanavalin A. Its carbohydrate content amounted to 8–9% of the total protein and was found to be rich in mannose. The interaction of RC1 with galactose and lactose was measured in 0.05 M phosphate buffer containing 0.2 M NaCl (pH 6.8) by the method of conventional equilibrium dialysis. From the analysis of the binding data according to Scatchard method the association constant (Ka) at 5°C was calculated as 3.8 mM−1 and 1.2 mM−1 for lactose and galactose, respectively. In both cases the number of binding sites per molecule of RC1 with molecular weight of 120000 was found to be 2. From the temperature-dependent Ka values for the binding of lactose, the values of –5.7 kcal/mol and –4.3 cal × mol−1× K−1 were calculated for ΔH and ΔS, respectively.
The addition of concanavalin A to RC1 or vice versa led to the formation of the insoluble complex RC1· ConA4 containing one molecule of RC1 and one molecule of tetrameric concanavalin A (ConA4) which could be dissociated upon addition of concanavalin A-specific sugars. The complex formation results in a time-dependent appearance of turbidity in the time range from 10s to 10 min. From the measurement of the time-dependent appearance and disappearance of the turbidity the formation (kf) and dissociation (kd) rate constants were calculated as 3 mM−1× s−1 and 0.07 ks−1 respectively. The ratio kf/kd (43μM −1), that corresponds to the association constant of complex RC1· ConA4, is higher than that of mannoside · ConA4 and thereby suggests that protein-protein interaction contributes significantly in stabilising glycoprotein · lectin complexes. The relevance of this finding to the understanding of the chemical specificities that are involved in a model cell-lectin interaction is discussed
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