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D D Roberts

Publications and source records attributed to D D Roberts.

103 records · Page 6Linked to original sources
PubMed1984

Isolation from lima bean lectin of a peptide containing a cysteine residue essential for carbohydrate binding activity.

The location and amino acid sequence surrounding a cysteine residue required for carbohydrate binding in the lima bean lectin (LBL) was determined. Following selective conversion of the sulfhydryl group to its S-cyano derivative, LBL was cleaved at the essential cysteine residue to give two fragments, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in two buffer systems to have molecular masses of 16.5-19 kDa and 10.5-11 kDa. The larger fragment, which contained the glycosyl moiety of the lectin, was shown by sequence analysis to contain the NH2-terminal sequence of LBL. The smaller COOH-terminal fragment was found to contain the cysteine residue involved in the intersubunit disulfide bond of LBL. Digestion of LBL with pepsin and trypsin yielded four peptides containing the essential cysteine. Sequencing of the three major peptides gave a single consensus sequence, Val-Glu-Phe-Asp-Thr-Cys-His-Asn-Leu-Asp-, for the primary sequence surrounding the cysteine. The peptide sequence and site of cyanylation cleavage were used to predict alignment of the LBL peptide with the primary sequence of concanavalin A. Maximum homology was found with a sequence in concanavalin A beginning at valine 7. Implications of this alignment to the function of the cysteine in carbohydrate and metal ion binding of LBL, and for conservation of carbohydrate binding site residues in legume lectins are discussed.

Amino Acid Sequence↗
PubMed1984

Reexamination of the carbohydrate binding stoichiometry of lima bean lectin.

The carbohydrate binding stoichiometry of lima bean lectin component III was reexamined using equilibrium dialysis and quantitative affinity chromatography following limited chemical modification. Equilibrium dialysis employing methyl[2-14C]benzamido-2-deoxy-alpha-D-galactopyranoside as ligand demonstrated that the lectin tetramer bound 4 mol of sugar with Kassoc = 1.44 +/- 0.13 X 10(3) M-1 (T = 5 degrees C, pH 7.0, ionic strength 0.1). The previous report of two sites/tetramer [Bessler, W. and Goldstein, I. J. (1974) Arch. Biochem. Biophys. 165, 444] appears to be the result of partial inactivation of the lectin due to oxidation of essential thiol groups. Following limited chemical modification of the thiol groups by methyl methanethiosulfonate, multiple intermediate forms with reduced affinity for Synsorb A were obtained. The number and hemagglutinating activities of these intermediates provided further support for the presence of four carbohydrate binding sites on lima bean lectin component III.

Binding Sites↗
PubMed1983

Adenine binding sites of the lectin from lima beans (Phaseolus lunatus).

A single high-affinity binding site for adenine and related compounds was identified in the lima bean lectin (LBL) component III tetramer. This site is identical with the high affinity site for 2,6-toludinyl-naphthalenesulfonate described previously (Roberts, D. D., and Goldstein, I. J. (1982) J. Biol. Chem. 257, 11274-11277). [14C]Adenine was bound with high affinity (Kd = 1.2 +/- 0.1 X 10(-5) M, T = 25 degrees C) and a high degree of specificity in that hypoxanthine and guanine were very poor ligands for this site. Specificity was also observed for free purine bases relative to nucleosides or nucleotides. A number of N6 derivatives of adenine with cytokinin activity were found to bind to LBL, with relative affinities decreasing in the order: N6 - benzyladenine greater than kinetin greater than zeatin greater than N6 - [delta 2-isopentenyl]adenine greater than dihydrozeatin greater than zeatin riboside. Evidence was also obtained for heterotropic interaction between the adenine binding site and a second class of hydrophobic sites present on each subunit of LBL. Binding of adenine and N6-benzyladenine to LBL was found to produce a 2.3- and 3.8-fold increase, respectively, in the affinity of the lectin subunit hydrophobic sites for 1,8-anilinonaphthalenesulfonate. 1,8-Anilinonaphthalenesulfonate, in turn, enhanced the affinity of LBL for adenine, demonstrating that binding of ligands to the two classes of hydrophobic sites is thermodynamically linked. Equilibrium dialysis also revealed high affinity binding sites for [14C]adenine on the lectins from Dolichos biflorus, Phaseolus vulgaris, and soybean (Glycine max).

Adenine↗
PubMed1983

Binding of hydrophobic ligands to plant lectins: titration with arylaminonaphthalenesulfonates.

Binding of the hydrophobic ligands 1,8-anilinonaphthalenesulfonic acid (ANS) and 2,6-toluidinylnaphthalenesulfonic acid (TNS) to a variety of plant lectins was studied by lectin-induced alteration of the fluorescence spectra of the two ligands. With one exception, all legume lectins examined bound ANS, with affinity constants ranging from 10(3) to 10(4) M-1. Similar ANS binding was noted for some nonlegume lectins. Titration of the five isolectins from Phaseolus vulgaris with ANS indicated positive cooperative binding of ANS to the two isolectins E4 and E3L1. Titrations with TNS revealed high-affinity sites for this ligand in a number of lectins. Addition of haptenic sugars did not inhibit binding of ANS, suggesting that the hydrophobic binding sites of lectins are independent of the carbohydrate binding sites.

Anilino Naphthalenesulfonates↗
PubMed1982

Hydrophobic binding properties of the lectin from lima beans (Phaseolus lunatus).

Hydrophobic binding to the lectin from lima beans (Phaseolus lunatus) was studied by lectin-induced alterations in the fluorescence and absorption spectra of several hydrophobic ligands. The fluorescence of 1,8-anilinonaphthalenesulfonic acid (ANS) was greatly enhanced in the presence of lima bean lectin with concomitant shift of the fluorescence emission maximum from 520 to 469 nm. Similar enhancement was seen with 2,6-toluidinylnaphthalenesulfonic acid (TNS) with a shift of emission from 500 to 423 nm, and with an uncharged analogue, N-phenyl-1-naphthylamine. Fluorescence titrations with ANS and rose bengal yielded affinity constants of 3.9 X 10(3) and 6 X 10(5) M-1, respectively. Fluorescence titration with TNS indicated binding heterogeneity and yielded intrinsic association constants of 7.8 X 10(4) and 2.2 X 10(3) M-1 assuming a model with two classes of independent sites. The high affinity binding had an apparent stoichiometry of 1.08 sites/lectin tetramer. Equilibrium dialysis for ANS and TNS confirmed the results of fluorescence titration and gave stoichiometries for ANS and low affinity TNS binding of one site/subunit. Neither chemical modification of thiol groups known to be essential for carbohydrate binding nor titration in the presence of haptenic sugar affected the binding of ANS or TNS to the lectin. These results indicated that the carbohydrate and hydrophobic binding sites of lima bean lectin are independent and noninteracting.

Anilino Naphthalenesulfonates↗
PubMed1982

Subunit heterogeneity in the lima bean lectin.

Three forms of lectin (components I, II, and III) from lima beans (Phaseolus lunatus) have been purified on an affinity support containing the synthetic type A blood group trisaccharide alpha-D-GalNAc-(1 leads to 3)-[alpha-L-Fuc-(1 leads to 2)]-beta-D-Gal-(1 leads to). Conversion of components I and II to component III has been achieved by reduction in 10(-2) M dithiothreitol. Isoelectric focusing of lima bean lectin in the presence of 8 M urea and beta-mercaptoethanol revealed charge heterogeneity of the lectin subunits. Three major subunit classes of apparent pI 7.05, 6.65, and 6.45, designated alpha, beta, and alpha', respectively, were identified; they occur in a relative abundance of 2:5:3. Green lima beans harvested before maturity lacked the alpha' subunit (pI 6.45) which appears to accumulate during seed maturation. The three subunits are glycoproteins of identical size and immunochemical reactivity. Identical NH2-terminal sequences were found for the three subunits. Amino acid analysis and tryptic peptide mapping indicated that the observed charge heterogeneity is probably due to differences in the primary structure of the subunits. Studies of subunit composition of charge isolectins provided evidence of nonrandom subunit assembly. A model is proposed involving pairing of a pI 6.65 subunit with either a pI 7.06 or 6.45 subunit to form dimeric units. Possible roles for subunit heterogeneity and ordered subunit assembly in determining the metal and sugar binding properties of lima bean lectin are discussed.

Amino Acid Sequence↗
PubMedDate not supplied

Thrombospondin binds falciparum malaria parasitized erythrocytes and may mediate cytoadherence.

Plasmodium falciparum infected erythrocytes containing mature trophozoites and schizonts sequester along venular endothelium and are not in the peripheral circulation of patients with malaria. Knobs appear on infected erythrocytes and are the points of attachment to endothelium. Sequestration may protect the parasite from splenic destruction and may play a role in the pathogenesis of cerebral malaria. Correlates of sequestration have been developed in vitro using cultured human endothelium and an amelanotic melanoma cell line. Knobless strains (K-) of P. falciparum fail to sequester in vivo and to bind to cells in vitro. We now present evidence that the receptor for cytoadherence is the glycoprotein, thrombospondin. Aotus monkey or human erythrocytes containing knobby (K+) but not Aotus erythrocytes containing knobless strains of P. falciparum bind to immobilized thrombospondin. Neither binds to the adhesive proteins laminin, fibronectin, factor VIII/von Willebrand factor or vitronectin. Both soluble thrombospondin and anti-thrombospondin antibodies inhibit binding of parasitized Aotus erythrocytes to immobilize thrombospondin and to melanoma cells which secrete thrombospondin.

Animals↗