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Two mannose-binding lectins, Allium sativum agglutinin (ASA) I (25 kDa) and ASAIII (48 kDa), from garlic bulbs have been purified by affinity chromatography followed by gel filtration. The subunit structures of these lectins are different, but they display similar sugar specificities. Both ASAI and ASAIII are made up of 12.5-and 11.5-kDa subunits. In addition, a complex (136 kDa) comprising a polypeptide chain of 54 ؎ 4 kDa and the subunits of ASAI and ASAIII elutes earlier than these lectins on gel filtration. The 54-kDa subunit is proven to be alliinase, which is known to form a complex with garlic lectins. Constituent subunits of ASAI and ASAIII exhibit the same sequence at their amino termini. ASAI and ASAIII recognize monosaccharides in mannosyl configuration. The potencies of the ligands for ASAs increase in the following order: mannobiose (Man␣1-3Man) < mannotriose (Man␣1-6Man␣1-3Man) Ϸ mannopentaose < < Man 9 -oligosaccharide. The addition of two GlcNAc residues at the reducing end of mannotriose or mannopentaose enhances their potencies significantly, whereas substitution of both ␣1-3-and ␣1-6-mannosyl residues of mannotriose with GlcNAc at the nonreducing end increases their activity only marginally. The best manno-oligosaccharide ligand is Man 9 GlcNAc 2 Asn, which bears several ␣1-2-linked mannose residues. Interaction with glycoproteins suggests that these lectins recognize internal mannose as well as bind to the core pentasaccharide of N-linked glycans even when it is sialylated. The strongest inhibitors are the high mannose-containing glycoproteins, which carry larger glycan chains. Indeed, invertase, which contains 85% of its mannose residues in species larger than Man 20 GlcNAc, exhibited the highest binding affinity. No other mannose-or mannose/glucose-binding lectin has been shown to display such a specificity.The majority of the well characterized plant lectins have been isolated from the seeds of dicotyledonous species. But lectins of non-seed origin from other species are also emerging as promising tools chiefly because of two reasons: (i) a good number of them might contain novel sugar-binding sites; and (ii) they can provide valuable information regarding the biological roles of plant lectins, which to a large extent still remain elusive. In the recent past, there have been several reports of non-seed lectins from monocotyledonous families (1-3), especially Amaryllidaceae. The most remarkable property of these lectins is that they show strict specificity for mannose (2, 4, 5), unlike other mannose/glucose-binding plant lectins. Hence, they are being used extensively as affinity ligands for the purification of glycoproteins, viz. IgM, ␣ 2 -macroglobulin, haptoglobin, and ␤-lipoprotein (3, 6).Van Damme et al. (3) examined a number of species (including Allium sativum) from the family Alliaceae (which is taxonomically close to the family Amaryllidaceae) and found them to accumulate mannose-binding lectins. They observed that lectins from both families share many common properties like their ...

DNA Topoisomerase I (TopoI) in eubacteria is the principle DNA relaxase, belonging to Type 1A group. The enzyme from Mycobacterium smegmatis is essential for cell survival and distinct from other eubacteria in having several unusual characteristics. To understand genome-wide TopoI engagements in vivo, functional sites were mapped by employing a poisonous variant of the enzyme and a newly discovered inhibitor, both of which arrest the enzyme activity after the first transestrification reaction, thereby leading to the accumulation of protein-DNA covalent complexes. The cleavage sites are subsets of TopoI binding sites, implying that TopoI recruitment does not necessarily lead to DNA cleavage in vivo. The cleavage protection conferred by nucleoid associated proteins in vitro suggest a similar possibility in vivo. Co-localization of binding and cleavage sites of the enzyme on transcription units, implying that both TopoI recruitment and function are associated with active transcription. Attenuation of the cleavage upon Rifampicin treatment confirms the close connection between transcription and TopoI action. Notably, TopoI is inactive upstream of the Transcription start site (TSS) and activated following transcription initiation. The binding of TopoI at the Ter region, and the DNA cleavage at the Ter indicates TopoI involvement in chromosome segregation, substantiated by its catenation and decatenation activities.

The genotoxic effects of the herbicide dicamba have been studied by measuring 1) the unwinding rate of liver DNA from intraperitoneally (i.p.) treated rats (fluorimetric assay); 2) DNA repair as unscheduled DNA synthesis (UDS) induced in cultured human peripheral blood lymphocytes (HPBL); and 3) sister chromatid exchanges (SCE) in HPBL. Results show that dicamba is capable of inducing DNA damage since it significantly increases the unwinding rate of rat liver DNA in vivo and also induces UDS in HPBL in vitro in the presence of exogenous metabolic activation (S-9 mix). Furthermore, dicamba causes a very slight increase in SCE frequency in HPBL in vitro.

The S protein-S peptide interaction is a model system to study binding thermodynamics in proteins. We substituted alanine at position 4 in S peptide by alpha-aminoisobutyric acid (Aib) to investigate the effect of this substitution on the conformation of free S peptide and on its binding to S protein. The thermodynamic consequences of this replacement were studied using isothermal titration calorimetry. The structures of the free and complexed peptides were studied using circular dichroic spectroscopy and X-ray crystallography, respectively. The alanine4Aib replacement stabilizes the free S peptide helix and does not perturb the tertiary structure of RNase S. Surprisingly, and in contrast to the wild-type S peptide, the DeltaG degrees of binding of peptide to S pro, over the temperature range 5-30 degrees C, is virtually independent of temperature. At 25 degrees C, the DeltaDeltaG degrees, DeltaDeltaH degrees, DeltaDeltaS and DeltaDeltaCp of binding are 0.7 kcal/mol, 2.8 kcal/mol, 6 kcal/mol x K and -60 kcal/mol x K, respectively. The positive value of DeltaDeltaS is probably due to a decrease in the entropy of uncomplexed alanine4Aib relative to the wild-type peptide. The positive value of DeltaDeltaH: degrees is unexpected and is probably due to favorable interactions formed in uncomplexed alanine4Aib. This study addresses the thermodynamic and structural consequences of a replacement of alanine by Aib both in the unfolded and complexed states in proteins.

؊1 lower than that of mannotriose. This indicates that, while Man␣3Man and Man␣6Man interact with the lectin exclusively through their nonreducing end monosaccharide with the subsites specific for the ␣1,3 and ␣1,6 arms, the mannotriose interacts with the lectin simultaneously through all three of its mannopyranosyl residues. This study thus underscores the distinction in the recognition of this common oligosaccharide motif in comparison with that displayed by other lectins with related specificity.Carbohydrates conjugated to proteins and lipids play key structural and functional roles in essentially all living organisms. Recognition of glycoconjugates is an important event in biological systems and is frequently in the form of carbohydrate-protein interactions. The study of how biological molecules interact with one another is fundamental to understanding the chemistry of life. Among the carbohydrate binding proteins, lectins are a group of proteins or glycoproteins which stereospecifically bind carbohydrates (1). N-Linked oligomannose-type carbohydrates constitute one class of oligosaccharide chains associated with cellular glycoproteins. The oligosaccharide chains of many of the glycoproteins appear to function as receptors for lectins in a variety of biological recognition processes, such as fertilization, embryogenesis, cell migration, organ formation, immune defense, protein folding, signal transduction, and apoptosis (2-4). Detailed insights into the specificity of carbohydrate-protein interactions, however, require not only analytical data such as inhibition assays but also thermodynamic data on the complexes. Titration microcalorimetry provides a powerful tool for investigating the binding thermodynamics of macromolecule-ligand interactions and provide important insights on the nature and magnitude of forces involved therein (5-11).Artocarpin, a mannose-specific lectin isolated from jack fruit seeds is a homotetrameric protein devoid of covalently attached carbohydrates and consists of four isolectins with pI in the range of 5-6.5. Artocarpin is of considerable interest because of its potent and selective mitogenic effect on distinct T and B-cell functions, more so because of its B-cell maturation mitogenic activity (12, 13). Earlier investigations of its carbohydrate binding specificity revealed that among monosaccharides, mannose is preferred over glucose. Among mannooligosaccharides, mannotriose (Man␣1-3[Man␣1-6]Man), and mannopentaose were noted as the strongest ligands followed by Man␣1-3Man. Substitution of both the ␣1-3 or ␣1-6 linked mannosyl residues of mannotriose by GlcNAc in ␤1-2 linkage diminishes their inhibitory potencies (9). In this investigation, isothermal titration calorimetry was employed to determine the thermodynamics of the carbohydrate-artocarpin binding reaction in terms of the binding constant (K b ) and change in the free energies, enthalpies, and entropies, i.e. ⌬G b 0 , ⌬H b 0 , and ⌬S b 0 . EXPERIMENTAL PROCEDURESMaterials and Sample Preparation-Glucose (Glc), Mannose (Man...

Jacalin and artocarpin, the two lectins from jackfruit (Artocarpus integrifolia) seeds, have different physicochemical properties and carbohydrate-binding specificities. However, comparison of the partial amino-acid sequence of artocarpin with the known sequence of jacalin indicates close to 50% sequence identity. Artocarpin crystallizes in two forms, both monoclinic P21, with one and two tetramic molecules, respectively, in the asymmetric units of form I (a = 69.9, b--73.7, c=60.6A and fl=95.1 °) and form II (a=87.6, b = 72.2, c = 92.6 .& and fl = 101.1°). Both the crystal structures have been solved by the molecular replacement method using the known structure ofjacalin as the search model and one of them partially refined, confirming that the two lectins are indeed homologous.