Department of Biochemistry

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Browsing Department of Biochemistry by Subject "Affinity chromatography"
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    Affinity purification, physicochemical and immunological characterization of a galactose-specific lectin from the seeds of Dolichos lablab (Indian lablab beans)
    ( 2006-02-01) Lavanya Latha, Vakada ; Nagender Rao, Rameshwaram ; Nadimpalli, Siva Kumar
    A galactose-specific lectin has earlier been isolated from the seeds of Dolichos lablab in our laboratory by conventional protein purification methods. We now established conditions to bind the lectin on Sepharose-galactose gel in the presence of 1.5 M ammonium sulfate in Tris-buffered saline, pH 7.4. It can be specifically eluted with 0.3 M galactose. The purified lectin is a glycoprotein, binds to Con A, agglutinates erythrocytes, and has an apparent native molecular weight of 120 ± 5 kDa. In SDS-PAGE under reducing conditions, it dissociates into two subunits of molecular mass (Mr) 31 and 29 kDa. Among a number of sugars tested for inhibitory activity of the lectin, galactose was found to be a potent inhibitor. Rabbit polyclonal antibody to the purified lectin specifically reacted with the lectin subunits in Western blot analysis and additionally, an antibody raised to the isolated 31 kDa subunit show reactivity with both the subunits. Amino terminal sequences of both the subunits are identical. The purified lectin is stable up to 40°C with a pH optimum of 7.4. The lectin has a high content of acidic amino acids and lacks sulfur-containing amino acids. Chemical modification of the lectin with group-specific reagents indicates the possible role of histidine, lysine, and tyrosine residues in lectin activity. © 2005 Elsevier Inc. All rights reserved.
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    An efficient method for the purification and quantification of a galactose-specific lectin from vegetative tissues of Dolichos lablab
    ( 2008-01-15) Rameshwaram, Nagender Rao ; Nadimpalli, Siva Kumar
    The affinity purified galactose-specific seed lectin from Dolichos lablab, designated as DLL-II, is a tetrameric protein with an apparent native molecular mass of 120 kDa that is composed of two non-identical subunits of 31 and 29 kDa, respectively, associated non-covalently. The stems and leaves of the D. lablab plant also contain a galactose-specific lectin that cross-reacts with the seed lectin antiserum (antiserum raised against the 31 kDa subunit of DLL-II). Anti-lectin antibodies have been purified from this antiserum using a gel containing purified DLL-II lectin. Lectin specific antibodies have been used to develop simple and efficient immuno-affinity matrix, which allowed the purification of the lectin from stems and leaves of the D. lablab. The vegetative lectin (DLL-VL) exhibits similar electrophoretic properties as the seed lectin. Using these antibodies, an ELISA method was developed that allowed quantification of the lectin in the vegetative tissues (stems, leaves and roots) at concentrations of 0.5-50 ng. MS and database analysis of the tryptic peptides of the purified subunits of the DLL-VL suggested the purified protein to be a lectin. © 2007 Elsevier B.V. All rights reserved.
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    Evolution of mannose 6-phosphate receptors (MPR300 and 46): Lysosomal enzyme sorting proteins
    ( 2010-02-01) Nadimpalli, S. K. ; Amancha, P. K.
    Lysosomal enzymes undergo phosphorylation on their mannose residues in the Golgi apparatus and are recognized by two distinct type I transmembrane glycoproteins designated as the mannose 6-phosphate receptors; MPR300, (Mr 300 kDa) and MPR46, (Mr 46 kDa) that internally transport them to the lysosomes. In humans, absence of this recognition system leads to severe lysosomal storage disease, emphasizing their essential role in the biogenesis of lysosomes. Among the two receptors only MPR46 shows an absolute requirement for divalent metal ions. Only MPR300 is known to be a multifunctional protein that also binds many other ligands such as the human IGF-II, thyroglobulin, retinoic acid, granzyme A and B. In mammals, the extracytoplasmic domain of MPR300 protein is comprised of 15 repetitive cassettes which share significant similarity with each other and also with the single cassette that constitutes the extracytoplasmic domain of MPR46. Therefore it became necessary to understand the evolution of these receptors. Homologous proteins were affinity purified from different non-mammalian vertebrates such as birds, reptiles, amphibians, fish and also from the invertebrates, echinodermates (starfish) and molluscs (unio). Cloning and sequencing of both receptors from different mammals, chicken, fish and MPR46 from starfish revealed that these proteins exhibit similar structural domains as the mammalian receptors. α-fucosidase characterized from the molluscs exhibits specific interaction with the putative MPR300 protein from the same species. Available evidence suggests evolutionary conservation of both receptors from molluscs, as below these species no receptors that bind phosphomannan have been identified. © 2009 Bentham Science Publishers Ltd.
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    Mannose 6-phosphate receptor proteins from reptiles and amphibians: Evidence for the presence of MPR 300 and MPR 46
    ( 1997-01-01) Nadimpalli, Siva Kumar ; Hille-Rehfeld, Annette ; Von Figura, Kurt
    Two mannose 6-phosphate receptors (MPR 300 and MPR 46) are involved in transport of lysosomal enzymes. Both receptors are expressed in all mammalian species studied so far and in chicken. Here we present the first report on affinity purification of both MPRs from the liver tissues of reptiles and amphibians using Sepharose divinyl sulfone phosphomannan at pH 7.0. MPR 300 from both species show similar electrophoretic mobility as mammalian MPR 300 and cross-react with an antibody directed against MPR 300 from goat liver. Furthermore, MPR 46 from reptilian liver and amphibian oocytes cross-react with peptide-specific antibodies against the cytoplasmic domain of human MPR 46 (anti-MSC1).
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    Mannose 6-phosphate receptors (MPR 300 and MPR 46) from a teleostean fish (trout)
    ( 1999-01-01) Nadimpalli, Siva Kumar ; Yerramalla, Udaya Lakshmi ; Hille-Rehfeld, Annette ; Von Figura, Kurt
    Mannose 6-phosphate receptors (MPRs) are known to occur in mammals, birds, reptiles and amphibians. Here we provide evidence for the presence of two MPRs in fish, the earliest vertebrates. Using phosphomannan-Sepharose affinity chromatography, MPR 300 was purified from liver membrane extract of trout. The purified trout liver MPR 300 showed similar electrophoretic mobility as the goat liver receptor and a pH optimum of 7.0 for binding to phosphomannan. The presence of MPR 46 in fish was shown by metabolically labelling embryonic fish cells (Xiphophorus) and immunoprecipitation with an antibody against the cytoplasmic tail of human MPR 46 (anti-MSC1). This antibody had recently been shown to immunoprecipitate MPR 46 also from reptiles and amphibians. Copyright (C) 1999 Elsevier Science Inc.
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    Purification in high yield and characterisation of a new galactose, specific lectin from the seeds of Trichosanthes cucumerina
    ( 1998-02-10) Padma, P. ; Komath, Sneha Sudha ; Nadimpalli, Siva Kumar ; Swamy, Musti J.
    Ten Cucurbitaceae species have been investigated for the presence of seed lectins of which only two species, Trichosanthes cucumerina and T. palmata, have displayed agglutination activity which was inhibited by galactose. The lectin from T. cucumerina seeds has been purified in high yield (~350 mg lectin/100 g deshelled seeds) by affinity chromatography on cross-linked guar gum. The purified T. cucumerina seed lectin (TCSL) moved as a single symmetrical peak on gel filtration on Sephadex G-150 with an apparent molecular weight of 62 (±5) kDa and gave a single band on PAGE under non-denaturing conditions. In SDS-PAGE, TCSL gave a single band at 69 kDa in the absence of 2-mercaptoethanol, whereas in the presence of 2- mercaptoethanol two bands corresponding to 41 and 22 kDa were observed, suggesting that the lectin is made up of two nonidentical subunits that are linked by one or more disulphide bridges. TCSL is a glycoprotein with about 3.0% covalently bound neutral sugar. The lectin cross-reacted with rabbit antiserum raised against the Trichosanthes anguina (snake gourd) seed lectin (SGSL), yielding a single precipitin line and SGSL cross-reacted with the anti-TCSL antiserum raised in rabbits, indicating that the two lectins are antigenically very similar. This was further confirmed by Western blot analysis where the two subunits of TCSL were found to react with both anti- TCSL and anti-SGSL antisera and vice versa. On the other hand, TCSL did not cross-react with the antiserum raised against Momordica charantia lectin and vice versa, suggesting that these two cucurbit seed lectins are antigenically dissimilar. Haemagglutination-inhibition data show that TCSL is specific for the β-anomer of galactose with MeβGal and lactose being the best mono- and disaccharide inhibitors, respectively.
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    β-N acetylhexosaminidase from Dolichos lablab seeds: Purification, biochemical characterization and interaction studies with the Dolichos lablab lectins
    ( 2011-12-01) Gnanesh Kumar, B. S. ; Lavanya Latha, V. ; Siva Kumar, N.
    Seeds of the Dolichos lablab (Indian lablab beans) have been shown by us to contain two distinct lectins, viz., glucose/mannose specific lectin (DLL-I) and a galactose specific lectin(DLL-II) that have been well characterized, hi the present study we purified (β-N-acetylhexosaminidase from these seeds by using conventional protein purification followed by Con A-Sepharose chromatography. The purified enzyme is a glycoprotein and migrates as a single band on SDS-PAGE with an apparent molecular mass of ~60 kDa. Furthermore, when the protein bodies from the seeds were isolated, the enzyme was found to be located in the protein bodies together with the lectins and other glycosidases such as the a-mannosidase. The enzyme specifically binds onto DLL-I affigel at pH 5.0 and not at pH 8.0 and showed no such interaction with the DLL-II. The protein body membrane contains a protein of 97 kDa which might possibly represent a receptor for the lectin/enzyme.