scholarly journals Some properties of the lectin from Datura stramonium (thorn-apple) and the nature of its glycoprotein linkages

1981 ◽  
Vol 197 (2) ◽  
pp. 345-353 ◽  
Author(s):  
Nila N. Desai ◽  
Anthony K. Allen ◽  
Albert Neuberger
Keyword(s):  
Amino Acids ◽  
Polypeptide Chain ◽  
Gel Filtration ◽  
Datura Stramonium ◽  
Inhibitory Effect ◽  
Plant Cell Walls ◽  
Thorn Apple ◽  
A Subunit ◽  
Sepharose 4B ◽  
Derivatives Of

The lectin from Datura stramonium (thorn-apple; Solanaceae) has been purified by affinity chromatography and shown to be a glycoprotein containing about 40% (w/w) of carbohydrate. The most abundant amino acids are hydroxyproline, cystine, glycine and serine. Results obtained by gel filtration in 6m-guanidinium chloride on Sepharose 4B suggest that it has a subunit mol.wt. of about 30000 and that it probably associates into dimers. The lectin is inhibited specifically by chitin oligosaccharides and bacterial-cell-wall oligosaccharides, but only weakly by N-acetylglucosamine. Glycopeptides from soya-bean (Glycine max) lectin and fetuin are also strong inhibitors of Datura lectin, indicating that it interacts with internal N-acetylglucosamine residues. Its specificity is similar to, but not identical with, that of potato (Solanum tuberosum) lectin. After prolonged proteolytic digestion of reduced and S-carboxymethylated or S-aminoethylated derivatives of the lectin, glycopeptides of mol.wt. of about 18000 were isolated. The glycopeptides contained all the carbohydrate and hydroxyproline of the original glycoprotein, and lesser amounts of serine, S-carboxymethylcysteine and other amino acids. The arabinose residues of the glycoprotein are present as β-l-arabinofuranosides linked to the polypeptide chain through the hydroxyproline residues, and can be removed by mild acid treatment; the ratio of arabinose to hydroxyproline is 3.4:1. Some of the serine residues of the polypeptide chain are substituted with one or two α-galactopyranoside residues, most of which can be removed by the action of α-galactosidase. The galactose residues are more easily removed from the acid-treated glycopeptide (from which arabinose has been removed) than from the complete glycopeptide, indicating a steric hindrance of the galactosidase action by the adjacent chains of arabinosides. There is a slow release of galactose residues by a process of β-elimination in 0.5m-NaOH (pH13.7) from the complete glycopeptide, and a fairly rapid release of galactose by this process from the acid-treated glycopeptide, which lacks arabinose. This is probably due to the inhibitory effect of the negative charge on the adjacent arabinofuranoside residues. The similarities and differences between the lectins from Datura and potato are discussed, as are their structural resemblance to glycopeptides that have been isolated from plant cell walls.

scholarly journals Structural studies of the carbohydrate moieties of lectins from potato (Solanum tuberosum) tubers and thorn-apple (Datura stramonium) seeds

1982 ◽  
Vol 201 (1) ◽  
pp. 199-208 ◽  
Author(s):  
David Ashford ◽  
Nila N. Desai ◽  
Anthony K. Allen ◽  
Albert Neuberger ◽  
Malcolm A. O'Neill ◽  
...  
Keyword(s):  
Solanum Tuberosum ◽  
Chemical Ionization ◽  
Optical Rotation ◽  
Gel Filtration ◽  
Datura Stramonium ◽  
Plant Cell Walls ◽  
Methylation Analysis ◽  
Thorn Apple ◽  
Optical Rotations ◽  
Potato Lectin

1. Methylation analysis of potato (Solanum tuberosum) lectin and thorn-apple (Datura stramonium) lectin confirmed previous conclusions that both glycoproteins contained high proportions of l-arabinofuranosides and lesser amounts of d-galactopyranosides. The arabinofuranosides are present in both lectins as short unbranched chains containing 1→2- and 1→3-linkages, which are known to be linked to hydroxyproline. Galactopyranosides are present as monosaccharides, which are known to be attached to serine, in potato lectin and as both the monosaccharide and the 1→3-linked disaccharide in Datura lectin. 2. Alkaline digestion of potato lectin and subsequent separation of the components by gel filtration led to the isolation of four fractions corresponding to the mono-, di-, tri- and tetra-arabinosides of hydroxyproline. The latter two fractions accounted for over 70% of the total hydroxyproline. 3. Methylation analysis was used to show that the triarabinoside contained only 1→2-linkages between sugars, but that the tetra-arabinoside contained both 1→2- and 1→3-linkages. Direct-insertion mass spectrometry of these compounds using electron impact and chemical ionization, in a comparison with other known structural patterns, was used to determine the sequences of the sugars, which were Araƒ1→2Araƒ1→2Araƒ1→Hyp and Araƒ1→3Araƒ1→2Araƒ1→2Araƒ 1→Hyp. 4. On the basis of optical rotation it had previously been suggested [Allen, Desai, Neuberger & Creeth (1978) Biochem. J.171, 665–674] that all the arabinose of potato lectin was present as the β-l-furanoside. However, measurement of the optical rotations of the hydroxyprolyl arabinosides showed that whereas the diarabinoside had a molar rotation ([m]) value close to that predicted, the triarabinoside was more dextrorotatory and the tetra-arabinoside was less dextrorotatory than expected. Possible explanations for these findings are that, although the di- and tri-arabinosides contain exclusively β-arabinofuranosides, in the tri-arabinoside, interactions between pentose units lead to an enhanced positive rotation. The tetra-arabinoside, however, is proposed to contain a single α-arabinofuranoside residue, which is responsible for the lower than expected positive rotation. The observed rotation of the tetra-arabinoside was found to be close to the theoretical value predicted on that basis. Furthermore, the action of a specific α-arabinofuranosidase on the tetrasaccharide was to remove a single arabinose residue, presumably the terminal non-reducing sugar, and to produce a product that was indistinguishable on electrophoresis from the triarabinoside. Changes in rotation were compatible with this assumption. 5. It is concluded that the structures of the hydroxyprolyl tri- and tetra-arabinosides of potato lectin are: βAraƒ1→2βAraƒ1→2βAraƒ1→Hyp and αAraƒ1→3βAraƒ1→2βAraƒ 1→2βAraƒ1→Hyp. These are identical with compounds that have been isolated from the insoluble hydroxyproline-rich glycoproteins of plant cell walls.

scholarly journals Easy assembly of ligands for glycosidase affinity chromatography

1990 ◽  
Vol 270 (2) ◽  
pp. 539-540 ◽  
Author(s):  
R C Bernotas ◽  
B Ganem
Keyword(s):  
Amino Acids ◽  
Affinity Chromatography ◽  
Enzyme Purification ◽  
Reductive Amination ◽  
Water Soluble ◽  
High Yield ◽  
Glycosidase Inhibitors ◽  
Neutral Ph ◽  
Sepharose 4B ◽  
Derivatives Of

An improved, high-yield synthesis of the corresponding N-carboxypentyl derivatives of three iminoalditol glycosidase inhibitors has been developed for affinity chromatography enzyme purification. Reductive amination of 1-deoxynojirimycin (or its D-manno or D-galacto analogues) with methyl 5-formylvalerate and NaBH3CN at neutral pH afforted an aminoester which upon hydrolysis with aqueous 5% HCl gave the desired aminoacid in 97% overall yield. These amino acids could then be covalently attached using water-soluble carbodi-imide to 6-aminohexyl Sepharose 4B.

STUDIES ON THE INTERACTION OF LONG-ACTING THYROID STIMULATOR (LATS) WITH SOLUBLE THYROID FRACTIONS

1971 ◽  
Vol 68 (4) ◽  
pp. 625-644 ◽  
Author(s):  
N. Amino ◽  
K. Miyai ◽  
M. Azukizawa ◽  
Y. Kumahara
Keyword(s):  
Inhibitory Activity ◽  
Acid Treatment ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Inhibitory Effect ◽  
Human Thyroid ◽  
Thyroid Cell ◽  
Long Acting ◽  
Sepharose 4B

ABSTRACT The specificity, stability and reversibility of the in vitro interaction of LATS with soluble human thyroid fractions was studied. With regard to tissue specificity, the cell sap obtained from human liver, spleen, kidney, and muscle did not inhibit the LATS activity while the same amount of thyroid cell sap significantly inhibited it. When the LATS inhibitory activity in thyroid subcellular fractions was compared, the microsomal fraction was more active than cell sap or solubilized microsomes in terms of milligram of protein, but the cell sap had considerable activity as based on the original thyroid weight. Lyophilization of cell sap did not reduce the LATS inhibitory activity, but treatment with 2 m NaSCN and 6 m urea apparently destroyed this capacity. Acid treatment of cell sap at pH 2.5 and at 3.0 completely destroyed its ability to inhibit LATS activity. Inhibition of LATS activity was roughly proportional to the amount of thyroid cell sap. Human TSH, on the other hand, was not inhibited by cell sap which had a significant inhibitory effect on LATS. LATS activity was more effectively inhibited when a mixture of LATS-IgG and thyroid cell sap was incubated for 96 hours than for 12 hours. The inhibition of LATS activity by thyroid cell sap was partially but significantly reversed by acid treatment, as observed in experiment using microsomes. When thyroid cell sap was fractionated by gel filtration on Sepharose 4B, LATS inhibitory activity was distributed in all the fractions including the 27S to 4S proteins. In DEAE-cellulose column chromatography, LATS inhibitory activity tended to be eluted at a higher ionic strength. In each fraction of Sepharose 4B and DEAE-cellulose, LATS inhibitory activity was found to be unrelated to the thyroglobulin content. It is believed that the inhibition of LATS activity by thyroid cell sap is compatible with an antigen-antibody reaction and that the LATS inhibitor may not be a thyroglobulin itself but a more negatively charged heterogeneous substance.

scholarly journals The amino acid sequence of dromedary pancreatic ribonuclease.

1975 ◽  
Vol 147 (3) ◽  
pp. 505-511 ◽  
Author(s):  
G W Welling ◽  
G Groen ◽  
J J Beintema
Keyword(s):  
Amino Acids ◽  
Polypeptide Chain ◽  
Proteolytic Enzymes ◽  
Gel Filtration ◽  
Paper Electrophoresis ◽  
Pancreatic Tissue ◽  
British Library ◽  
Peptide Bonds ◽  
Pancreatic Ribonuclease ◽  
External Loop

Dromedary (Camelus dromedarius) RNAase (ribonuclease) was isolated from pancreatic tissue by affinity chromatography. Peptides obtained by digestion with different proteolytic enzymes and CNBr were isolated by gel filtration, preparative high-voltage paper electrophoresis and paper chromatography. Peptides were sequenced by the dansyl-Edman method. All peptide bonds were overlapped by one or more peptides. The polypeptide chain consists of 123 amino acids. A deletion (position 39) was observed in an external loop of the polypeptide chain (residues 35-40), as was found earlier to horse RNAase (Scheffer & Beintema, 1974). A heterogeneity was found at position 103 (glutamine and lysine). Dromedary RNAase differs at 23-32% of the positions from all other pancreatic RNAases sequenced to date. In evolutionary terms this indicates that dromedary RNAase has evolved independently during the larger part of the evolution of the mammals. Detailed evidence for the sequence has been deposited as Supplementary Publication SUP 50046 (14 pages) at the British Library (Lending Division), Boston Spa, Wetherby, W. Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1975) 145, 5.

scholarly journals Dissociation of fibronectin from gelatin-agarose by amino compounds

1978 ◽  
Vol 175 (1) ◽  
pp. 333-336 ◽  
Author(s):  
M Vuento ◽  
A Vaheri
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Human Plasma ◽  
Gel Filtration ◽  
Basic Amino Acid ◽  
Amino Sugars ◽  
Neutral Ph ◽  
Acidic Amino Acids ◽  
Amino Compounds ◽  
Derivatives Of

Soluble fibronectin of human plasma was specifically dissociated at neutral pH from gelatin-agarose by several cationic amino compounds, notably the polyamines spermine, spermidine and putrescine, the basic amino acid arginine, and amino sugars. The neutral and acidic amino acids and the N-acetylated derivatives of amino sugars tested were ineffective. Gel-filtration experiments demonstrated that [14C]spermidine bound to fibronectin but not to gelatin.

scholarly journals Purification of an exo-β-D-glucanase from cell-free extracts of Candida utilis

1976 ◽  
Vol 159 (3) ◽  
pp. 555-562 ◽  
Author(s):  
V Notario ◽  
T G Villa ◽  
J R Villanueva
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Gel Electrophoresis ◽  
Candida Utilis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Acidic Amino Acids ◽  
Sepharose 4B ◽  
Hydrolysis Of

β-Glucanase present in cell-free extracts from Candida utilis was isolated and purified 562-fold by procedures that include adsorption on DEAE-Sephadex A-50 and filtration through columns of Sephadex G-50, G-100 and G-200, Bio-Gel P-10, and Concanavalin A-Sepharose 4B. The purified enzyme appeared homogeneous on polyacrylamide-gel electrophoresis and in ultracentrifugation studies (S20,w = 1.74S). The enzyme behaved as an acidic glycoprotein (pI4.1) with 68% carbohydrate and a high content of acidic amino acids. The mol.wt. was estimated to be 20000 from gel filtration and polyacrylamide-gel electrophoresis and 36000 from sedimentation experiments. Studies on the hydrolysis of different substrates showed that the enzyme is an unspecific β-glucanase able to break down both (1 leads to 3)-eta- and (1 leads to 6)-β-linkages by an exo-splitting mechanism. Glucono-δ-lactone, Zn2+ and Hg2+ inhibited the enzyme activity.

FORMATION OF UREA IN LIVER HOMOGENATES FROM CITRULLINE AND VARIOUS N-ALKYL DERIVATIVES OF ASPARTIG ACID

1963 ◽  
Vol 41 (1) ◽  
pp. 2297-2305 ◽  
Author(s):  
R. Charbonneau ◽  
L. Berlinguet
Keyword(s):  
Amino Acids ◽  
Carboxylic Acid ◽  
Aspartic Acid ◽  
Metabolic Pathways ◽  
Inhibitory Effect ◽  
Urea Synthesis ◽  
Alkyl Derivatives ◽  
Amino Acid Analogues ◽  
Liver Homogenates ◽  
Derivatives Of

The effects of various N-alkylated derivatives of aspartic acid on the synthesis of urea by rat liver homogenates have been studied. At 5 × 10−3 M concentration, N-methyl, N-ethyl, N-isopropyl, and N-cyclohexyl aspartic acids are not utilized and have no effect on the formation of urea. At this concentration, N-allyl-DL-aspartic acid inhibits the formation of endogenous urea by 77%. At concentrations of 2.5 to 7.5 × 10−2 M, N-methyl, N-ethyl, and N-isopropyl aspartic acids slightly increase the formation of endogenous urea; this is about 15% of the value obtained when aspartic acid alone is added at the same concentration. In the case of simple N-alkylated aspartic acids, liver homogenates are able to cleave the alkylated chain with the result that a small amount of urea synthesis is possible. N-allylaspartic acid totally inhibits the formation of urea from aspartic acid at a relatively low concentration of 6.2 × 10−3 M. N-cyclohexylaspartic acid has also an inhibitory effect which is ten times less pronounced than that of the N-allyl derivative.Natural amino acids such as DL- and L-valine, DL- and L-leucine, DL- and L-lysine, DL-alanine and glycine, at concentrations of 1.2 to 5 × 10−2 M, also inhibit the formation of urea. This inhibition is probably due to the fact that other metabolic pathways, used by these amino acids, have priority over the formation of urea. Amino acid analogues, such as 1-aminocyclopentane carboxylic acid and 1-amino-2-methylcyclopentane-carboxylic acid, do not have any effect on the synthesis of urea.A free amino group in the aspartic acid molecule seems to be essential for the synthesis of argininosuccinic acid.

scholarly journals Navy (haricot)-bean (Phaseolus vulgaris) lectin. Isolation and characterization of two components from a toxic agglutinating extract

1974 ◽  
Vol 139 (2) ◽  
pp. 421-429 ◽  
Author(s):  
A. T. Andrews
Keyword(s):  
Amino Acids ◽  
Phaseolus Vulgaris ◽  
Aspartic Acid ◽  
Polypeptide Chain ◽  
Isolation And Characterization ◽  
Tetrameric Structure ◽  
A Subunit ◽  
Terminal Amino ◽  
Protein Components

Two protein components were isolated in a highly purified state from a toxic fraction from the navy (haricot) bean (Phaseolus vulgaris). One, a lectin, strongly agglutinated horse erythrocytes and leucocytes, agglutination being readily observable with both types of cell at a lectin concentration of 4μg/ml. The other component (component 1), although possessing some similarity in composition, was thought to be non-agglutinating or, at most, only very weakly so. Component 1 had a mol.wt. of about 143000 and a subunit mol.wt. of about 37000, suggesting a tetrameric structure probably with identical subunits. Alanine was the only N-terminal amino acid identified and the molecule was notable in being devoid of tryptophan and cysteine, low in methionine and high in leucine, glutamic acid and aspartic acid. The lectin was somewhat smaller (mol.wt. about 114000) and apparently also composed of four identical subunits of mol.wt. about 30000. Dansylation showed that arginine occupied the N-terminus of the polypeptide chain. Aspartic acid, serine, threonine and leucine were the predominant amino acids of the lectin, and the sulphur-containing amino acids were entirely absent. Both constituents were glycoproteins and the compositions of the carbohydrate portions (4.9% for component 1 and 8.1% for the lectin) were generally similar, consisting of mannose and glucosamine with smaller amounts of glucose and traces of xylose and arabinose.

scholarly journals Analysis of Soluble Fibrin Complexes in Patients with Intra Vascular Coagulation

1977 ◽  
Author(s):  
W. Edgar ◽  
C. McKillop ◽  
P. W. Howie ◽  
C. D. Forbes ◽  
C. R. M. Prentice
Keyword(s):  
Major Part ◽  
Polypeptide Chain ◽  
Gel Filtration ◽  
Chain Structure ◽  
Agarose Gel ◽  
Soluble Fibrin ◽  
Α Chain ◽  
Sepharose 4B ◽  
Soluble Complexes

We have analysed the polypeptide chain structure of soluble fibrin complexes in patients with pre-eclampsia and during defibrination with ancrod (Arvin). In pre-eclampsia soluble fibrin measured by fibrinogen-sepharose 4B chromatography was increased in comparison with normal pregnant women. The isolated fibrin contained intact α, β and δ chains but no crosslihked γ chains. A correlation (r = 0.959 P <0.001) was found between the concentration of soluble fibrin and soluble complexes as measured by agarose gel filtration, suggesting these complexes consist mainly of fibrin-fibrinogen dimers. In vitro, the binding of soluble fibrin to fibrinogen-sepharose depended on the structure of the complexes and temperature. At 37°C intact fibrin, prepared with ancrod, was bound to the column, but fibrin lacking intact α chain could be eluted at 37°C. The soluble fibrin complexes in patients having therapy with ancrod could be separated into two;the greater part had reduced α chain and were eluted from the column at 3 7°C, whereas the smaller part had intact α chain and remained bound at 37°C. Bio-Gel A5m chromatography carried out at 37°C and 20°C also indicated that the major part of soluble complexes produced during ancrod defibrination were unstable at 37°C.

FORMATION OF UREA IN LIVER HOMOGENATES FROM CITRULLINE AND VARIOUS N-ALKYL DERIVATIVES OF ASPARTIG ACID

1963 ◽  
Vol 41 (11) ◽  
pp. 2297-2305 ◽  
Author(s):  
R. Charbonneau ◽  
L. Berlinguet
Keyword(s):  
Amino Acids ◽  
Carboxylic Acid ◽  
Aspartic Acid ◽  
Metabolic Pathways ◽  
Inhibitory Effect ◽  
Urea Synthesis ◽  
Alkyl Derivatives ◽  
Amino Acid Analogues ◽  
Liver Homogenates ◽  
Derivatives Of

The effects of various N-alkylated derivatives of aspartic acid on the synthesis of urea by rat liver homogenates have been studied. At 5 × 10−3 M concentration, N-methyl, N-ethyl, N-isopropyl, and N-cyclohexyl aspartic acids are not utilized and have no effect on the formation of urea. At this concentration, N-allyl-DL-aspartic acid inhibits the formation of endogenous urea by 77%. At concentrations of 2.5 to 7.5 × 10−2 M, N-methyl, N-ethyl, and N-isopropyl aspartic acids slightly increase the formation of endogenous urea; this is about 15% of the value obtained when aspartic acid alone is added at the same concentration. In the case of simple N-alkylated aspartic acids, liver homogenates are able to cleave the alkylated chain with the result that a small amount of urea synthesis is possible. N-allylaspartic acid totally inhibits the formation of urea from aspartic acid at a relatively low concentration of 6.2 × 10−3 M. N-cyclohexylaspartic acid has also an inhibitory effect which is ten times less pronounced than that of the N-allyl derivative.Natural amino acids such as DL- and L-valine, DL- and L-leucine, DL- and L-lysine, DL-alanine and glycine, at concentrations of 1.2 to 5 × 10−2 M, also inhibit the formation of urea. This inhibition is probably due to the fact that other metabolic pathways, used by these amino acids, have priority over the formation of urea. Amino acid analogues, such as 1-aminocyclopentane carboxylic acid and 1-amino-2-methylcyclopentane-carboxylic acid, do not have any effect on the synthesis of urea.A free amino group in the aspartic acid molecule seems to be essential for the synthesis of argininosuccinic acid.

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