X-ray fibre diffraction study of conformational changes in hyaluronate induced in the presence of sodium,potassium and calcium cations

Hyaluronate purified from all cations by ion exchange chromatography was introduced to the cations sodium, potassium and calcium in a controlled way. The conformations formed in the presence of these ions were studied as a function of ionic strength, hydrogen ion activity, humidity and temperature using X-ray fibre diffraction. In sodium hyaluronate above pH 4.0 a contracted helix is found which approximates to a four-fold helix with an axial rise per disaccharide of 0.84 nm. There is no requirement for water molecules in the unit cell as the Na⁺ can be coordinate by the hyaluronate chains alone. On crystallizing hyaluronate below pH 4.0 an extended 2-fold helix with an axial rise per disaccharide of 0.98 nm is formed. In the presence of potassium above pH 4.0 a conformation similar, but not identical, to that of sodium was found where the helix backbone is again four-fold with an axial rise per disaccharide h=0.90 nm. To maintain the coordination of the potassium ion, four water molecule/disaccharide are required and on removal of these the conformation is destabilized going to a new helix where n = 4 and h = 0.97 nm. Below pH 4.0 the conformation is a contracted 4-fold helix with h = 0.82 nm. In this structure two antiparallel chains intertwine to form a double helix. The packing of the double helical units is stabilized by water molecules, the unit cell requiring 8 water molecules/disaccharide. Formation of the calcium hyaluronate complex above pH 3.5 yields a three-fold helix with h = 0.95 nm. The requirement for water in the unit cell to maintain full crystallinity is high, at 9 water molecules/disaccharide; however, on removal of this water, though the crystallinity is disrupted, the conformation remains constant. The acid form of calcium-hyaluronate yields an equivalent conformation to that of sodium under the same condition, i.e. a helix with n = 2, h = 0.98 nm. The presence of small quantities of calcium in what are otherwise potassium or sodium solutions of hyaluronate yield the 3-fold conformation for hyaluronate. Thus calcium has an important role to play in deciding the dominating conformation present in hyaluronate. The variety of conformations yielded by the different cations indicates a subtle interaction between hyaluronate and its environment, in which the balance between the cations will control to some degree the interactions between hyaluronate chains and thus affect the mechanical properties of the matrix which they form. The conformations of individual chains are all stabilized in varying degrees by intra-chain hydrogen bonds.     

Electron microscopy of alpha-latrotoxin from the venom of the black widow spider Latrodectus mactans tredecimguttatus]

Two-dimensional crystals of alpha-latrotoxin from the venom of black widow spider (Latrodectus mactans tredecimguttatus) were studied by the negative staining electron microscopy. Two-dimensional crystals were obtained by adsorption of the protein solution with a high Mg2+ concentration on carbon-coated electron microscopy grids. The crystals were about 0.4 mkm in size, had the unit cell parameters: a = b = 15.55 nm, gamma = 90 degrees, p4 plane group symmetry. The contour map of a stain-excluding region of such crystals was calculated by the Fourier-filtering procedure at about 4 nm resolution. The calculation of molecular weight of the unit cell, with the symmetry p4 taken into account, showed that alpha-latrotoxin particles, revealed by negative staining, consisted of 4 or 8 protomers.     

Polymer length of teichuronic acid released from cell walls of Micrococcus luteus.

Teichuronic acid released from its phosphodiester linkage to peptidoglycan in the cell walls of Micrococcus luteus by mild acid treatment is resolved into a ladderlike series of bands by electrophoresis on polyacrylamide gels in the presence of borate. Each band of the ladder differs from its nearest neighbor by one disaccharide repeat unit, ----4)-2-acetamido-2-deoxy-beta-D-mannopyranuronosyl-(1----6)- alpha-D-glucopyranosyl-(1-. Acid-fragmented teichuronic acid, after conversion to the phenylamine derivative, was fractionated by preparative-scale molecular sieve column chromatography, which produced a series of elution peaks. Fast-atom-bombardment mass spectrometry of the smallest member of the series determined its molecular weight and established its identity as the phenylamine derivative of one disaccharide repeat unit of teichuronic acid. Homologous fractions of the same series were used to index the ladder of bands obtained by polyacrylamide gel electrophoresis from samples containing a more extensive distribution of polymer lengths. Nearly native teichuronic acid consists of polymers with a broad range of molecular sizes ranging from 20 to 55 disaccharide units. The most abundant species are those which have 25 to 40 repeat units. Prolonged treatment of teichuronic acid with the acid conditions used to release it from peptidoglycan causes gradual fragmentation of the teichuronic acid.     

Structure of an S layer on a pathogenic strain of Aeromonas hydrophila.

Negative staining revealed a tetragonal surface array (S layer) on all the members of a serogroup of Aeromonas hydrophila which possess high virulence for fish. The S layers were similar on all the strains examined, with unit cell dimensions of approximately 12 nm. A single representative strain, strain TF7, was selected for further analysis. Freeze-cleaved and etched preparations and sections for electron microscopy showed that the S layer was the outermost component of the cell envelope. This was confirmed by observation of thin sections. Computer-generated enhancements of the negatively stained micrographs showed the subunit organization to a resolution of less than 4 nm. Two structural units of identical lattice constants alternated in the array in both axes, and one of them was apparently dominant as the center of mass. The lesser unit was rotated 20 degrees from the dominant axes of symmetry and was formed by the junction of linker projections from a corner of the four components of the dominant unit. This interpretation was supported by finding that the array consists of a single polypeptide (molecular weight, 52,000). The unit cell as defined showed p4 symmetry, and a = b = 12.2 nm.     

Characterization of a low-sulfated chondroitin sulfate from the body of Viviparus ater (mollusca gastropoda). Modification of its structure by lead pollution

A chondroitin sulfate was purified from the body of Viviparus ater (Mollusca gastropoda) and analyzed for molecular mass, constituent disaccharides, and structure by 1H NMR and 1H 2D NMR. A quite unique glycosaminoglycan species was isolated having a high molecular mass (greater than 45,000) and low charge density, about 0.60, due to the presence of 42% non-sulfated disaccharide, 5% 6-sulfated disaccharide, 48% 4-sulfated disaccharide, and 5% 4,6-disulfated disaccharide. Specimens of Mollusca were also submitted to lead exposure for different times, and the effect on chondroitin sulfate structure was studied. After 96 h treatment a strong decrease in chondroitin sulfate content was observed with a significant modification of its structure producing a more desulfated polymer, in particular in position 4 of the galactosamine unit. Simultaneously, the amount of unsaturated non-sulfated disaccharide increased with an overall decrease of the charge density.     

Chondroitin 4-O-sulfotransferase-1 modulates Wnt-3a signaling through control of E disaccharide expression of chondroitin sulfate

Wnt-3a is a ligand that activates the beta-catenin-dependent pathway in Wnt signaling, which is implicated in numerous physiological events such as morphogenesis. So far, heparan sulfate (HS) proteoglycans have been highlighted as a low affinity receptor for morphogens containing Wnts. Here we show the importance of chondroitin sulfate (CS) proteoglycans in the efficient signaling of Wnt-3a and the structural features of CS required for the regulation of Wnt-3a signaling. Wnt-3a signaling was depressed in a mouse L cell mutant, called sog9, which is defective in the EXT1 gene encoding the HS-synthesizing enzyme and the chondroitin 4-O-sulfotransferase (C4ST-1) gene compared with parental L cells. The transfection of sog9 cells with C4ST-1 resulted in the recovery of Wnt-3a signaling, whereas the expression of EXT1 in sog9 cells could not restore Wnt-3a signaling. In addition, the expression level of introduced C4ST-1 correlated with the recovery of Wnt-3a signaling accompanied by the increased expression of the E disaccharide unit of CS. Interestingly, molecular interaction analyses using Biacore revealed that squid CS-E (rich in the E disaccharide unit) bound strongly to Wnt-3a (K(d)=13.2 nm) to the same extent as heparin from bovine lung (K(d)=8.43 nm). In contrast, other CS isoforms as well as HS isolated from bovine kidney showed little binding activity to Wnt-3a. Moreover, exogenously added CS-E potently inhibited the accumulation of beta-catenin induced by Wnt-3a. These results suggest that CS-E-like structures synthesized by C4ST-1 participate in Wnt-3a signaling and modulate the physiological events caused by Wnt-3a signals.     

Characterization of a novel linkage unit between ribitol teichoic acid and peptidoglycan in Listeria monocytogenes cell walls

The structure of the linkage unit between ribitol teichoic acid and peptidoglycan in the cell walls of Listeria monocytogenes EGD was studied. A teichoic-acid--glycopeptide preparation isolated from lysozyme digests of the cell walls of this strain contained mannosamine, glycerol, glucose and muramic acid 6-phosphate in an approximate molar ratio of 1:1:2:1, together with large amounts of glucosamine and other components of teichoic acid and glycopeptides. A teichoic-acid-linked sugar preparation, obtained by heating the cell walls at pH 2.5, also contained glucosamine, mannosamine, glycerol and glucose in an approximate molar ratio of 25:1:1:2. Part of the glucosamine residues were shown to be involved in the linkage unit. Thus, on mild alkaline hydrolysis, the teichoic-acid-linked sugar preparation gave a disaccharide characterized as N-acetylmannosaminyl(beta 1----4)-N-acetylglucosamine [ManNAc(beta 1----4)GlcNAc] in addition to the ribitol teichoic acid moiety, whereas the teichoic-acid - glycopeptide was separated into disaccharide-linked glycopeptide and the ribitol teichoic acid moiety by the same procedure. Furthermore, Smith degradation of the cell walls gave a characteristic fragment, EtO2-P-Glc(beta 1----3)Glc(beta 1----1/3)Gro-P-ManNAc(beta 1----4)GlcNAc (where EtO2 = 1,2-ethylenediol and Gro = glycerol). The results lead to the conclusion that in the cell walls of this organism, the ribitol teichoic acid chain is linked to peptidoglycan through a novel linkage unit, Glc(beta 1----3)Glc(beta 1----1/3)Gro-P-(3/4)ManNAc-(beta 1----4)GlcNAc.     

Molecular organization of heparan sulphate from human skin fibroblasts.

The molecular structure of human skin fibroblast heparan sulphate was examined by specific chemical or enzymic depolymerization and high-resolution separation of the resulting oligosaccharides and disaccharides. Important features of the molecular organization, disaccharide composition and O-sulphate disposition of this heparan sulphate were identified. Analysis of the products of HNO2 hydrolysis revealed a polymer in which 53% of disaccharide units were N-acetylated and 47% N-sulphated, with an N-/O-sulphate ratio of 1.8:1. These two types of disaccharide unit were mainly located in separate domains. Heparitinase and heparinase scission indicated that the iduronate residues (37% of total hexuronate) were largely present in contiguous disaccharide sequences of variable size that also contained the majority of the N-sulphate groups. Most of the iduronate residues (approx. 70%) were non-sulphated. About 8-10% of disaccharide units were cleaved by heparinase, but only a minority of these originated from contiguous sequences in the intact polymer. Trisulphated disaccharide units [alpha-N-sulpho-6-sulphoglucosaminyl-(1----4)-iduronate 2-sulphate], which are the major structural units in heparin, made up only 3% of the disaccharide units in heparan sulphate. O-Sulphate groups (approx. 26 per 100 disaccharide units) were distributed almost evenly among C-6 of N-acetylglucosamine, C-2 of iduronate and C-6 of N-sulphated glucosamine residues. The results indicate that the sulphated regions of heparan sulphate have distinctive and potentially variable structural characteristics. The high content of non-sulphated iduronate in this heparan sulphate species suggests a conformational versatility that could have important implications for the biological properties of the polymer.     

Three-dimensional structure of phenylalanyl-transfer RNA synthetase from Thermus thermophilus HB8 at 0.6-nm resolution.

The three-dimensional structure of the heterodimeric alpha 2 beta 2 enzyme phenylalanyl-tRNA synthetase from Thermus thermophilus HB8 has been determined by X-ray crystallography, using the multiple-isomorphous-replacement method at 0.6 nm resolution. Trigonal crystals of space group P3(2)21 have cell dimensions a = b = 17.6 nm and c = 14.2 nm. Assuming one heterodimeric molecule/asymmetric unit, the ratio of unit cell volume/molecular mass was V = 0.00244 nm3/Da, which is in the middle of the range normally observed. However, after a rotation-function calculation and measurement of the density of the native crystals, we postulate the existence of only the alpha beta dimer in the asymmetric units. This implies 73% solvent content in the unit cell. Three heavy-atom derivatives [K2PtCl4, KAu(CN)2 and Hg(CH3COO)2] and the solvent-flattening procedure were used for electron-density-map calculations. This map confirmed our hypothesis and revealed a remarkably large space filled by solvent, with alpha beta dimer only in the asymmetric unit. The phenylalanyl-tRNA synthetase from T. thermophilus molecule has a 'quasi-linear' subunit organization. As can be concluded at this level of resolution, there is no contact between small alpha subunits in the functional heterodimer.     

Hyaluronic acid: molecular conformations and interactions in two sodium salts.

A detailed structure for the tetragonal form (a = b = 0.989 nm, c, fibre axis, = 3.394 nm) of sodium hyaluronate has been obtained by analysing X-ray fibre diffraction data using new molecular modelling techniques. Two polysaccharide chains pass through each unit cell, one at the corner and one at the centre. The chains are anti-parallel to one another. Each chain is a left-handed, 4-fold helix of disaccharide units. There are intramolecular hydrogen bonds stabilising each glycosidic linkage. Octahedrally co-ordinated sodium ions link, by O … Na⁺ … O bridges, neighbouring polysaccharide chains that are further linked by hydrogen bonds. No double-helix model (as originally proposed for this structure) has been found to be free of unacceptable non-bonded contacts or to fit the diffraction intensities as closely.The tetragonal form, which is stable at zero relative humidity, contains no detectable water molecules. At higher relative humidities a related orthorhombic form is observed in which only the a dimension of the lattice is different (a = 1.153 nm, b = 0.989 nm, c = 3.386 nm). In this form the hyaluronate helix is 2-fold with tetrasaccharide units conformationally similar to the 4-fold helix of the tetragonal form. The Na⁺ … O binding and hydrogen bonds lost on expansion of the tetragonal lattice are all replaced in the orthorhombic structure by bridges through water molecules, four of which associated with each tetrasaccharide.     

Effects of chondroitin sulfate and its oligosaccharides on toll-like receptor-mediated IL-6 secretion by macrophage-like J774.1 cells

The effects of two types of chondroitin sulphate (CS), CS-A and CS-C, their oligosaccharides (oligo-CSs), and disaccharides (Di-CSs) on toll-like receptor (TLR)-mediated secretion of interleukin (IL)-6 were compared using macrophage-like cell line J774.1. IL-6 secretion in the J774.1 cells was markedly increased by Pam3CS4, LPS, and CpG, the ligands to TLR1/2, 4, and 9 respectively. Among these three ligands, CpG-induced IL-6 was most clearly suppressed by CSs and their digests. Suppression of IL-6 secretion by smaller sized CS-A was stronger than that by intact CS-A, whereas no such size-dependent suppression was apparent for CS-C. Di-4S, the disaccharide unit of the CS-A digest, also showed much stronger suppression than Di-6S, the disaccharide unit of the CS-C digest, and the non-sulfated disaccharide unit, Di-0S. The suppressing activity of oligo-CSs, particularly Di-CSs, against TLR-mediated inflammation was dependent on the CS structure, including the sulfation site.     

Effects of Chondroitin Sulfate and Its Oligosaccharides on Toll-Like Receptor-Mediated IL-6 Secretion by Macrophage-Like J774.1 Cells

The effects of two types of chondroitin sulphate (CS), CS-A and CS-C, their oligosaccharides (oligo-CSs), and disaccharides (Di-CSs) on toll-like receptor (TLR)-mediated secretion of interleukin (IL)-6 were compared using macrophage-like cell line J774.1. IL-6 secretion in the J774.1 cells was markedly increased by Pam3CS4, LPS, and CpG, the ligands to TLR1/2, 4, and 9 respectively. Among these three ligands, CpG-induced IL-6 was most clearly suppressed by CSs and their digests. Suppression of IL-6 secretion by smaller sized CS-A was stronger than that by intact CS-A, whereas no such size-dependent suppression was apparent for CS-C. Di-4S, the disaccharide unit of the CS-A digest, also showed much stronger suppression than Di-6S, the disaccharide unit of the CS-C digest, and the non-sulfated disaccharide unit, Di-0S. The suppressing activity of oligo-CSs, particularly Di-CSs, against TLR-mediated inflammation was dependent on the CS structure, including the sulfation site.     

The three-dimensional structures of S-layers of two novel Eubacterium species isolated from inflammatory human processes

The three-dimensional structures of the crystalline surface layers of two species of Eubacteria have been determined by electron microscopy and computerized image processing. The S-layer of Eubacterium sp. ES4C has tetragonal symmetry, with a unit cell spacing of 10.6 nm and a thickness of 9.5 nm, while that of Eubacterium sp. AHN 990 has hexagonal symmetry a = b = 15.7 nm and a thickness of 13 nm. The resolutions in the reconstructions were 2.5 nm and 1.8 nm, respectively. The reconstruction of the S-layer of strain ES4C reveals a distinct domain structure: a major tetramer, arms connecting adjacent unit cells, and a minor tetramer. The S-layer of strain AHN 990, on the other hand, has a rather complex arrangement, centred around the six-fold axis.     

Crystallization of a complex between ribonuclease T1 and 2'-guanylic acid

Ribonuclease T1 was crystallized under various conditions. Form I crystals were produced by microdialysis against 53% (v/v) 2-methyl-2,4-pentanediol in 0.01 M sodium acetate, 0.05% 2'-guanylic acid (2'GMP) and 0.02% NaN3 (pH 6.2-7.2). These crystals are tetragonal, space group P41212 and contain two molecules per asymmetric unit; cell dimensions are a = b = 5.86 nm, c = 13.28 nm. Form IIa and form IIb crystals were obtained by microdialysis from a buffer of 0.01-0.05 M sodium acetate, 0.25-0.5% 2'GMP, 0.02% NaN3 and 2-5 mM calcium acetate (pH 4.0-4.4) in the presence of 50-75% (v/v) 2-methyl-2,4-pentanediol. These crystals are orthorhombic, space group P212121, and contain one molecule per asymmetric unit; cell dimensions are a = 4.66 nm, b = 5.02 nm, c = 4.04 nm (form I) and alpha = 4.44 nm, b = 5.00 nm, c = 4.03 nm (form II). Using high-performance liquid chromatography, it could be shown for all crystal forms that 2'-GMP is bound in the crystals. The molecular ratio between RNase T1 and 2'GMP was 0.9 for form II crystals and thus agreed with a 1:1 enzyme-nucleotide complex. Heavy-atom derivatives were produced with lead acetate for form IIa crystals and with uranyl acetate for from IIb crystals. Three-dimensional X-ray analysis of the RNase-T1 x 2'GMP complex is under way.     

Inactivation of unbound rat liver glucocorticoid receptor by N-alkylmaleimides at sub-zero temperatures

Biosynthetically radiolabelled heparan sulphate proteoglycans have been isolated from the growth medium and the cell lysate of a human neuroblastoma cell line (CHP100). Chromatography on Sepharose CL-4B identified two heparan sulphate proteoglycans in the medium (Kav 0.220 and 0.389), whereas in the cell lysate the major proteoglycan species were more heterogenous and of a smaller overall molecular size (Kav 0.407) than the medium-derived counterparts. Chromatography on Sepharose CL-6B of free heparan sulphate glycosaminoglycan chains showed that the majority of cell-layer-derived material heparan sulphate 2, Kav = 0.509) was smaller than medium heparan sulphates (heparan sulphate 1 and heparan sulphate 2, Kav 0.230 and 0.317). Analysis of the patterns of polymer sulphation by nitrous acid treatment, gel chromatography and high-voltage electrophoresis established that in each heparan sulphate fraction there was on average 1.1 sulphate residues per disaccharide with an N:O sulphate ratio of 1.1. Heparan sulphate in the medium had a high proportion of di-O-sulphated disaccharides in regions of the chain with repeat disaccharide sequences of structure GlcA-GlcNSO3, whereas cell-associated material was enriched in di-O-sulphated tetrasaccharides of alternating sequences GlcA-GlcNAc-GlcA-GlcNSO3. The identification of several populations of heparan sulphate proteoglycans differing in molecular size and glycosaminoglycan fine structure may reflect the functional diversity of this family of macromolecules in the nervous system.     

Model for the Structure of the Shape-Maintaining Layer of the Escherichia coli Cell Envelope

The surface area per repeating murein unit (i.e. per molecule of diaminopimelate) has been determined for the cell envelopes of the Escherichia coli strains K-12 and W. This area was constantly found to be 1.3 nm(2). Using this value and other previously determined properties of E. coli murein, a three-dimensional model of murein is proposed. The model specifies a monomolecular layer in which disaccharide units are each 1.03 nm long, and the polysaccharide chains, all parallel, are 1.25 nm apart. The cross-linking peptide side-chains have the same atomic coordinates and are arranged above or below the polysaccharide chains.     

Molecular replacement studies on crystal structure of allophycocyanin from red algae Porphyra yezoensis

Using the crystal structure of allophycocyanin from cyanobacterium Spirulina platensis (APC-SP) as a search model,the crystal structure of allophycocyanin from red algae Porphyra yezoensis (APC-PY) has been studied by molecular replacement methods.The APC-PY crystals (Form 3) belong to the space group of R32,cell dimensions a=b= 10.53 nm,c=18.94 nm,α= β= 90°,γ=120°;there is one αβ monomer in each crystallographic asymmetric unit in the cell.The translation function search gave a unique peak with a correlation coefficient (Cc) of 67.0% and an R-factor of 36.1% for reflection data from 1.0 to 0.4 nm.Using the results by molecular replacement,the initial model of APC-PY was built,and the coincidence of the chromophore in APC-PY initial model with its 2Fo-Fc OMIT map further confirms the results by molecular replacement.     

Crystallization and subunit structure of canine myeloperoxidase

Three crystal forms of canine myeloperoxidase are described. An orthorhombic form in space group P2(1)2(1)2(1) has unit cell dimensions: a = 108.3 A (1 A = 0.1 nm) b = 205.9 A and c = 139.9 A. A trigonal form in space group P3(1)21 or P3(2)21 has unit cell dimensions: a = b = 138.9 A and c = 145.2 A. A monoclinic form in space group C2 has unit cell dimensions: a = 117.2 A, b = 96.9 A, c = 131.4 A and beta = 116.3 degrees. Unusual features in the diffraction patterns of the monoclinic form place restrictions on the molecular packing in the crystal. The proposed model for the molecular packing requires that the myeloperoxidase molecule consist of two identical or near-identical halves. In the intact molecule these halves may be related either by a crystallographic dyad axis or by an approximate dyad axis in which one subunit is translated relative to the other by 3.2 A along the symmetry axis. The trigonal crystal form appears most suitable for high-resolution X-ray structural analysis.     

Characterization of chondroitin sulfate from deer tip antler and osteogenic properties

Deer antler is a highly regenerative tissue that involves cellular differentiation, osteogenesis and ossification processes. Chondroitin sulfate is the major glycosaminoglycan contained in antler connective tissue and has been isolated from cartilaginous antler by 4 M GuHCl extraction, gradient ultracentrifugation and chromatography techniques. We examined the disaccharide composition by 2-AB labeling and anion exchange HPLC analysis of the three resultant fractions (high, medium and low density fractions). The high density fraction consists of A-unit and D-unit disaccharide in the ratio of 1:1, whereas, the CS disaccharide composition ratio of A- unit:C-unit:D-Unit:E-unit contained in medium and low density fractions are 3:4:3:1 and 2:2:2:1, respectively. The only intact CS oligosaccharides of the medium density fraction upregulated gene expression of bone-specific proteins of a human osteoblastic cell line (hFOB1.19). Thus, CS oligosaccharides from cartilaginous deer antler, with their oversulfated chondroitin sulfate composition, demonstrated the physiological properties and may be good candidates for osteogenetic agents in humans.     

The structure and antiviral activity of ribavirin analogs. III. Molecular and crystal structure of 1-(2-hydroxyethoxymethyl)-1,2,4-triazole-carboxamide

The crystal and molecular structure of a ribavirin acyclic analogue, 1-(2-hydroxyethoxymethyl)-1,2,4-triazole-3-carboxamide, has been determined by X-ray diffraction method. The space group is P1, unit cell parameters: a = 5,237, b = 6,960, c = 11,483 A, alpha = 93,89, beta = 97,43, gamma = 94,26 degrees; Z = 2. The structure was solved by the direct method and refined by least-squares procedure to R = 3.7%. Two molecular conformers statistically coexist in the unit cell, differing in the hydroxyethoxymethyl group conformation. Trans-conformation about O4'-C4' bond and gauche about C4'-C5' bond are observed in both molecules. C1'-O4' bond is approximately perpendicular to the aglicon.