Determination of the primary and fine structures of galactomannan from seeds of Gleditsia triacanthos f. intermis L

This was the first study to isolate galactomannan, a 660-kDa polysaccharide, from the seeds of Gleditsia triacanthos f. inermis L. (yield 15.4%). Its aqueous solutions were optically active ([alpha] D = +31.0 degrees) and highly viscous ([eta] = 578 ml/g). The analysis of this heteropolysaccharide using chemical, enzymatic, and chromatographic procedures, as well as IR- and 13C-NMR spectroscopy, showed that is consists of D-mannopyranose and D-galactopyranose residues (molar ratio 2.42:1). Its main chain is comprised of 1,4-beta-D-mannopyranose residues, 41% of which is substituted at C-6 with single residues of alpha-D-galactopyranose. The probability of occurrence of differently substituted mannobiose units in the chain, determined experimentally, was 0.16 for the unit Man-Man, 0.50 for the units Gal(Man-Man) and (Man-Man)Gal, and 0.34 for the dissubstitued unit Gal(Man-Man)Gal.     

Composition and structure of galactomannan from the seed of Gleditsia ferox Desf

Galactomannan, a heteropolysaccharide with a molecular weight of 1660 kDa, was isolated form the seed of Gleditsia ferox Desf., introduced in Russia, with a yield of 18.9%. Its aqueous solutions were optically active ([alpha]D = +30.5 degrees) and highly viscous ([eta] = 1430 ml/g). Analysis of the heteropolysaccharide using chemical, enzymatic, and chromatographic procedures showed that it consists of D-mannopyranose and D-galactopyranose residues (molar ratio, 2.54:1). The main chain of this galactomannan consists of 1,4-beta-D-mannopyranose residues, 39.2% of which are substituted at C6 with single residues of alpha-D-galactopyranose. The probability of occurrence of mannobiose units differentially substituted with galactose was determined by 13C-NMR data and equaled, respectively, 0.37, 0.47, and 0.16 for non-substituted Man-Man units, monosubstituted Gal(Man-Man) and (Man-Man)Gal units taken together, and for the disubstituted Gal(Man-Man)Gal units.     

Fractional Isolation and Study of the Structure of Galactomannan from Sophora (Styphnolobium japonicum) Seeds

Two fractions (1 and 2) of the galactomannan from seeds of sophora (Styphnolobium japonicum) were isolated using cold and hot aqueous extraction with a total yield of 12.88%. The two fractions differed by the ratio between mannose (Man) and galactose (Gal) residues (4.8 : 1 and 5.3 : 1, respectively) and molecular weight (1190 and 1400 kDa, respectively). Aqueous solutions of these fractions were optically active ([]_D = +4.80° and –3.36°, respectively) and highly viscous ([] 1028.8 and 1211.2 ml/g). ¹³C NMR spectra of both fractions were identical with respect to the number and positions of signals, which indicates that their primary structures were identical. Using chemical and spectroscopic (IR and NMR) methods, it was shown that the galactomannan has a main chain consisting of 1,4--D-mannopyranose, some residues of which (16 and 17% in fractions 1 and 2, respectively) are -galactosylated at the C-6 position. Frequencies of differently substituted mannobiose blocks in the chain, calculated for fraction 1 using NMR spectroscopic data, were 0.13 for the disubstitited blocks Gal(Man–Man)Gal, 0.37 for the sum of monosubstituted blocks Gal(Man–Man) and (Man–Man)Gal, and 0.50 for the unsubstituted block Man–Man.     

Galactomannan from the seeds of Ural licorice (Glycyrrhiza uralensis Fisch.)

Galactomannans from the seeds of Ural licorice (Glycyrrhiza uralensis Fisch.) obtained by hot water extraction of freshly ripened (GGu-1) and overwintered (GGu-2) seeds were studied. GGu-1 and GGu-2 (yield, 1.98 and 1.99% of the seed weight) had molecular weights of 1379 and 877 kDa, respectively; their solutions were characterized by high viscosity ([eta] 1193.1 and 765.8 mg/g, respectively) and optical activity ([alpha]D +64.8 and +65.6 deg, respectively). Their galactose-to-mannose ratio was 1 : 1.52 and 1 : 1.50, respectively. According to IR and 13C NMR spectroscopic data and methylation analysis, the polymeric chains of GGu-1 and GGu-2 are comprised of 1,4-beta-D-mannopyranose residues substituted at C-6 with single alpha-D-galactopyranose residues. The content of mannobiose units Man-Man, (Gal)Man-Man / Man-Man(Gal), and (Gal)Man-Man(Gal) differentially substituted with galactose in macromolecules GGu-1 and GGu-2 was 25.2, 18.4 and 55.9% for GGu-1 and 26.5, 32.5, and 41.0% for GGu-2.     

Galactomannan from the seeds of Ural licorice (<Emphasis Type="Italic">Glycyrrhiza uralensis</Emphasis> Fisch.)

Galactomannans from the seeds of Ural licorice (Glycyrrhiza uralensis Fisch.) obtained by hot water extraction of freshly ripened (GGu-1) and overwintered (GGu-2) seeds were studied. GGu-1 and GGu-2 (yield, 1.98 and 1.99% of the seed weight) had molecular weights of 1379 and 877 kDa, respectively; their solutions were characterized by high viscosity ([η 1193.1 and 765.8 mg/g, respectively) and optical activity ([α_D, +64.8 and +65.6 deg, respectively). Their galactose-to-mannose ratio was 1: 1.52 and 1: 1.50, respectively. According to IR and ¹³C NMR spectroscopic data and methylation analysis, the polymeric chains of GGu-1 and GGu-2 are comprised of 1,4-β-D-mannopyranose residues substituted at C-6 with single α-D-galactopyranose residues. The content of mannobiose units Man-Man, (Gal)Man-Man/Man-Man(Gal), and (Gal)Man-Man(Gal) differentially substituted with galactose in macromolecules GGu-1 and GGu-2 was 25.2, 18.4 and 55.9% for GGu-1 and 26.5, 32.5, and 41.0% for GGu-2.     

Galactomannan from the seeds of chinese honey locust (Gleditsia sinensis Lam.)

By the hot water extraction method, galactomannan was extracted (4.5% yield of the seed mass) from the seeds of Chinese honey locust (Gleditsia sinensis Lam). It had a molecular weight of 1230 kDa, and its solutions had a high viscosity [η] of 1064 ml/g and optical activity [α]_D of +21.4°. The polysaccharide consists of mannose and galactose residues in the molar ratio 2.69: 1. In the galactomannan macromolecule the backbone is formed by 1,4-β-D-mannopyranose residues, 37% of which are substituted by α-D-galactopyranose at C6. By ¹³C-NMR-spectroscopy, fragments of differently galactose-substituted mannobiose units were found to be in the galactomannan being studied: Man-Man, (Gal)Man-Man, and Man-Man(Gal) in the ratio of 0.23: 0.47: 0.30.     

NMR spectroscopy and chemical studies of an arabinan-rich system from the endosperm of the seed of Gleditsia triacanthos

Exhaustive extraction of the endosperm from the seed of Gleditsia triacanthos using water at room temperature and 50 degrees C left a residue, which was further extracted at 95 degrees C. Precipitation of this extract with 2-propanol yielded major amounts of galactomannan components, while the supernatant was mainly composed of arabinose-rich constituents. Two fractions were obtained by anion-exchange chromatography. The fraction that eluted with water is an arabinan with (1-->5) alpha-L linkages and branching mainly on C-2, accompanied with equal amounts of a low-galactose galactomannan oligosaccharide, and a small proportion of a beta-(1-->4)-galactan. The fraction eluted with an increased ionic strength consists mainly of a similar arabinan, and lower proportions of a high-galactose galactomannan, galactan, and protein. The arabinan moiety in both fractions was characterized by chemical analysis and 1D and 2D NMR spectroscopic techniques.     

Determination of the Primary and Fine Structures of a Galactomannan from the Seed of Gleditsia triacanthos f. inermis L.

Galactomannan, a polysaccharide with a molecular weight of 660 kDa, was isolated for the first time from the seed of Gleditsia triacanthos f. inermis (yield, 15.4%). Its aqueous solutions were optically active ([] _D = +31.0°) and highly viscous ([] = 578 ml/g). Analysis of this heteropolysaccharide using chemical, enzymatic, and chromatographic procedures, as well as IR and ¹³C NMR spectroscopy, showed that it consists of D-mannopyranose and D-galactopyranose residues (molar ratio, 2.42 : 1). The main chain of this galactomannan comprises 1,4--D-mannopyranose residues, 41% of which are substituted at C6 with single residues of -D-galactopyranose. The probability of occurrence in the chain of mannobiose units substituted otherwise, determined experimentally, was 0.16 for the Man–Man unit, 0.50 for the Gal(Man–Man) and (Man–Man)Gal units, and 0.34 for the disubstituted Gal(Man–Man)Gal unit.     

Composition and structure of a galactomannan macromolecules from seeds of Astragalus lehmannianus Bunge

The composition and structure of a galactomannan from seeds of Astragalus lehmannianus, an endemic legume species, is reported for the first time. The purified galactomannan (yield, 4.8%) contained 55% D-mannose and 45% D-galactose and had a molecular weight of 997.03 kDa. Its aqueous solutions were optically active and highly viscous (the specific rotation, [alpha]D, equaled +81.3 degrees; the characteristic viscosity, [eta], 868.4 ml/g). Chemical, chromatographic, and spectral (IR and 13C-NMR spectroscopy) methods were used to demonstrate that the main chain of the molecule is formed by residues of 1,4-beta-D-mannopyranose, 78% of which are substituted at position 6 with single alpha-D-galactopyranose. The distribution of galactose along the chain was calculated from NMR spectra: frequencies of occurrence, per pair of neighboring mannose units, of (1) two substituents, (2) one substituent, and (3) no substituents were 65.3, 31.5, and 3.2%, respectively. The specific rotation of galactomannans was shown to correlate with their content of galactose.     

Tobacco transgenic lines that express fenugreek galactomannan galactosyltransferase constitutively have structurally altered galactomannans in their seed endosperm cell walls

Galactomannans [(1-->6)-alpha-D-galactose (Gal)-substituted (1-->4)-beta-D-mannans] are major cell wall storage polysaccharides in the endosperms of some seeds, notably the legumes. Their biosynthesis in developing legume seeds involves the functional interaction of two membrane-bound glycosyltransferases, mannan synthase (MS) and galactomannan galactosyltransferase (GMGT). MS catalyzes the elongation of the mannan backbone, whereas GMGT action determines the distribution and amount of Gal substitution. Fenugreek (Trigonella foenum-graecum) forms a galactomannan with a very high degree of Gal substitution (Man/Gal = 1.1), and its GMGT has been characterized. We now report that the endosperm cell walls of the tobacco (Nicotiana tabacum) seed are rich in a galactomannan with a very low degree of Gal substitution (Man/Gal about 20) and that its depositional time course is closely correlated with membrane-bound MS and GMGT activities. Furthermore, we demonstrate that seeds from transgenic tobacco lines that express fenugreek GMGT constitutively in membrane-bound form have endosperm galactomannans with increased average degrees of Gal substitution (Man/Gal about 10 in T(1) generation seeds and about 7.5 in T(2) generation seeds). Membrane-bound enzyme systems from transgenic seed endosperms form galactomannans in vitro that are more highly Gal substituted than those formed by controls under identical conditions. To our knowledge, this is the first report of structural manipulation of a plant cell wall polysaccharide in transgenic plants via a biosynthetic membrane-bound glycosyltransferase.     

Unique conformer selection of human growth-regulatory lectin galectin-1 for ganglioside GM1 versus bacterial toxins

Endogenous lectins induce effects on cell growth by binding to antennae of natural glycoconjugates. These complex carbohydrates often present more than one potential lectin-binding site in a single chain. Using the growth-regulatory interaction of the pentasaccharide of ganglioside GM(1) with homodimeric galectin-1 on neuroblastoma cell surfaces as a model, we present a suitable strategy for addressing this issue. The approach combines NMR spectroscopic and computational methods and does not require isotope-labeled glycans. It involves conformational analysis of the two building blocks of the GM(1) glycan, i.e., the disaccharide Galbeta1-3GalNAc and the trisaccharide Neu5Acalpha2-3Galbeta1-4Glc. Their bound-state conformations were determined by transferred nuclear Overhauser enhancement spectroscopy. Next, measurements on the lectin-pentasaccharide complex revealed differential conformer selection regarding the sialylgalactose linkage in the tri- versus pentasaccharide (Phi and Psi value of -70 degrees and 15 degrees vs 70 degrees and 15 degrees, respectively). To proceed in the structural analysis, the characteristic experimentally detected spatial vicinity of a galactose unit and Trp68 in the galectin's binding site offered a means, exploiting saturation transfer from protein to carbohydrate protons. Indeed, we detected two signals unambiguously assigned to the terminal Gal and the GalNAc residues. Computational docking and interaction energy analyses of the entire set of ligands supported and added to experimental results. The finding that the ganglioside's carbohydrate chain is subject to differential conformer selection at the sialylgalactose linkage by galectin-1 and GM(1)-binding cholera toxin (Phi and Psi values of -172 degrees and -26 degrees, respectively) is relevant for toxin-directed drug design. In principle, our methodology can be applied in studies aimed at blocking galectin functionality in malignancy and beyond glycosciences.     

Composition and Structure of Galactomannan from the Seed of Gleditsia ferox Desf.

Galactomannan, a heteropolysaccharide with a molecular weight of 1660 kDa, was isolated from the seed of Gleditsia ferox Desf., introduced in Russia, with a yield of 18.9%. Its aqueous solutions were optically active ([]_D = +30.5°) and highly viscous ([] = 1430 ml/g). An analysis of the heteropolysaccharide using chemical, enzymatic, and chromatographic procedures showed that it consists of D-mannopyranose and D-galactopyranose residues (molar ratio, 2.54 : 1). The main chain of this galactomannan consists of 1,4--D-mannopyranose residues, 39.2% of which are substituted at C6 with single residues of -D-galactopyranose. The probability of occurrence of mannobiose units differentially substituted with galactose was determined by ¹³C-NMR data and equaled, respectively, 0.37, 0.47, and 0.16 for non-substituted Man–Man units, monosubstituted Gal(Man–Man) and (Man–Man)Gal units taken together, and for the disubstituted Gal(Man–Man)Gal units.     

Galactomannan from ambiguous Crazyweed (Oxytropis ambigua (Pall) DC)

A galactomannan with a molecular weight of 735 kDa was first isolated and purified from seeds of ambiguous crazyweed Oxytropis ambigua (Pall) DC. (family Leguminosae) with a yield of 3.6%. Its aqueous solutions displayed an optical activity ([alpha]D = 73.32 degrees) and high viscosity ([eta] = 644 ml g-1). Chemical analysis and 13C-NMR spectroscopy revealed the presence in the heteropolysaccharide of D-mannopyranose and D-glucopyranose at a molar ratio of 1.39:1. The linear backbone of its macromolecule consists of 1.4-beta-D-mannopyranose residues. Single beta-D-galactose residues substitute 72% of mannoses to form branches.     

Transfer specificity of detergent-solubilized fenugreek galactomannan galactosyltransferase

The current experimental model for galactomannan biosynthesis in membrane-bound enzyme systems from developing legume-seed endosperms involves functional interaction between a GDP-mannose (Man) mannan synthase and a UDP-galactose (Gal) galactosyltransferase. The transfer specificity of the galactosyltransferase to the elongating mannan chain is critical in regulating the distribution and the degree of Gal substitution of the mannan backbone of the primary biosynthetic product. Detergent solubilization of the galactosyltransferase of fenugreek (Trigonella foenum-graecum) with retention of activity permitted the partial purification of the enzyme and the cloning and sequencing of the corresponding cDNA with proof of functional identity. We now document the positional specificity of transfer of ((14)C)Gal from UDP-((14)C)Gal to manno-oligosaccharide acceptors, chain lengths 5 to 8, catalyzed by the detergent-solubilized galactosyltransferase. Enzymatic fragmentation analyses of the labeled products showed that a single Gal residue was transferred per acceptor molecule, that the linkage was (1-->6)-alpha, and that there was transfer to alternative Man residues within the acceptor molecules. Analysis of the relative frequencies of transfer to alternative Man residues within acceptor oligosaccharides of different chain length allowed the deduction of the substrate subsite recognition requirement of the galactosyltransferase. The enzyme has a principal recognition sequence of six Man residues, with transfer of Gal to the third Man residue from the nonreducing end of the sequence. These observations are incorporated into a refined model for enzyme interaction in galactomannan biosynthesis.     

Novel Schizosaccharomyces pombe N-linked GalMan9GlcNAc isomers: role of the Golgi GMA12 galactosyltransferase in core glycan galactosylation

Schizosaccharomyces pombe synthesizes very large N-linked galactomannans, which are elongated from the Man9GlcNAc2 core that remains after the trimming of three Glc residues from the Glc3Man9GlcNAc2 originally transferred from dolichyl pyrophosphate to nascent proteins in the endoplasmic reticulum. Prior to elongation of the galactomannan outer chain, the Man9GlcNAc2 core is modified into a family of Hex10-15GlcNAc2 structures by the addition of both Gal and Man residues (Ziegler et al. (1994) J. Biol. Chem., 269, 12527-12535). To understand the pathway of Man9GlcNAc2 modification, the Hex10GlcNAc-sized pool was isolated by Bio-Gel P-4 gel filtration from the endo H-released N-glycans of S.pombe glycoproteins. This pool yielded four major fractions, a, b, c, and g, on preparative high pH, anion exchange chromatography, that represented 10, 29, 46, and 13% of the total Hex10GlcNAc present, respectively. Structures of the glycan isomers present in each fraction were determined by one- and two-dimensional 1H NMR spectroscopy techniques. Fraction a is principally (approximately 93%) a Man10GlcNAc with a new alpha1,2-linked Man cap on the upper-arm of Man9GlcNAc. Fraction b contained two isomers of GalMan9GlcNAc in which an alpha1,2-linked terminal Gal had been added either to the upper (b1, 30%) or middle-arm (b2, 70%) of Man9GlcNAc. The gma12 - alpha1,2-galactosyltransferase-negative S. pombe strain (Chappell et al. (1994) Mol. Biol. Cell., 5, 519-528) did not make fraction b implying that the gma12p galactosyltransferase is responsible for synthesis of both isomers b1 and b2. Isomer c is Man10GlcNAc in which a new branching alpha1, 6-linked Man had been added to the lower-arm alpha1,3-linked core residue as found earlier in Saccharomyces cerevisiae and Pichia pastoris. Fraction g had less than molar stoichiometry of both Gal and Glc. The major isomer (g1, 85%) is the Man9GlcNAc core with an alpha1,3-linked branching Gal on the penultimate 2-O-substituted Man of the lower arm. This residue is also found on a novel O-linked oligosaccharide recently described in S.pombe; Manalpha1,2(Galalpha1, 3)Manalpha1,2Mannitol (Gemmill and Trimble (1999) Glycobiology, 9, 507-515). The second isomer (g2, 15%) is the partially processed Glc2Man9GlcNAc intermediate. Defining these Hex10GlcNAc structures provides a starting point for understanding the enzymology of N-linked galactomannan core heterogeneity seen on S.pombe glycoproteins.     

Biosynthesis of the O-glycosidically linked oligosaccharide chains of fetuin. Indications for an alpha-N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase with a narrow acceptor specificity in fetal calf liver

Fetal calf liver microsomes were found to be capable of sialylating 14C-galactosylated ovine submaxillary asialomucin. The main oligosaccharide product chain could be obtained by beta-elimination under reductive conditions and was identified as NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAcol (where GalNAcol represents N-acetylgalactosaminitol) by means of high performance liquid chromatography (HPLC) analysis and methylation. The branched trisaccharide Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)-GalNAcol and the disaccharide NeuAc alpha 2 leads to 6GalNAcol were not formed. Very similar results were obtained when asialofetuin and antifreeze glycoprotein were used as an acceptor. When 3H-sialylated antifreeze glycoprotein ([3H]NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc-protein) was incubated with fetal calf liver microsomes and CMP-[14C]NeuAc, a reduced tetrasaccharide could be isolated. The structure of this product chain appeared to be [3H]NeuAc alpha 2 leads to 3Gal beta 1 leads to 3([14C]NeuAc alpha 2 leads to 6)GalNAcol, as established by means of HPLC analysis, specific enzymatic degradation with Newcastle disease virus neuraminidase, and periodate oxidation. These data indicate that fetal calf liver contains two sialyltransferases involved in the biosynthesis of the O-linked bisialotetrasaccharide chain. The first enzyme is a beta-galactoside alpha 2 leads to 3 sialyltransferase which converts Gal beta 1 leads to 3 GalNAc chains to the substrate for the second enzyme, a (NeuAc alpha 2 leads to 3Gal beta 1 leads to 3)GalNAc-protein alpha 2 leads to 6 sialyltransferase. The latter enzyme does not sialylate GalNAc or Gal beta 1 leads to 3GalNAc units but is capable of transferring sialic acid to C-6 of GalNAc in NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc trisaccharide side chains, thereby dictating a strictly ordered sequence of sialylation of the Gal beta 1 leads to 3 GalNAc units in fetal calf liver.     

Galactomannans with novel structures from the lichen Roccella decipiens Darb

Two homogeneous galactomannan fractions were isolated from the lichen, Roccella decipiens, one (FP) containing Man and Gal in an 81:19 molar ratio and the other (RFS), having Man, Gal, and Glc in a 43:56:1 molar ratio. FP consisted of a main chain with (1-->4)-linked alpha-D-Manp units, most of which were substituted at O-2 with side chains consisting of nonreducing end-, 2-O- and 6-O-substituted alpha-Manp units. The latter appeared to be substituted by single-unit beta-D-Galf nonreducing ends. RFS contained a similar alpha-D-Manp core structure, but with side chains containing nonreducing end, 5-O-, 6-O-, and 5,6-di-O-substituted beta-D-Galf units. Such polysaccharide structures have not been previously reported.     

Xyloglucan from the leaves of Hymenaea courbaril

A fucosylated xyloglucan was isolated from the leaves of Hymenaea courbaril by alkaline extraction, followed by ethanol precipitation and ion-exchange chromatography. The isolated polysaccharide showed Glc:Xyl:Gal:Fuc in molar ratio of 8:5:2.5:1 and (D)(25) +40.5 degrees. Composition and linkage analyses, supported by NMR spectroscopic measurements, showed that the polysaccharide has a glucan backbone which is highly substituted at O-6 with D-xylopyranose residues, about a half of which are substituted at O-2 by D-galactopyranosyl units. Some of the galactose residues are further substituted by L-fucopyranose at O-2. The M(r), as determined by HPSEC, was 49,500.     

N-linked oligosaccharide changes with oncogenic transformation require sialylation of multiantennae

Glycopeptides derived from NIH 3T3 fibroblasts and these cells transformed by transfection with human DNA containing oncogene H-ras were analyzed by 500-MHz 1H-NMR spectroscopy and binding to immobilized lectins. The cells were metabolically labeled with D-[3H]glucosamine or L-[3H]fucose and the glycopeptides included in Bio-Gel P-10 (Mr 5000-3500) were separated into neutral and charged fractions on DEAE-cellulose. The major portion (80%) of these [3H]fucose glycopeptides from the non-transformed NIH 3T3 fibroblasts were neutral or contained one or two charged residues, whereas 90% of the glycopeptides from the transformed cells contained two or more charged residues. The structure of the predominant neutral glycopeptide from the non-transformed NIH 3T3 cells was determined by 1H-NMR spectroscopy to be tetraantennary containing terminal Gal alpha 1----3. (formula; see text) This structure was verified by binding to the immobilized alpha-Gal-specific lectin, Griffonia simplicifolia I and leukoagglutinating phytohemagglutinin from Phaseolus vulgaris (L-PHA), which binds certain tri- or tetraantennary glycopeptides. In contrast, the structure derived by NMR spectroscopy of one of the predominant charged glycopeptides from the transformed cells was triantennary containing terminal NeuNAc alpha 2----3 in addition to Gal alpha 1----3. (formula; see text) In attempting to verify this structure by lectin-binding properties it was found that removal of NeuNAc alpha 2----3 reduced the affinity to L-PHA - agarose. The other major glycopeptides of the transformed cells which were more charged also cotained NeuNAc alpha 2----3 but no NeuNAc alpha 2----6 or Gal alpha 1----3. A tentative structure was proposed for the major glycopeptide of the first charged class from NIH 3T3 cells on the basis of lectin-binding properties and the NMR spectrum which showed, in addition to NeuNAc alpha 2----3, the presence of NeuNAc alpha 2----6 and Gal alpha 1----3. On the basis of the NMR spectrum and other results, it is concluded that the presence of tetraantennary oligosaccharides are not sufficient for the transformed oligosaccharide phenotype. Rather, the tri- or tetraantennae must be sialylated in alpha 2----3 linkage, on more than one antennae, when properties of transformation are expressed in NIH 3T3 cells. Prior to transformation the tetraantennary oligosaccharides of these cells are terminated in alpha-Gal residues, whereas after transformation alpha-Gal residues appear to be replaced by NeuNAc alpha 2----3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Deuterium NMR investigation of ether- and ester-linked phosphatidylcholine bilayers

Deuterium nuclear magnetic resonance (2H NMR) spectra of specifically head-group- and chain-deuterated ester- and ether-linked phosphatidylcholine bilayers were studied as a function of temperature over the range -33 to 50 degrees C. Head-group-deuterated dihexadecylphosphatidylcholine ([alpha-2H2]DHPC) bilayers yield line shapes and spin-lattice relaxation times similar to those observed for its ester-linked counterpart, dipalmitoylphosphatidylcholine ([alpha-2H2]DPPC), in the high-temperature ripple and L alpha bilayer phases. These results indicate the ether linkage has no effect on the dynamics or the orientational order at the alpha-C2H2 segment of the phosphocholine head group. At all temperatures, the 2H NMR spectra of chain-deuterated 1,2[1',1'-2H2]DHPC bilayers exhibit a reduced spectral width compared to 1,2[2',2'-2H2]DPPC bilayers. The most significant feature of the deuterated alkyl chain spectrum of DHPC at 45 degrees C is the observation of four separate quadrupolar splittings from the alpha-methylene segments of the alkyl chains, in comparison to the three quadrupolar splittings reported previously from the alpha-methylene segments of the acyl chains of DPPC. Spin-lattice relaxation experiments performed on DHPC suggest an assignment of the two smaller and the two larger quadrupolar splittings to separate alkyl chains, respectively. Low-temperature (T less than or equal to -20 degrees C) gel-phase spectra of deuterated head-group [alpha-2H2]DHPC remain an order of magnitude narrower than those observed for [alpha-2H2]DPPC.(ABSTRACT TRUNCATED AT 250 WORDS)