By copolymerization of acrylamide and allyl glycosides of various sugars, O-glycosyl derivatives of polyacrylamide copolymers were prepared. The sugar content of the copolymers can be varied in the range 0--40%, their sedimentation coefficient shows the vales of 2.5-5.7 S; the molecular weight of an O-alpha-D-mannopyranosyl polyacrylamide copolymer (29% mannose, so20,w = 2.9 S) was estimated as 44 500. Copolymers with incorporated glycosyl residues interacting specifically with lectins yield precipitates with them upon immunodiffusion in cellulose acetate. The quantitative precipitin curves obtained with these copolymers are similar to those produced by quantitative precipitation of lectins with natural polysaccharides. The copolymers may serve as model substances of natural polysaccharides. 相似文献
Hydrophilic water-insoluble gels suitable for affinity chromatography of lectins have been prepared by copolymerization of acrylamide, N,N′-methylene bisacrylamide and alkenyl 1-thioglycosides. Water-soluble copolymers of analogous type have been obtained by omitting the cross-linking agent, N,N′-methylene bisacrylamide.In affinity chromatography of the Ricinus communis lectin it could be shown that the capacity for the lectin of the water insoluble copolymers was more than four times higher in copolymers having the ligand attached through a methylene bridge than in derivatives with a nonamethylene spacer.None of the insoluble copolymers prepared could be shown usable as affinity adsorbent for glycosidases though the corresponding soluble copolymers inhibited the activity of the enzymes. 相似文献
A number of lectins has been purified by affinity chromatography on O-glycosyl polyacrylamide gels. The lectins isolated (and the particular sugar ligands used in the affinity carriers) are as follows: Anguilla anguilla, serum (α-L-fucosyl-), Vicia cracca, seeds; Phaseolus lunatus, seeds; Glycine soja, seeds; Dolichos biflorus, seeds; Maclura pomifera, seeds; Sarothamnus scoparius, seeds; Helix pomatia, ablumin glands; Clitocybe nebularis, fruiting bodies (all ); Ricinus communis, seeds (β-lactosyl-); Onomis spinosa, root; Fomes fomentarius, fruiting bodies; Marasmius oreades, fruiting bodies (all ), Canavalia ensiformis, seeds, (i.e., concanavalin A) ().Physicochemical properties of Glycine soja, Dolichos bifloras, Phaseolus lunatus, Helix pomatia and Ricinus communis lectins correponded well to properties of the preparations studied earlier by other workers. For the other purified lectins the essential physicochemical data (sedimentation coefficient, molecular weight, subunit composition, electrophoretic patterns, amino acid composition, carbohydrate content, isoelectric point) were established and their precipitating, hemagglutinating and mitogenic activities determined. 相似文献
Free monosaccharides can be used for direct glycosylation of Spheron, a spherical macroporous hydroxyyalkyl methacrylate-ethylene dimethacrylate copolymer, in a reaction that proceeds at room temperature in dioxane medium under catalysis of dry HCl or BF3. Derivatives of L-fucose, D-galactose, D-glucose, D-mannose, and thus prepared from Spheron beads have been shown to be efficient affinity carriers in isolation of lectins from seeds of Canavalia ensiformis D.C. (concanavalin A), Dolichos biflorus L., Glycine soja (L.) Sieb. et Zucc., Lens esculenta Moench, Ricinus communis L., Ulex europaeus L. and from albumin glands of the garden snail Helix pomatia L. 相似文献
Lectins were specifically adsorbed from solution onto metallized glass slides coated with polysacchride, glycopeptide and glycoprotein films. The degree of interaction was determined by measuring the thickness of the bound lectin layer with an ellipsometer after washing and drying the slide. The binding of concanavalin A (tetrameric) and succinyl concanavalin A (dimeric) to a yeast mannan film was studied as a function of lectin concentration, temperature, rinsing time and the extent of stirring of the slide. The maximum thickness of bound concanavalin A and succinyl concanavalin A was 11 and 3.8 nm, respectively. The method permitted the measurement of the association constants for both lectins (1.0 · 107 M?1 for concanavalin A, 2 · 106 M?1 for succinyl concanavalin A) and the detection of 0.6 pmol concanavalin A. The same sensitivity was observed with anti-mannan antibodies. The binding of both lectins was shown to be specific using sugar haptens. When compared with methyl α-D-mannoside, the affinity of concanavalin A for D-mannose and D-glucose was 14 and 3%, respectively. A film of mucin glycopeptide (universal adsorbent) interacted similarly with concanavalin A, Ricinus communis I, soya bean and wheat germ lectins. However, films of glycoproteins such as fetuin, ceruloplasmin and Aspergillus niger β-D-galactosidase interacted to different degrees with these lectins. The relative affinity of wheat germ agglutinin for and for chitin-derived oligosaccharides was also determined. When films of sialoglyproteins were treated with neuraminidase, the thickness of the bound peanut agglutinin layer increased. Although this method cannot determine quantitatively the sugar composition of the film, it permits rapid estimation of the interaction of lectins with polysaccharides and glycoproteins, usingg little material. 相似文献
A method was developed for quantitative determination of 5α,7α-dihydroxy-11-ketotetranor-prostane-1,16-dioic acid, the major urinary metabolite of prostaglandins F1α and F2α in man. The method was based on the use of the O-methyloxime derivative of [5β-3H; 10,10,12,12-2H4]5α,7α-dihydroxy-11-ketotetranor-prostane-1,16-dioic acid as internal standard and determination of ratios between unlabeled and deuterium-labeled molecules by multiple-ion analysis. Excretion values found for healthy human subjects were: males, (n = 10, mean value, 24.0 ± 17.2 (SD) μg) and females, (n = 10, mean value, 10.5 ± 2.1 (SD) μg). 相似文献
We have investigated the interaction of three lectins, differing in their sugar specificities, with the surface of the three differentiation stages of Trypanosoma cruzi. The Scatchard constants for each lectin and parasite stage imply that differentiation of T. cruzi is accompanied by changes in the cell surface saccharides. Trypomastigotes obtained from two different sources do not differ appreciably as to the number and affinity of binding sites for the three lectins employed, suggesting a similar cell-surface saccharide composition. These conclusions are reinforced by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the 131I-labeled surface glycoproteins, following isolation by affinity chromatography. The surface membrane of trypomastigotes, the infective stage to T. cruzi for mammalian cells, possesses a specific glycoprotein of apparent Mr 85 000 (Tc-85) which is absent from the other two stages and can be isolated by affinity chromatography on wheat germ agglutinin-Sepharose columns. This glycoprotein also binds to concanavalin A, but not to Lens culinaris lectin. The binding of Tc-85 to wheat germ agglutinin is unnafected by treatment of either the isolated glycoprotein or intact living trypomastigotes with neuraminidase. Since inhibits internalization of trypomastigotes by cultured mammalian cells, it is suggested that Tc-85 might be involved in adhesion and/or interiorization of T. cruzi into mammalian cells, possibly via recognition of an ubiquitous host-cell surface receptor activity. 相似文献
[1-14C]acetate was readily incorporated into unsaturated fatty acids by leaf slices of spinach, barley and whole cells of and . In these systems the [14C] label in newly synthesized oleate and linoleate was approximately equally distributed in the C1–9 and the C10–18 fragments obtained by reductive ozonolysis of these acids, whereas in a-linolenic acid over 90% of the total [14C] was localized in the C1–9 fragment. While [1-14C]oleic acid was converted by whole cells of to [1-14C]linoleic and [1-14C]linolenic acids, [U-14C]oleic acid yielded [U-14C]linoleic acid but a-linolenic acid was labeled only in the carboxyl terminal carbon atoms. When spinach leaf slices were supplied with carboxyl labeled octanoic, decanoic, dodecanoic, tetradecanoic and octadecanoic acids, only the first three acids were converted to a-linolenic acids while the last two acids were ineffective. Thus we suggest that (a) linoleic acid is not the precursor of a-linolenic acid and (b) 12:3(3, 6, 9) is the earliest permissible trienoic acid which is then elongated to a-linolenic acid. 相似文献
Motility of a marine alkali-tolerant bacterium, Vibrio alginolyticus, can be observed in the presence of high concentrations of a protonophorous uncoupler, CCCP. Motility in the CCCP-containing media is completely inhibited by decrease in extracellular [Na+] or by monensin-induced increase in intracellular [Na+]. A mutant has been selected that grows only in media supplemented with a substrate such as acetate requiring no ΔNa to be transported into the cell. Motility of the mutant was found to be completely inhibited by CCCP. Cyanide, CCCP and vanadate added separately or in twos inhibit motility only partially. The three poisons added together completely paralyse the cells. In this inhibitor cocktail, arsenate can substitute for CCCP + vanadate; cyanide can be replaced by anaerobiosis. It is concluded that (i) ΔNa rather than ΔNH powers the flagellar motor of V. alginolyticus in the presence of CCCP, and (ii) in addition to the Na+-motive respiratory chain [Tokuda, H. and Unemoto, T. (1982) J. Biol. Chem. 257, 10007–10014] there is a vanadate and arsenate-sensitive oxygen-independent mechanism of ΔNa generation, presumably an ion-motive ATPase. A suggestion is put forward that circulation of Na+ can replace that of H+ in V. alginolyticus, ΔNa being formed by the Na+-motive respiratory chain and utilized by Na+-solute symporters, the Na+-driven flagellar motor and maybe by a reverse ion-motive ATPase. 相似文献
A panel of five plant lectins with different binding specificities was used to determine if plant lectins could bind specifically to membrane-associated glycolipids. and wheat germ agglutinins both bound specifically to mixed brain gangliosides and globoside I from human erythrocytes. Wheat germ agglutinin also bound to ganglioside GM1 and human erythrocyte ceramide trihexoside, but not to ceramide dihexoside, mono-, or digalactosyl diglycerides. Concanavalin A bound to liposomes with or without glycolipid substituents, and this binding was partially inhibited by α-methyl mannoside. This study indicates that lectins can specifically recognize and bind to certain glycolipids in membranes. 相似文献
Extracts of the cohesive forms of the cellular slime molds Dictyostelium discoideum, Dictyostelium mucoroides and Dictyostelium purpureum contain lectin activity, assayed as hemagglutination activity. The lectin activity from each species binds quantitatively to Sepharose 4B and can be eluted with d-galactose. The resultant purified lectins are abundant proteins representing, in the case of D. purpureum, up to 5% of the total soluble protein of cohesive cells. The preparations from each species are similar but distinct in amino acid composition and other properties. Each purified preparation gives rise to two protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the major band representing as little at 77% (D. purpureum) and as much as 96% (D. mucoroides) of the total protein in the two bands. The molecular weights of the pair of bands were different for each species, ranging between about 23 000 and 26 000. The two bands are believed to represent subunits of lectins made up of either one or a combination of these two proteins. The apparent molecular weights of the purified lectin activities determined by sucrose density gradient centrifugation were all in the range of 100 000. was a potent inhibitor of the hemagglutination activity of each preparation; but there were some differences in the relative inhibitory potency of a number of other saccharides. Antiserum raised against each preparation, as well as univalent antibody fragments derived from these antisera, reacted best with the antigens to which they were raised; but showed some cross reaction measured both by precipitin reactions and by inhibition of hemagglutination activity of the purified lectins. The differences between the lectins from the different species could be trivial; but they also could be important for defining specific properties of these three species which reliably segregate into colonies of a single species when grown in mixed culture. 相似文献
A-Side (4-R)-(4-2H)-reduced nicotinamide adenine dinucleotide (NADD) was prepared by a stepwise oxidation of ethanol-d6 to acetate in the presence of NAD, alcohol dehydrogenase, and aldehyde dehydrogenase. The B-side (4-S) isomer of NADD was prepared using the glucose dehydrogenase activity of glucose-6-phosphate dehydrogenase to oxidize to oxidize glucose-1-d in 40% dimethyl aulfoxide. Subsequent purifieation of the reduced nucleotides was achieved using a column of strongly basic polystyrene macroporous resin (AG MP-1) eluted with 0.2 m LiCl, pH 10, and applying the pooled NADD peak to a polyacrylamide gel (Bio-Gel P-2) column. The final ratio obtained for these preparations was below 2.3. Preparation of the deuterated reduced nucleotides in this manner allows production of specifieally deuterated substrates by coupled enzymatic synthesis. L-Malate-2-d was prepared by coupled synthesis of A-side NADD to the reduction of oxaloacetate by the A-side enzyme malate dehydrogenase. 相似文献
Yeast phosphofructokinase (PFK) exists in two forms, an ATP-sensitive form, PFKs, and a desensitized form, PFKd(MgF+). PFKs exhibits sigmoidal kinetics with respect to Fru-6-P, whereby the S0.5, Fru-6-P is determined by [ATP]. This form of PFK is inhibited by ATP and citrate and allosterically activated by Fru-6-P and AMP. NH4+ activates PFKs and enhances its affinity for substrate Fru-6-P (1–3).PFKd(MgF+) in contrast is not inhibited by ATP and citrate, nor activated by Fru-6-P and AMP. Kinetics of the reaction with PFKd(MgF+) with respect to Fru6-P are hyperbolic, with of S0.5, Fm-6-P for PFKs. NH4+ strongly activates this form.In terms of the model of Monod et al. (4), PFKd(MgF+) corresponds to a fixed R-conformation, while PFKs is a limiting T-conformation. 相似文献
The carotenoids of selected Cryptophyceae, Rhodomonas D3 and Cryptomonas ovata, have been examined by methods including HPLC, mass spectrometry 1H NMR and circular dichroism. has been assigned to monadoxanthin from 1H NMR and CD data; β,?-carotene possessed the common . The quantitative distribution pattern of carotenoids in Cryptophyceae established here and previously, totalling five species' is discussed in chemosystematic context. β,?-Carotene (3–8% of total) is the major carotene, accompanied by ?,?-carotene (0.2%), β,β-carotene (0–1%) and lycopene (0-trace). Zeaxanthin (2%) was identified in C. ovate. The diacetylenic alloxanthin is the major carotenoid (70–88%), and the monoacetylenic crocoxanthin (5–15%) and monadoxanthin (0–16%) less abundant. No epoxidic or allenic carotenoids could be detected. The biosynthetic precursor of acetylenic carotenoids in this primitive algal class is discussed. The significance of Cryptophyceae in the marine food chain is commented on, using alloxanthin as an indicator. 相似文献
Binding activity for the cage convulsant [35S]--butylbicyclophosphorothionate, which appears to label a site closely associated with the chloride ionophore of the GABAA/benzodiazepine receptor complex has been solubilized from rat cerebral cortex using the zwitterionic detergent CHAPS. Of several detergents screened, only CHAPS and CHAPSO were capable of solubilizing the binding activity with good recovery. The pharmacologic specificity of soluble [35S]--butylbicyclophosphorothionate binding is very similar to the membrane state. In both the membrane and soluble state, [35S]--butylbicyclophosphorothionate binding is enhanced by anions which support inhibitory post-synaptic potentials (“Eccles anions”), suggesting that [35S]--butylbicyclophosphorothionate may label chloride channels thought to be involved in these potentials. Since this solubilization procedure also preserves GABA and benzodiazepine binding and their regulation by drugs such as barbiturates, purification and isolation of the macromolecular complex including chloride channel and GABA-benzodiazepine sites may be feasible. 相似文献
Four subunits of the acetylcholine receptor molecule, obtained from the electric organ of , have been isolated using polyacrylamide gel electrophoresis, and assayed by titration with a fluorescent lanthanide, terbium, and by affinity-labeling with -(-maleimido)benzyl [trimethyl-3H] ammonium iodide. The site with which the activator-analogue affinity label reacts, as well as the terbium-binding sites, are mainly associated with the smallest of the subunits of an apparent molecular weight of 40,000. Calcium competes with terbium for these binding sites. The affinity for terbium is the same in the intact molecule as in the subunit (KTb ? 19 ± 1 μM), but the affinity for calcium decreases by a factor of 4 (KCa ? 4 mM) in the subunit. Hydrolysis of the receptor, catalyzed by trypsin and chymotrypsin, to peptides with an apparent molecular weight of 8000 or less, does not affect the terbium-binding sites. These experiments indicate that the binding sites for neural activators and for calcium are associated with the same subunit, and that the terbium- and calcium-binding sites reflect structural properties of the polypeptide chain rather than the three-dimensional structure of the protein. 相似文献
PGA1 was incubated with rabbit renal cortical homogenates containing HSA (0–4.5%). The ability of this tissue to readily metabolize PGA1 progressively decreased with increasing HSA levels in the incubates The rate of disappearance of 3H-PGA1 was twice as rapid in rats treated with salicylic acid (S. A.) in comparison to control animals; since only 30% of the injected radioactivity was bound to the plasma of the S.A. treated rats, as compared to 90% bound to control plasma, an association may exist between the degree of binding of 3H-PGA1 and its rate of clearance. The studies indicate that PGA1 interaction with HSA decreases its metabolism , and slows down its clearance , implicating HSA as a possible factor in prostaglandins metabolism . 相似文献
The polyether bridged diphosphines, (n = 1,2) have been prepared in 60–70% yield by reduction of the corresponding diphosphinedioxides with Si2Cl6 or (i-Bu)2AlH. These diphosphinedioxides have been prepared in 75–90% yield by reaction of two equivalents of the appropriate with one equivalent of di- and triethylene glycol ditosylate.In general, reaction between the diphosphines, Rh(COD)acac and HClO4 gives a mixture of species, cis-[Rh(COD)()] [ClO4] being the main complex. This complex reacts with CO to η3-trans- [)] [ClO4]. 相似文献