Lectin receptors in Trypanosoma cruzi an N-acetyl-d-glucosamine-containing surface glycoprotein specific for the trypomastigote stage

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 M_r 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 $\text{N-}\text{acetyl}\text{-}\text{d}\text{-}\text{glucosamine}$ 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 $\text{N-}\text{acetyl}\text{-}\text{d}\text{-}\text{glucosamine}\text{-}\text{specific}$ receptor activity.     

The linkage of teleost skin keratan sulfate to protein

The linkage of teleost skin keratan sulfate to protein was investigated. Afeter its exhaustive digestion with pronase, peptidokeratan sulfate was obtained with aspartic acid as the predominant amino acid. The N-terminal of the amino acid residues of the preparation was dansylated, and the carbohydrate-peptide linkage fragment was isolated, with the aid of fluorescence, by sequential digestion with Flavobacterium endo-β-galactosidase, β-galactosidase, β-N-acetylhexosaminidase and endo-β-N-acetylglucosaminidase D, followed by Bio-Gel p-4 column chromatography. The structure of the dansylated fragment thus obtained was identified dansylated asparaginyl $\text{N-}\text{acetyl-}\text{D}\text{-glucosamine}$. Treatment of the dansylated keratan sulfate peptide with almond glycopeptidase, which specially cleaves thet asparaginyl $\text{N-}\text{acetyl-}\text{D}\text{-glucosamine}$ linkage in the glycoproteins, also showed asparaginyl $\text{N-}\text{aceytyl-}\text{D}\text{-glucosamine}$ linkage to be in the core region of this keratan sulfate. We conclude that teleost skin keratan sulfate is bound to protein via an N-glycosyl linkage between $\text{N-}\text{acetyl-}\text{D}\text{-glucosamine}$ and asparagine. The keratan sulfate core apparently consist of trimannosyl-N,N′-diacetylchitobiose units, considering the specificity of endo-β-N-acetylglucosaminidase D.     

The nature of mannose-containing material which accumulates in cultured fibroblasts from patients with mannosidosis

Fibroblasts from a patient with mannosidosis were grown in a medium containing a radioactive monosaccharide (D[U-¹⁴C]mannose or $\text{N-}\text{acetyl}\text{-}\text{D}\text{-[1-14}\text{C}\text{]-}\text{glucosamine}$). An accumulation of radioactive material was observed. It was possible to prevent the accumulation to a certain degree by the addition of human liver α-D-mannosidase to the fibroblast medium. After six days of fibroblast culture the majority of the accumulated material had a molecular weight in the oligosaccharide range and was stationary during high-voltage electropresis. Paper chromatography of the stationary material separated three radioactive compounds with the same chromatographic mobilities as the oligosaccharides $\text{α-}\text{D}\text{-}\text{Man}\text{-(1 → 3)-β-}\text{D}\text{-}\text{Man}\text{-(1 → 4)-}\text{D}\text{-}\text{GlcNAc}\text{(I), α-}\text{D}\text{-}\text{Man}\text{-(1 → 2)- α-}\text{D}\text{-}\text{Man}\text{-(1 → 3)-β-}\text{D}\text{-}\text{Man}\text{-(1 → 4)-}\text{GlcNAc}$ (II), and $\text{α-}\text{D}\text{-}\text{Man}\text{-(1 → 2)-α-}\text{D}\text{-}\text{Man}\text{- (1 → 2)-α-}\text{D}\text{-}\text{Man}\text{-(1 → 3)-β-}\text{D}\text{-}\text{Man}\text{-(1 → 4)-}\text{GlcNAc}$ (III) previously isolated from the urine of patients with mannosidosis. Degradation of the three radioactive compounds with jack bean α-mannosidase gave D-mannose and a disaccharide (containing D-mannose and $\text{N-}\text{acetyl}\text{-}\text{D}\text{-}\text{glucosamine}$). Thus the three main compounds observed in the fibroblast from patients with mannosidosis are most probably identical to the oligosaccharides I–III.     

Emission-wavelength-dependent decay of the fluorescent probe N-phenyl-1-naphthylamine

We have measured the fluorescence decay of $\text{N-}\text{phenyl}\text{-1-}\text{naphthylamine}$ using the phase-modulation method, in several solvent systems and egg phosphatidylcholine vesicles. The decay is monoexponential in pure solvents (both polar and non-polar) of low viscosity. In polar viscous solvents or in non-polar solvents containing an added polar solute, the decay is heterogeneous and emission wavelength dependent. In such cases, dielectric relaxation and/or excited-state complexing give rise to a shift of the emission spectrum on the nanosecond time scale. Emission-wavelength-dependent decay was also observed when $\text{N-}\text{phenyl}\text{-1-}\text{naphthylamine}$ was bound to egg phosphatidylcholine vesicles. From these results as well as the position of the emission spectral maximum, we conclude that $\text{N-}\text{phenyl}\text{-1-}\text{naphthylamine}$ probes the ester-carbonyl region of the phospholipid acyl chains, where it undergoes an excited-state reaction. This result contradicts the often made assumption that $\text{N-}\text{phenyl}\text{-1-}\text{naphthylamine}$ probes the deeper hydrocarbon region of the bilayer.     

Enantiomer selection in the nucleophilic addition reaction of optically active amines to α-amino acid N-carboxyanhydrides

The enantiomer selection in the nucleophilic addition reaction of optically active amines such as α-amino acid esters to phenylalanine and $\text{N-}\text{methylphenylalanine}\text{N-}\text{carboxyanhydride}$ in $\text{m-}\text{dimethoxybenzene}$ as a solvent has been investigated. Stereoselectivity between the amines and the $\text{N-}\text{carboxyanhydrides}$ was found to change markedly according to the reaction conditions. This experimental finding is in contrast to the idea hitherto accepted that in the nucleophilic addition-type polymerization of α-amino acid $\text{N-}\text{carboxyanhydride}$ the growing chain end reacts preferentially with one of the enantiomorphic $\text{N-}\text{carboxyanhydrides}$ having the same configuration, and indicates the importance of the investigation of stereoselectivity in the $\text{N-}\text{carboxyanhydride}$ polymerization using suitable model reactions. Most $\text{(S)-α-}\text{amino}$ acid esters reacted preferentially with $\text{(R)-}\text{phenylalanine}\text{N-}\text{carboxyanhydride}$, and this type of stereoselectivity increased with the $\text{N-}\text{methylation}$ of $\text{N-}\text{carboxyanhydride}$ and with increasing bulkiness of the C^α substituent of α-amino acid esters (alanine < norleucine < leucine < valine). The relationship observed between the stereoselectivity and the structures of amines and $\text{N-}\text{carboxyanhydrides}$ was explained satisfactorily in terms of the transition state model in which the interaction of $\text{N-}\text{carboxyanhydride}$ nitrogen and α-amino acid ester carbonyl as well as the interaction of $\text{N-}\text{carboxyanhydride}$ carbonyl and α-amino acid ester nitrogen was taken into account. $\text{(S)-}\text{Proline}$ ethyl ester did not show enantiomer selectivity toward phenylalanine $\text{N-}\text{carboxyanhydride}$, but reacted preferentially with $\text{(S)-(N)-}\text{methylphenylalanine}\text{N-}\text{carboxyanhydride}$. for the reaction of proline ester with $\text{N-}\text{carboxyanhydride}$ a transition-state model was proposed, which was different from the transition state model proposed for other α-amino acid esters. Some experiments were carried out to examine the transition-state models proposed. The implications of the present investigation in stereoselectivity in the nucleophilic addition-type polymerization of $\text{N-}\text{carboxyanhydride}$ hitherto reported are discussed.     

13C magnetic resonance study of the ionization of N-acetyl-dl-proline in aqueous solution

NMR titration curves have been recorded for all the ¹³C resonances of cis and trans$\text{N-}\text{acetyl}\text{-}\text{dl}\text{-}\text{proline}$ in ²H₂O. the measured pK_2H values are 3.4 ± 0.8 and 4.13 ± 0.08 respectively; the free energy of ionization for the trans isomer being (3.8 kJ/mole) greater than for the cis. The ionization shifts of the two isomers differ significantly only at the acetyl carbonyl and C_γ positions. It is suggested that these are related to conformational changes which stabilize the trans form at low p²H.     

Studies on lectins XL. O-glycosyl derivatives of spheron in affinity chromatography of lectins

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, $\text{N-}\text{acetyl}\text{-}\text{D}\text{-}\text{galactosamine}$ and $\text{N-}\text{acetyl}\text{-}\text{D}\text{-}\text{glucosamine}$ 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.     

Effect of N-ethylmaleimide on leucine transport in chang liver cells. I. Stimulatory effect on Na+-independent transport at low concentrations of leucine

Pretreatment of Chang liver cells with $\text{N-}\text{ethylmaleimide}$ (0.5 or 1 mM) stimulated Na⁺-independent uptake of leucine at low concentrations (?1 mM). The stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on the uptake of leucine measured in Na⁺-replete medium was completely blocked by the addition of $\text{b-2-}\text{aminobicyclo}\text{[2,2,1]}\text{heptane}\text{-2-}\text{carboxylate}$ (5 mM), which shows that the L system participates in the stimulation. The Na⁺-dependent uptake of glycine was depressed by $\text{N-}\text{ethylmaleimide}$ pretreatment. The stimulation of the Na⁺-independent component of leucine uptake continued for at least 30 min after $\text{N-}\text{ethylmaleimide}$ treatment, while the inhibition of glycine uptake was progressive with time and the Na⁺-dependent uptake of leucine became depressed later, after the treatment. It has been demonstrated that treatment of cells with $\text{N-}\text{ethylmaleimide}$ is capable of increasing the Na⁺-independent influx of leucine and at the same time slightly decreasing the efflux of it. These results suggest that $\text{N-}\text{ethylmaleimide}$ attacks the Na⁺-independent system of amino acid transport at the reactive SH groups(s) of relevant protein(s) in favor of specific activation of that system in this cell.     

Acid glycohydrolase in Chinese hamster with spontaneous diabetes VII. The lack of short-term glucose-effect in cultured kidney cells

An epithelial cell line, designated CHK-AC_E, was established from the kidney of a spontaneously diabetic Chinese hamster from the highly inbred AC line. CHK-AC_E was separated into two sublines, CHK-AC_E-100 and CHK-AC_E-400, by successive passages in 100 and 400 mg/dl glucose respectively. Extra- and intracellular activities of $\text{N-}\text{acetyl}\text{-β-}\text{D}\text{-}\text{glucosaminidase}$ and β-D-galactosidase were measured in these cultures after exposure to varying concentrations of glucose (100, 200, 300 and 400 mg/dl) for one passage and 10% heated fetal calf serum for 6.5 h before enzyme measurements were taken; no apparent dependence on medium-glucose concentration was found. In serum-free medium, the time-dependent release of both $\text{N-}\text{acetyl}\text{-β-}\text{D}\text{-}\text{glucosaminidase}$ and β-D-galactosidase was sustained for up to 24 h; no significant difference in their activities was found between CHK-AC_E-100 cultures grown in 100 and 400 mg/dl glucose for one passage.     

Properties of a trypsin and chymotrypsin inhibitor secreted by larval Taenia pisiformis

Living metacestodes of Taenia pisiformis maintained in vitro discharge into the surrounding medium a protease inhibitor, which has been purified from the medium by affinity chromatography on bovine α-chymotrypsin immobilized to CNBr-activated Sepharose 4B. The purified inhibitor was shown to inactivate the hydrolysis of $\text{N-α-}\text{benzoyl}\text{-}\text{L}\text{-}\text{arginine}$ ethyl ester and $\text{N-}\text{benzoyl}\text{-}\text{L}\text{-}\text{tyrosine}$ ethyl ester, respectively, by trypsin and chymotrypsin of bovine, rabbit and dog origin, and also the hydrolysis of casein by both bovine trypsin and bovine α and β chymotrypsins, but it did not affect the enzymic activity of subtilisin, elastase, collagenase, pepsin, rennin and papain. The inhibitor withstood heating at 100°C for up to 30 min, was stable in the pH range of 1.5–8.0, was unaffected by 8 M-urea or 0.2 M-2-mercaptoethanol, and had a molecular weight of about 7000 as calculated from its gel chromatographic behaviour. The inhibitor specifically inhibits either trypsin or chymotrypsin with the formation of stable enzyme inhibitor complexes that are not dissociated by 4 M-KCl. Inhibition of trypsin and chymotrypsin is non-competitive and is linear with inhibitor concentration up to 70–80% inhibition. Inhibitory activities toward both enzymes are functions of the same binding site of the inhibitor molecule. Complex formation between the inhibitor and the enzymes is timedependent; it requires 3–4 min for completion.     

Membrane lipid modifications: Biosynthesis and identification of phosphatidyl-N-methyl-N-isopropylethanolamine in rat liver microsomes

In previous studies on the modification of polar head groups of membrane phospholipids with the unnatural base analog, $\text{N-}\text{isopropylethanolamine}$, we reported an unidentified phospholipid in addition to $\text{phosphatidyl}\text{-N-}\text{isopropylethanolamine}$ in the various membrane fractions of rat liver. The structure of this phospholipid has now been identified as $\text{phosphatidyl}\text{-N-}\text{methyl}\text{-N-}\text{isopropylethanolamine}$ by nuclear magnetic resonance spectroscopy, and by chromatographic and enzymic analysis. In addition, we found that when rats were injected intraperitoneally with the $\text{N-}\text{methyl}\text{-N-}\text{isopropylethanolamine}$, 19% of teh liver microsomal phospholipid was $\text{phosphatidyl}\text{-N-}\text{methyl}\text{-N-}\text{isopropylethanolamine}$.     

Studies on avian heart pyruvate kinase during development.

The cytokinin-active nucleoside 6-(4-hydroxy-3-methyl-$\text{cis}$-2-butenylamino)-9-β-$\text{D}$-ribofuranosylpurine, i.e. ribosyl-$\text{cis}$-zeatin, has been isolated from an hydrolysate of tRNA from $\text{Corynebacterium fascians}$. The identification of ribosyl-$\text{cis}$-zeatin is based on biological activity, liquid chromatographic mobility and uv spectrum of the purified material as well as the mass spectrum and gas chromatographic mobility of its trimethylsilylated derivative.     

Lysosomal enzyme secretion by macrophages during intracellular storage of particles

Cultured mouse peritoneal macrophages containing previously endocytosed zymosan or small-fibre asbestos (but not latex or sucrose) were shown to release selectively into the medium the lysosomal hydrolase β-N-acetylglucosaminidase. Thus macrophage lysosomal enzyme secretion was experimentally dissociated from endocytosis (as the residual external particles were washed away from the cells). The cells remained viable, and total activities of both $\text{N-}\text{acetyl}\text{-β-}\text{D}\text{-}\text{glucosaminidase}$ and of lactate dehydrogenase (a cytosol enzyme) rose with time. The relevance of such secretion by macrophages containing stored materials to chronic inflammatory processes is discussed.     

Structural studies on the extracellular polysaccharide of the red alga, Porphyridium cruentum

Partial acid hydrolyzates of the extracellular polysaccharide from Porphyridiunm cruentum yield three disaccharides and two uronic acids. These constitute all of the uronic acid in the polymer. The novel disaccharides are 3-O-(α-$\text{D}$-glucopyranosyl- uronic acid)-$\text{L}$-galactose, 3-O-(2-O-methyl-ca-glucopyranosyluronic acid)-$\text{D}$- galactose, and 3-0-(2-0-methyl-a-$\text{D}$-glucopyranosyluronic acid)-$\text{D}$-glucose. The polyanion of high molecular weight contains $\text{D}$- and $\text{L}$-galactose, xylose, $\text{D}$-glucose, $\text{D}$-glucuronic acid and 2-O-methyl-D-glucuronic acid, and sulfate in molar ratio (relative to $\text{D}$-glucose) of 2.12:2.42:1.00:1.22:2.61. Preliminary periodate-oxidation studies suggest that the hexose and uronic acids are joined to other residues by ( 1→3) glycosidic linkages. About one-half of the xylose residues are (1→3)-linked.     

Hydrodynamic properties of monomeric cytochromes P-450LM2 and P-450LM4 in n-octylglucoside solution

Sedimentation equilibrium and sedimentation velocity measurements were carried out on cytochrome P-450_LM2 from phenobarbital-treated rabbit liver and on cytochrome P-450_LM4 from 5,6-benzoflavone-treated rabbit liver in the presence of the nonionic detergent $\text{1-O-}\text{n}\text{-}\text{octyl}\text{-β-}\text{D}\text{-}\text{glucopyranoside}$. P-450_LM2 was monomeric with a molecular weight of 48,800 and a Stokes radius of 3.1 nm in 7 g/l detergent and P-450_LM4 was monomeric with a molecular weight of 49,800 and a Stokes radius of 2.6 nm at 5 g/l detergent. Both particles were spherical in shape under these conditions. Neither cytochrome was irreversibly denatured at these detergent concentrations as indicated by the ability to form substantial amounts (>60%) of the CO adduct with an absorption maximum at 451 nm (P-450_LM2) or 448 nm (P-450_LM4) when diluted into detergent-free buffer containing CO and sodium dithionite.     

Biosynthesis of N-methyl-l-glucosamine from d-glucose by Streptomyces griseus

Biosynthesis of $\text{N-}\text{methyl}\text{-}\text{l}\text{-}\text{glucosamine}$ moiety of streptomycin from d-glucose by Streptomyces griseus was studied. A mixture of d-[1-¹⁴C]glucose and d-[6-³H]glucose was given to the culture of S. griseus. The ³H/¹⁴C ratio found in $\text{N-}\text{methyl}\text{-}\text{d}\text{-}\text{glucosamine}$ further supports a mechanism that the conversion of d-glucose to l-hexose is carried out without scission of carbon skeleton. When d-[1-¹⁴C]glucose and d-[3-³H]glucose were used, the fall of ³H/¹⁴C ratio in $\text{N-}\text{methyl}\text{-}\text{l}\text{-}\text{glucosamine}$ showed that the hydrogen atom at C-3 plays a rôle in such a transformation.     

A trypsin and chymotrypsin inhibitor from Taenia pisiformis

The somatic extract of mature T. pisiformis has been demonstrated to contain a potent inhibitor capable of inactivating the esterolysis of $\text{N-α-}\text{benzoyl-}\text{L}\text{-arginine ethyl ester}$ and $\text{N-}\text{benzoyl-}\text{L}\text{-tyrosine ethyl ester}$ by trypsin and chymotrypsin, respectively, of bovine, dog and rabbit origin, but not affecting the caseinolytic activity of subtilisin and elastase. The protease inhibitor, partially purified by trichloroacetic acid treatment, Sephadex G-100 column chromatography and affinity chromatography on CNBr-activated Sepharose 4B-bovine chymotrypsin conjugate, was soluble in 5% trichloroacetic acid, stable to heating at 100°C for up to 30 min, tolerated the pH range of 1.5–9.0, and was unaffected by 8 m-urea or 0.2 M-2-mercaptoethanol. The molecular weight of the inhibitor was estimated to be 7000–7200 by Sephadex G-100 chromatography. Activity determinations on crystalline bovine trypsin and chymotrypsin revealed that both inhibitory actions are located on the same or closely adjacent sites of the inhibitor molecule. Complex formation between the inhibitor and mammalian trypsin and chymotrypsin required 3–4 min for completion.     

A soybean lectin having 4-O-Methyl-D-Glucuronic acid specificity

Evidence is presented for the presence of a new lectin activity in soybean seeds [$\text{Glycine}\text{max}$ (L.) Merrill] that has specificity towards the 4-O-methyl-$\text{D}$-glucurono-$\text{L}$-rhamnan exopolysaccharide produced by certain strains of $\text{Rhizobium}\text{japonicum}$. Bacterial agglutination and precipitin reactions revealed the lectin activity in phosphate-buffered saline extracts of seeds of all cultivars tested, including the “lectinless” varieties. Reaction of such extracts with carbohydrate haptens demonstrated that the specificity of the binding was towards 4-$\text{O}$-methyl-$\text{D}$-glucuronic acid, $\text{D}$-glucuronic acid and their methyl glycosides.