Alkali-labile oligosaccharides from bovine milk fat globule membrane clycoprotein

Phenol extraction of bovine milk fat globule membrane gave a glycoprotein fraction which, in sodium dodecyl sulphate electrophoresis, showed three major bands, all staining for both protein and carbohydrate. Alkaline borohydride treatment and desialylation of the glycoprotein fraction released the reduced disaccharide β-d-galactosyl(1 → 3)-$\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ (T-antigen), which was identified by gas chromatography using a standard. All of the disaccharide units in the native glycoprotein were shown to be substituted by sialic acid, and a tetrasaccharide containing the disaccharide plus two molecules of sialic acid was isolated following alkaline borohydride treatment of the glycoprotein and gel filtration. Periodate oxidation of native and desialylated glycoprotein, together with paper chromatography of alkali degraded oligosaccharide fragments, indicated that the major alkali-labile oligosaccharide of the glycoprotein fraction is a tetrasaccharide containing $\text{β-}\text{d}\text{-galactosyl(1 → 3)}\text{-N-}\text{acetyl-}\text{d}\text{-galactosamine}$ substituted by sialic acid at position C3 of the galactosyl and position C6 of the $\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ residue. Evidence was also obtained for the presence of small amounts of unsubstituted alkali-labile $\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ linked directly to protein in the native glycoprotein.Serological evidence using agglutinins from Vicia graminea, Arachis hypogoea and human anti-T serum confirmed the presence in the native glycoprotein of a sialic acid substituted T-antigen. Similar evidence using agglutinins from Helix pomatia and Cepaea hortensis also confirmed the presence of terminal alkali-labile $\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ in the native glycoprotein.     

Comparison of isolated peanut agglutinin receptor glycoproteins from human,bovine and porcine erythrocyte membranes

Affinity chromatography has been used to isolate and compare the peanut agglutinin receptors from neuraminidase-treated human, bovine and porcine erythrocyte membranes. Passage of Triton X-100-solubilised membrane material through either Sepharose- or acrylamide-based affinity columns resulted in the reversible binding of receptor molecules to the immobilised lectin. Elution with 0.2M galactose released specifically bound glycoprotein fractions, the composition and molecular weights of which were determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate.Carbohydrate analysis by gas chromatography identified these bound glycoprotein fractions as the major sources of the $\text{O-}\text{glycosidic-linked}$ disaccharide $\text{galactosyl}\text{-β-(1 → 3)-N-}\text{acetylgalactosamine}$ in these membranes. It is suggested that these isolated fractions represent a discrete population of glycoproteins within the membranes studied, which possess both $\text{O-}\text{glycosidic}$- and $\text{N-}\text{glycosidic-linked}$ carbohydrates.     

Mass spectrometry and 13C nuclear magnetic resonance spectroscopy of compounds modeling the glycopeptide linkage of glycoproteins

The properties of several compounds useful as models for three-dimensional conformational studies and the investigation of the chemical degradation of glycopeptide linkages both of the N- and O-glycosidic type are described. Using the method of differential chemical shift in H₂O and D₂O as solvents, the carbon NMR spectrum of N-acetylglucosaminylasparagine, 1-N-acetyl-β-d-glucopyranosylamine, and 1-N-acetyl-2-acetamido-β-d-glucopyranosylamine has been assigned. Electron impact mass spectra of the peracetylated derivatives of the latter two compounds show a peak apparently unique to glycopyranosylamides at $\text{m}\text{e}\text{= 269}$, no analog of which is observed in the mass spectra of other peracetylated sugars. As models of the α-O-glycosidic linkage, fully assigned carbon NMR spectra of α-methyl-N-acetylgalactosamine (GalNAc), α-methyl-3-O-methyl GalNAc,and -GlcNAc as well as the disaccharide Glc-β-1 → 3 GalNAc are reported. Because certain anomalies in the chemical shifts and ¹J_CH observed in the disaccharide and in O-glycosylated glycoproteins are not observed in the simple model compounds, they may result from conformational interactions in the glycopeptides.     

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.     

Steric factors involved in the action of glycosidases and galactose oxidase

α-(1→2)-$\text{L}\text{=}$-Fucosidase, β-$\text{D}\text{=}$-galactosidase and galactose oxidase are sterically hindered by certain types of branching in the oligosaccharide chains. 1) β-$\text{D}\text{=}$-Galactosidase will not cleave galactose when the penultimate sugar carries a sialic acid residue as in I. 2) Galactose Oxidase will not oxidize the galactose residue in trisaccharide I but will in II. Moreover, neither galactose nor $\text{N}$-acetylgalactosamine, glycosidically bound as in III, is susceptible to oxidation with galactose oxidase until the α-(1→2) linkage between them is cleaved by $\text{α-}\text{N}\text{-acetylgalactosaminidase}$. 3) α-(1→2)-$\text{L}\text{=}$-Fucosidase action is inhibited by $\text{α-(1→3)-}\text{N}\text{-acetylgalactosaminyl}$ or galactosyl residue, as in III and IV. Removal of the terminal sugars makes the fucosyl residue susceptible to fucosidase action.

     

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.     

Butyrophilin,an apical plasma membrane-associated glycoprotein characteristic of lactating mammary glands of diverse species

Lipid globule membranes were isolated from human and bovine milk and from the milk of sheep, goat, pig, rat and guinea pig, and their polypeptide compositions were analyzed. The major polypeptides with molecular weights similar to that of bovine butyrophilin were separated by gel electrophoresis, isolated and characterized with respect to isoelectric point, molecular weight, immunological cross-reactivity and peptide composition after proteolytic cleavage. We show that in all species examined these proteins are similar to bovine butyrophilin in (i) their relative insolubility in buffers of low and high ionic strength and in non-denaturing detergents, (ii) the occurrence of several isoelectric variants, and (iii) patterns of peptides obtained by protease digestion. It is concluded that closely related proteins are major constituents of the cytoplasmic coat structures associated with milk lipid globule membranes of many species, and we propose the name butyrophilins for this group of proteins. Bovine and human butyrophilins are glycosylated with relatively large amounts of glucosamine, mannose, glucose and galactose but little fucose, sialic acids or galactosamine. Most if not all of the sugar residues are associated with an acetone-soluble peptide fragment of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 12 000–16 000 focusing at about pH 4.0. We suggest that this fragment contains a membrane-spanning peptide sequence and is involved in the attachment of the cytoplasmic coat to the membrane of the milk lipid globule.     

Some differences in intracellular distribution and properties of the chymotrypsin-like esterase activity of human and rabbit neutrophils

The intracellular localization and properties of the chymotrypsin-like esterase activity ($\text{N-}\text{acetyl}\text{-}\text{DL}\text{-}\text{phenlylalanine}\text{β-}\text{naphthyl}$ esterase acitivity) of the rabbit peritoneal neutrophil has been studied and shown to differ from that of the human neutrophil.The major portion of the esterase activity in the rabbit neutrophil is in the 100 000 × g supernatant fraction with distinctly less activity in the lysosomal fraction. The 100 000 × g supernatant contained the highest relative specific activity of any of the subcellular fractions. Rabbit peripheral blood neutrophils gave the same distribution.The 100 000 × g supernatant esterase is 95% esterase 1 and 5% esterase 3, whereas, the lysosomal esterase is 78% esterase 1, 10–16% esterase 2 and 9% esterase 3 as defined by their ability to be inhibited by p-nitrophenyllethyl-5-chloropentylphosphonate. The 100 000 × g supernatant The 100 000 × g supernatant and lysosomal esterase activities further differ in their susceptibility to other inhibitors, their pH optima, ease of elution from DEAE and isoelectric points. Two molecular weight species of 174 000 and 70 000 were found in the 100 000 × g supernatant fraction and extracts of the lysosomal fraction but usually in differing proportions.In confirmation of others, essentially all of the chymotrypsin-like esterase activity ($\text{N-}\text{acetyl}\text{-}\text{DL}\text{-}\text{phenlylalanine}\text{β-}\text{naphthyl}$ esterase activity) of the human neutrophil is in the lysosomal fraction, unlike the rabbit cell. The human neutrophil esterase was less susceptible to inhibition by p-nitrophenylethyl-5-chloropentylphosphonate and diisopropylphosphofluoridate but more susceptible to soybean trypsin inhibitor than rabbit esterase activity. The pH optimum of the human neutrophil esterase differed from either the rabbit lysosomal or 100 000 × g supernatant esterase, as did the isoelectric point and molecular weights.     

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.     

Glycoprotein characteristics of the sodium channel saxitoxin-binding component from mammalian sarcolemma

The saxitoxin-binding component of the excitable membrane sodium channel exhibits glycoprotein characteristics as evidenced by its specific interaction with various agarose-immobilized lectins. The detergent-solubilized saxitoxin-binding component interacts quantitatively with immobilized wheat germ agglutinin and concanavalin A and fractionally with immobilized Lens culinaris hemagglutinin and Ricinus communis agglutinin. These lectins preferentially bind $\text{N-}\text{acetylglucosamine}$ and sialic acid (wheat germ agglutinin), mannose (concanavalin A and Lens cunilaris and galactose (Ricinus communis). Removal of terminal sialic acid residues by neuraminidase markedly decreases binding to immobilized wheat germ agglutinin but uncovers sites capable of interacting with lectins specific for galactose and $\text{N-}\text{acetylgalactosamine}$. $\text{β-N-}\text{acetylglucosaminidase}$, an exoglycosidase has no effect on the binding of the channel protein to wheat germ agglutinin. Similarly, phospholipase C has no effect on binding of the solubilized toxin binding component to this lectin. Neither wheat germ agglutinin nor concanavalin A free in solution alters the number of toxin binding sites or their affinity for toxin. The sodium channel saxitoxin-binding component appears to be a glycoprotein containing terminal sialic acid residues and internal mannose, galactose, $\text{N-}\text{acetylglucosamine}$, and $\text{N-}\text{acetylgalactosamine}$ residues. The toxin binding site is spatially separated from the binding sites for the lectins studied. The effect of these sugar moieties must be considered when evaluating the biophysical parameters of the sodium channel.     

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.     

Structure of the "keratosulfate-like" material in liver from a patient with G M1 -gangliosidosis ( -D-galactosidase deficiency)

Large amounts of a glycopeptide containing galactose, $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and threonine in the ratio 4:3:1:1, together with smaller amounts of mannose, fucose, sialic acid, sulfate, serine, and other amino acids were isolated from the liver of a patient with G_M1-gangliosidosis. Treatment with mild alkali and sodium borohydride indicated an $\text{O}$-glycosidic linkage between $\text{N}$-acetylgalactosamine and threonine. All the hexosamine residues were resistant to sodium metaperiodate whereas 2 out of 4 D-galactose residues were destroyed. Further studies indicated that one of the galactose residues was 1→3 linked to $\text{N}$-acetylgalactosamine (as in G_M1) and the other 1→4 linked to $\text{N}$-acetylglucosamine as found in skeletal keratosulfate.     

2′(3′)-O-l-phenylalanyl derivatives of N2,5′-anhydroformycin and N4,5′-anhydroformycin: new substrates for ribosomal peptidyltransferase with a fixed anti and syn conformation of the base

The $\text{2′(3′)-O-}\text{l}\text{-}\text{phenylalanyl}\text{-N}{}^2\text{,5′-anhydroformycin}$ (1c) and $\text{2′(3′)-O-}\text{l}\text{-}\text{phenylalanyl}\text{-N}{}^4\text{,5′-anhydroformycin}$ (2c), obtained by chemical synthesis, are substrates for ribosomal peptidyltransferase from Escherichia coli. Nucleoside 1c, which mimics an anti conformation of antibiotic formycin, has 80% of the acceptor activity of puromycin at 5 · 10^?4 M determined by the release of N-Ac-Phe residue from the 70 S $\text{ribosome}\text{-}\text{poly}\text{(}\text{U}\text{)-N-}\text{Ac}\text{-[}{}^{14}\text{C]}\text{Phe-tRNA}$ complex. The reaction product, $\text{2′(3′)-O-(N-}\text{acetyl}\text{)-}\text{l}\text{-}\text{phenylalanyl}\text{-}\text{l}\text{-}\text{phenylalanyl}\text{-N}{}^2\text{,5′-}\text{anhydroformycin}$ (1d), was characterized by paper electrophoresis before and after alkaline hydrolysis. By contrast, nucleoside 2c, which resembles a syn conformation of formycin, exhibited only 20% of the acceptor activity of puromycin at 5 · 10^∮4 M and essentially none in the concentration region between 1 · 10^?6 and 1 · 10^?4 M. The results which are in accord with previous models have shown that a substrate with its base in an anti conformation is preferable for the acceptor site of peptidyltransferase than the corresponding syn counterpart, Nevertheless, it is possible that an intermediate conformation, for example, high anti (amphi-minus), is an optimal arrangement for acceptor site substrates.     

Evaluation on the hydrolysis of methylumbelliferyl-tetra-N-acetylchitotetraoside by various glucosidases. A comparative study

• 1.1. In human plasma, an enzyme is present which hydrolyzes 4-methylumbelliferyl-tetra-N-acetylchitotetraoside. The function of this enzyme is unknown.
• 2.2. We have examined whether hyaluronidase, neutral endoglucosaminidase, $\text{N-}\text{acetyl}\text{-β-}\text{d}\text{-}\text{hexosaminidase}$, aspartylglucosaminidase, β-d-glucosidase, and chitobiase could hydrolyze MU-TACT. The results obtained are detailed below.
• 3.3. A purified commercial preparation of hyaluronidase does not hydrolyze MU-TACT.
• 4.4. Substrate specificity requirements, pH optimum and subcellular localization indicate that neutral endoglucosaminidase is distinguishable from MU-TACT hydrolase. Also commercial neutral endoglucosaminidase D and H have no affinity towards MU-TACT.
• 5.5. $\text{N-}\text{Acetyl}\text{-β-}\text{d}\text{-}\text{hexosaminidase}$ is different from MU-TACT hydrolase for the following reasons: (a) a purified enzyme preparation does not hydrolyze MU-TACT; (b) there is no correlation in the activity of the enzymes; (c) MU-TACT hydrolase is not deficient in cells of a patient with a deficiency of total $\text{N-}\text{acetyl}\text{-β-}\text{d}\text{-}\text{glucosaminidase}$; and (d) the 2 enzymes have very different Chromatographic characteristics and Con A binding properties.
• 6.6. Enzyme characteristics, substrate structural requirements and a lack of correlation with MU-TACT hydrolase activity suggest that aspartylglucosaminidase, β-d-glucosidase, and chitobiase are not involved in the hydrolysis of MU-TACT.
• 7.7. None of the enzymes which we have considered corresponds to MU-TACT hydrolase. The exact nature and the function of the enzyme remains an enigma.

     

Modification of sialic acid metabolism of murine erythroleukemia cells by analogs of N-acetylmannosamine

Two analogs of N-acetylmannosamine, $\text{2-}\text{acetamido}\text{-1,3,4,6-}\text{tetra}\text{-O-}\text{acetyl}\text{-2-}\text{deoxy-}\text{d}\text{-mannopyranose}$ (Ac₄-NAcMan) and the 2-trifluoroacetamido derivative (Ac₄F₃-NAcMan), were synthesized as potential inhibitors of the formation of sialic acid-containing glycoconjugates and were examined for their ability to modify the incorporation of $\text{N-[}{}^3\text{H]acetylmannosamine}$ into cellular glycoconjugates of Friend murine erythroleukemia cells. Ac₄F₃-NAcMan and Ac₄-NAcMan inhibited cellular replication in suspension culture at concentrations of 0.02 and 0.08 mM, respectively. The cytotoxicity of Ac₄-NAcMan was relatively reversible, whereas that produced by Ac₄F₃-NAcMan was not, as judged by measurement of the cloning efficiencies of cells exposed to these agents. The analogs inhibited incorporation of $\text{N-[}{}^3\text{H]acetylmannosamine}$ into ethanol-soluble and -insoluble materials. Separation of ethanol-soluble metabolites by HPLC demonstrated that Ac₄F₃-NAcMan caused accumulation of radioactivity from $\text{N-[}{}^3\text{H]acetylmannosamine}$ in CMP-N-acetylneuraminic acid (CMP-NeuNAc) equal to the decrease in macromolecular-bound ³H caused by this agent. In contrast, similar exposure to Ac₄-NAcMan produced a large increase in the amount of radioactivity in ethanol-soluble N-acetylneuraminic acid while decreasing the amount of label from $\text{N-[}{}^3\text{H]acetylmannosamine}$ in cellular CMP-NeuNAc, suggesting that the analogs differ in their biochemical sites of action. Treatment of cells with either analog increased the amount of neuraminidase-hydrolyzable sialic acid-like material on the cell surface; this appeared to be due to the incorporation of the analogs into cellular glycoconjugates, since incubation of cells with ³H-labeled analogs resulted in the appearance of radioactivity in cellular ethanol-insoluble and neuraminidase-hydrolyzable material. Incubation of cells with Ac₄-NAcMan labeled with ¹⁴C in the 4-O-acetyl group further demonstrated that incorporation occurred with approx. 50% retention of this substituent. Thus, both the amount and the nature of the surface sialic acid constituents of treated cells were altered, suggesting that these or similar analogs could potentially be used to modify cellular membrane function.     

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.     

Theoretical studies on the modes of binding of some of the derivatives of d-mannose to concanavalin A

The possible modes of binding for $\text{methyl}\text{-α-}\text{d}\text{-mannopyranoside}$, $\text{methyl}\text{-β-}\text{d}\text{-mannopyranoside}$, $\text{2-O-}\text{methyl}\text{-α-}\text{d}\text{-mannopyranoside}$, $\text{methyl-2-O-methyl}\text{-α-}\text{d}\text{-mannopyranoside}$ and $\text{methyl}\text{-α-}\text{d}\text{-N-}\text{acetylmannosamine}$ to concanavalin A have been investigated using theoretical methods. All these sugars, except $\text{methyl}\text{-α-}\text{d}\text{-N-}\text{acetylmannosamine}$, reach the active site of concanavalin A with a highly restricted number of binding orientations. Present investigations suggest that the failure of $\text{methyl}\text{-α-}\text{d}\text{-N-}\text{acetylmannosamine}$ to bind to concanavalin A is not so much due to steric factors as to repulsive electrostatic interactions. $\text{Methyl}\text{-2-O-}\text{methyl}\text{-α-}\text{d}\text{-mannopyranoside}$ can bind to concanavalin A in one mode whereas the other sugars can bind in more than one mode. The high potency of $\text{methyl}\text{-α-}\text{d}\text{-mannopyranoside}$ over $\text{methyl}\text{-β-}\text{d}\text{-mannopyranoside}$ is mainly due to the possibility of hydrophobic interactions of the α-methoxy group with Leu(99) or Tyr(100) and also due to the possibility of formation of better and more hydrogen bonds with the protein. A comparison of these data with those for the d-glucopyranosides suggests that the change of the hydroxyl at the C-2 atom from equatorial to axial orientation increases the stereochemically allowed region as well as the possible binding modes. From these studies it is also suggested that the overall shape of the oligosaccharides rather than the terminal or internal mannose alone affects the binding potency of saccharides to concanavalin A.     

Desensitization of the human neutrophil degranulation response: Studies with 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid

The human polymorphonuclear neutrophil degranulation response to 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid was completely desensitized by preincubating the cells with small amounts of this same fatty acid. Desensitization developed within 1 min, persisted in thoroughly washed cells, and was not due to inactivation of the stimulus. These desensitized cells, however, degranulated partially in response to the ionophore A23187 and normally in response to C5a, N-formyl-methionyl-leucyl-phenylalanine, 1-$\text{0}\text{-}\text{alkyl-2-}\text{0}\text{-}\text{acetyl}\text{-}\text{sn}\text{-}\text{glycero-3-phosphocholine}$, and phorbol myristate acetate. Thus, the dihydroxy fatty acid is a unique stimulus which degranulates and desensitizes neutrophils by pathways at least partially distinct from those utilized by the other stimuli. The fatty acid, although rapidly formed in degranulating neutrophils, is unlikely to be an essential or universal mediator of the degranulation response.     

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 $\text{phosphatidyl}\text{-N-}\text{methyl}\text{-N-}\text{isopropylethanolamine}$.     

Plasma membrane NADH dehydrogenase and Ca2+-dependent potassium transport in erythrocytes of several animal species

Ca²⁺-dependent K⁺ transport and plasma membrane NADH dehydrogenase activities have been studied in several ‘high-K⁺’ (human, rabbit and guinea pig) and ‘low-K⁺’ (dog, cat and sheep) erythrocytes. All the species except sheep showed Ca²⁺-dependent K⁺ transport. NADH-ferricyanide reductase was detected in all the species and showed positive correlation with the flavin contents of the membranes. NADH-cytochrome $\text{c}$ reductase was very low or absent in dog, sheep and guinea pig membranes. No correlation was found between NADH dehydrogenase and Ca²⁺-dependent K⁺ channel activities in the species studied. Nor were any of the above activities correlated with $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity.