Johan Järnefelt

Neutral and acidic glycopeptides in adult and developing rat brain

Abstract 1. 1. Glycopeptides were released from the lipid-free residue of rat brain by proteolytic digestion. Glycosaminoglycans and nucleic acids were removed from the digest by precipitation with cetyl pyridinium chloride. The glycopeptides were purified in a quantitative yield by gel filtration. 2. 2. A simple method was developed for fractionation of the glycopeptides by ion-exchange chromatography. Two distinct groups of glycopeptides were separated. 3. 3. The glycopeptide groups were characterized by gel filtration, ion-exchange chromatography and gas-liquid chromatographic analysis. The two groups of glycopeptides differed clearly in molecular size and sugar composition. The neutral glycopeptides, which account for 45% of the glycopeptide carbohydrate in rat brain, mostly contain mannose and N- acetylglucosamine in a ratio of 5:2, and have an average composition of 9–12 sugar residues per molecule. About 55% of the glycopeptide carbohydrate is found in the acidic glycopeptides, which contain fucose, mannose, galactose, N- acetylglucosamine , N- acetylgalactosamine and N- acetylneuraminic acid. The acidic glycopeptides, on average, are composed of 15–20 sugar residues per molecule. 4. 4. Qualitative and quantitative changes in the sugars composing the neutral and acidic glycopeptides and glycosaminoglycans were followed during the postnatal development of the rat brain between the ages of 0 and 60 days. There was a rise of about 50% in the glycopeptide content during the first four weeks to the adult level. The increase in the acidic glycopeptides was less great than in the neutral glycopeptides. N- acetylneuraminic acid and N- acetylgalactosamine differed from all other glycoprotein sugars in that their levels were highest between the ages of five and ten days. The amount of glycosaminoglycans decreased during development. This decrease was for the most part due to hyaluronic acid.

The red cell surface revisited

Abstract The erythrocyte surface is densely studded with carbohydrate, either as glycolipid or as glycoprotein. About half of this carbohydrate is in the polylactosamine-type structures, which are also principal carriers of ABH-blood group determinants. The high density of erythrocyte surface carbohydrate may have important consequences for the stability of the cell, but the more specific functional properties of the carbohydrates are unknown.

Methylation techniques in the structural analysis of glycoproteins and glycolipids.

Publisher Summary This chapter describes the methylation techniques used in the structural analysis of glycoproteins and glycolipids. The methylation reaction can be directly applied to the analysis of glycolipids, whereas glycoproteins are not generally methylated directly due to their low solubility in dimethyl sulfoxide. The carbohydrate moiety of the glycoprotein is first isolated as a glycopeptide after extensive proteolytic digestion, or as a reduced oligosaccharide after treatment with alkaline sodium borohydride. Completeness of the methylation reaction is a prerequisite for successful methylation analysis. The critical step in the permethylation procedure is the generation of the sugar alkoxides catalyzed by methylsulfinyl carbanion. It is to be expected that the extensive formation of the alkoxide species has occurred if an excess of the carbanion can still be detected in the mixture after the carbohydrates have reacted. Commercial tert -butoxide salt can be dissolved in dimethyl sulfoxide without any further steps. When this method is used, the intensities of the nonsugar signals in gas liquid chromatography (GLC) tend to be lowering than those seen when the reagent is prepared with the aid of sodium hydride. Methanolysis is also preferred in the analysis of N -acetyl- and N -glycolylneuraminic acid residues, which are commonly found in animal glycolipids and glycoproteins. The quantitation of the methylated monosaccharides is also elaborated in the chapter.

Lipid requirements of functional membrane structures as indicated by the reversible inactivation of (Na+-K+)-ATPase

Abstract 1. 1. Extraction of the (Na + -K + )-ATPase of the electric organ of Ellectrophorus electricus with hexane removes almost all of the cholesterol from the particles. ATPase activity and phospholipid content are left essentially intact. 2. 2. Subsequent removal of the major portion (70–80 %) of the phospholipids by hexane-ethanol extraction reduces the ATPase activity to about 10–15 % of the original. 3. 3. Recombination experiments show that cholesterol and phospholipids are taken up by the extracted proteins. Compared to the amounts originally present, the amounts of cholesterol bound are small, about 20 %, whereas the relative phospholipid concentration is returned to levels existing prior to hexane-ethanol extraction. 4. 4. In recombination experiments cholesterol has a considerably higher ability to reactivate the ATPase than phospholipids, especially if the relative amounts of bound lipids are considered.

Interaction of thallous ions with the cation transport mechanism of erythrocytes

Abstract 1. 1. Tl+ can replace K+ in the activation of the ATPase of fragmented red cell membranes. In the presence of Na+, equal activation is obtained with Tl+ concentrations only 1/10 of those of K+. In all other respects the ( Na + + K + )- and (Na + + Tl + )-ATPases were found to be identical. 2. 2. The transport of Tl+ into erythrocytes was found to be independent of ouabain and glucose. The stationary cell/medium distribution of 204Tl+ was only 1.8–2.0, i.e. close to reciprocal values of the passive distribution of small anions. The inward rate constants were 9.36 h−1 for 204Tl+ and 0.153 h− for K+. The corresponding outward rate constants were 4.68 h−1 and 0.0066 h−1, respectively. 3. 3. Tl+ could substitute for K+ in supporting the ouabain-sensitive outflux of 22Na+ from red cells. A similar outflux of 22Na+ was observed at 1 mM Tl+ and 10 mM K+. At higher concentrations Tl+, inhibited the ouabain-sensitive 22Na+ outflux. The inhibition was greater in fresh erythrocytes than in cells stored in the cold for about 1 week or more. The difference in the degree of the Tl+ inhibition may be attributed to the different Na+ concentrations in fresh and old erythrocytes. 4. 4. On the basis of the results obtained it is suggested that the mechanism of active ion transport involves a destruction of the ion hydration shells.

Factors affecting the relative magnitudes of the ouabain-sensitive and the ouabian-insensitive fluxes of thallium ion in erythrocytes

A maximal rate of the ouabain-sensitive 204-Tl influx in human erythrocytes can be attained at trace concentrations of Tl+ in Mg2+ isotonic media free of K+ and Na+. The maximal influx of Tl+ from isotonic Mg(NO3)2 at 20 degrees C and pH 7.4 was 0.45 mM.l(-1).h-1 with a Km of 0.025 mM. In contrast to the active influx of Tl+, the passive Tl+ fluxes were neither saturated nor influenced by external cations in the range of concentrations of Tl+ and K+ studied. The rate constants of Tl+ passive fluxes in human and cat erythrocytes can be related to pH by the equation log kin(OUT)= -A + B.pH, where A and B are empirical constants for particular conditions. The apparent activation energy was 16 and 11 kcal/mol in sulphate and nitrate media, respectively. Tl+ and the alkali metal cations seem to overcome a common barrier in the erythrocyte membrane. Nevertheless, the rate of the passive penetration of Tl+ is about two orders of magnitude faster than those of K+ or Rb+. An extra non-Coulombic interaction between Tl+ and membrane ligands appears to be involved providing an accumulation of Tl+ somewhere in the vicinity of the membrane barrier and increasing the diffusion fluxes of Tl+ in both directions.

Protein-bound oligosaccharides of cell membranes

Membrane glycoproteins contain several different types of carbohydrate side chain. Fractionation reveals four major classes, each having characteristic structural features. The relative abundance of the various carbohydrate structures is different in different tissues. This supports the view that glycoproteins, through their carbohydrate portions, express some of the cell-membrane specificity.

Some properties of brain cell suspensions prepared by a mechanical-enzymic method.

Abstract Rat brain tissue was dispersed in cell suspensions by applying enzymic digestion combined with high frequency shaking and a sequence of meshes. The enzymes used were (a) a combination of collagenase and hyaluronidase and (b) trypsin. Shaking was performed using a ball vibrator with a shaking rate of some 10,000/min. Samples of the cell suspension were examined by light microscopy after routine staining. Isolated cells and disintegrated cellular material were found. In trypsin-treated samples the nuclei did not stain and the level of DNA/mg protein was only some 40% of the collagenase-treated samples. Cellular integrity was assessed by measuring the distribution of inulin, sodium and potassium in the centrifuged cellular pellets and the bathing medium. Collagenase-hyaluronidase-treated cells were somewhat more intact. In metaboic studies respiration and amino acid incorporation into protein were measured in cell suspensions. These results were compared with values obtained by brain slices. Trypsin-treated suspensions yielded higher values than slices both in respiration and incorporation. Collegenase-hyaluronidase-treated suspensions were also active in respiration and incorporation of amino acids.

Thallium inhibition of ouabain-sensitive sodium transport and of the (Na++K+)-ATPase in human erythrocytes

The influence of Tl+ on Na+ transport and on the ATPase activity in human erythrocytes was studied. 0.1-1.0 mM Tl+ added to a K+-free medium inhibited the ouabain-sensitive self-exchange of Na+ and activated both the ouabain-sensitive 22Na outward transport and the transport related ATPase. 5-10mM external Tl+ caused inhibition of the ouabain-sensitive 22Na efflux as well as the (Na+ plus Tl+)-ATPase. Competition between the internal Na+ and rapidly penetrating thallous ions at the inner Na+-specific binding sites of the erythrocyte membrane could account for the inhibitory effect of Tl+. An increase of the internal Na+ concentration in erythrocytes or in ghosts protected the system against the inhibitory effect of high concentration of Tl+. A protective effect of Na+ was also demonstrated on the (Na+ plus Tl+)-ATPase of fragmented erythrocyte membranes studied at various Na+ and Tl+ concentrations.

Molecular nature of the blood-group ABH antigens of the human erythrocyte membrane

The oligosaccharide structures specifying the blood-group ABH determinants occur in the human erythrocyte membrane in different classes of compounds. The majority occur in a novel class of complex carbohydrate chains called the polyglycosyl chains. They are bound by an alkali-stable bond to glycoproteins (band 3, band 4.5) and occur also in glycolipids. Conventional glycosphingolipids as well as alkali-labile carbohydrate chains of glycoproteins (in the PAS-bands) are also carriers of the blood-group determinants.