Isolation from human urines of a mucin with blood group Sda activity

A mucin with Sd^a blood group activity was isolated from human group 0 urines by a multistep procedure including an affinity chromatography on $\text{Helix pomatia}$ - Ultrogel. About 8 mg of active material was obtained from 100 litres of urines. The purified substance of apparent molecular weight 340,000 dalton is not stained by Coomassie blue but gave a single periodic acid-Schiff positive band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analytical composition indicated the absence of mannose, a high content of N-acetylgalactosamine and a molar ratio galactose: N-acetylgalactosamine: N-acetylglucosamine: sialic acid of 2:2:1:2. Amino acid composition is typical of a mucin substance with high values of serine, threonine, proline and alanine. The urinary mucin inhibited human anti-Sd^a antibodies as strongly as the Tamm-Horsfall glycoprotein isolated from Sd(a+) urines. However, the two substances clearly have different composition and properties. It is suggested that oligosaccharide chains with Sd^a blood group activity might be carried by different glycoconjugates of human arines and tissues.     

A simultaneous assay system for N-acetylgalactosamine and N-acetylgalactosaminitol using gas chromatography/mass spectrometry

A procedure is described, using gas chromatography/mass spectrometry to identify and quantitate various sugar alditol acetates, which will simultaneously quantitate N-acetylgalactosamine and N-acetylgalactosaminitol. N-acetylgalactosamine is distinguished from N-acetylgalactosaminitol by reduction with sodium borodeuteride and determining relative intensities of the ion pairs $\text{m}\text{e}$ 84, 85; 102, 103; and 144, 145. The method is adequate for determining amounts of these sugars in the range of 40–1000 nmol.     

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.     

On the oligosaccharide moiety of angiotensin-converting enzyme.

Two glycopeptide fractions in a pronase digest of rabbit pulmonary angiotensin-converting enzyme were resolved by gel filtration. GP-I, the minor component (～1 mole/mol enzyme) contained mannose, galactose, glucose $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and sialic acid in an approximate molar ratio of 1:5:3:4:1:2 and molar equivalents of aspartic acid, threonine and serine. GP-II, the major oligosaccharide unit (～ 12 moles/mol enzyme, ～ 90% of total carbohydrate), contained fucose, mannose, galactose, $\text{N}$-acetylglucosamine, sialic acid and aspartic acid in a molar ratio of 1:4:4:4:1:1. Although accounting for about one-quarter of the weight of the enzyme, GP-II did not compete with the intact glycoprotein for binding to goat antienzyme antibodies. Some structural features of GP-II were deduced by periodate oxidation and digestion with various glycosidases.     

A model for the reaction pathways of the K+-dependent phosphatase activity of the (Na+ + K+)-dependent ATPase

$\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-dependent}$ ATPase preparations from rat brain, dog kidney, and human red blood cells also catalyze a K⁺-dependent phosphatase reaction. K⁺ activation and Na⁺ inhibition of this reaction are described quantitatively by a model featuring isomerization between E₁ and E₂ enzyme conformations with activity proportional to E₂K concentration:

Differences between the three preparations in $\text{K}{}_{0.5}$ for K⁺ activation can then be accounted for by differences in equilibria between E₁K and E₂K with dissociation constants identical. Similarly, reductions in $\text{K}{}_{0.5}$ produced by dimethyl sulfoxide are attributable to shifts in equilibria toward E₂ conformations. Na⁺ stimulation of K⁺-dependent phosphatase activity of brain and red blood cell preparations, demonstrable with KCl under 1 mM, can be accounted for by including a supplementary pathway proportional to E₁Na but dependent also on K⁺ activation through high-affinity sites. With inside-out red blood cell vesicles, K⁺ activation in the absence of Na⁺ is mediated through sites oriented toward the cytoplasm, while in the presence of Na⁺ high-affinity K⁺-sites are oriented extracellularly, as are those of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-dependent}$ ATPase reaction. Dimethyl sulfoxide accentuated Na⁺-stimulated K⁺-dependent phosphatase activity in all three preparations, attributable to shifts from the E₁P to E₂P conformation, with the latter bearing the high-affinity, extracellularly oriented K⁺-sites of the Na⁺-stimulated pathway.     

Biosynthesis of glycosoaminoglycans by microsomal preparations from cultured mastocytoma cells

Neoplastic mast cells of mice (including long-established and newly derived lines) were grown in large-volume suspension cultures to provide enough cells for preparation of microsomal fractions. Microsomal preparations from P815Y and P815S cells synthesized ¹⁴C-labelled glycosaminoglycan when incubated with UDP-[¹⁴C]glucuronic acid and UDP-N-acetylgalactosamine. No significant amount of ¹⁴C-labelled glycosaminoglycan was formed when UDP-N-acetylglucosamine was substituted for the UDP-N-acetylgalactosamine. Microsomal preparations from X163 cells synthesized ¹⁴C-labelled glycosaminoglycan when incubated with UDP-[¹⁴C]glucuronic acid and either UDP-N-acetylgalactosamine or UDP-N-acetylglucosamine. The ¹⁴C-labelled glycosaminoglycan formed in the presence of UDP-N-acetylgalactosamine was degradable by testicular hyaluronidase, indicating that it was chondroitin-like. The ¹⁴C-labelled glycosaminoglycan formed in the presence of UDP-N-acetylglucosamine was not degradable by testicular hyaluronidase. Microsomal preparations from P815S cells were tested for sulphating activity by incubation with adenosine 3′-phosphate 5′-sulphatophosphate, as well as UDP-[¹⁴C]glucuronic acid, and UDP-N-acetylgalactosamine. The resulting newly synthesized polysaccharide was shown by chondroitinase ABC digestion to be 70% chondroitin 4-sulphate and 30% chondroitin. The molecular size of this newly synthesized glycosaminoglycan was determined by gel filtration to be larger than 40000 mol.wt. In general, the glycosaminoglycan-synthesizing ability of the microsomal preparations appeared to reflect glycosaminoglycan synthesis by the intact cells.     

Isolation and purification of the sexual agglutination substance of mating type a cells in Saccharomyces cerevisiae

The substance responsible for the sexual agglutinability was successfully solubilized by a newly established autoclaving method from the surface of mating type $\text{a}$ cells of $\text{Saccharomyces cerevisiae}$ and purified by DEAE cellulose chromatography, gel filtration, affinity chromatography and electrophoresis. The substance was found to consist of at least two different glycoprotein subunits. The molecular weight of the substance was estimated to be about 23,000 daltons by gel filtration. The substance was univalent in its biological activity and specifically masked the sexual agglutinability of the mating type α cells. The substance formed a complementary complex with the agglutination substance from α cells in vitro.     

Isolation and partial characterization of a novel disulfoglycosphingolipid from rat kidney

A novel sulfoglycosphingolipid containing two sulfate ester groups was isolated from the lipid extract of rat kidney by a procedure involving mild alkaline methanolysis and column chromatographies on DEAE-Sephacel and silicic acid. The component carbohydrates were galactose, glucose and $\text{N}$-acetylgalactosamine in equimolar amounts. Infrared spectroscopy, permethylation study, periodate oxidation and solvolysis suggested that the sulfoglycolipid was GalNAc1-4Gal1-4GlcCer sulfated at the C3 hydroxyls of both galactose and $\text{N}$-acetylgalactosamine. The yield of this sulfoglycolipid was 11.2 nmol/g tissue.     

Surface carbohydrates of aged erythrocytes

Human erythrocytes, fractioned into populations of different density by ultracentrifugation in albumin gradients were examined to determine what changes in cell surface carbohydrates occur during their lifespan. In addition to changes occurring in $\text{N}$-acetylneuraminic acid ageing was accompanied by reduction in the $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and galactose content of erythrocyte membranes. These results show that extensive heterogeneity exists in the cell surface carbohydrate of the circulating population of erythrocytes and suggest clearance of neuraminidase treated erythrocytes may not be an adequate model for the removal of aged cells.     

Role of membrane proteins and lipids in water diffusion across red cell membranes

When human red cells are treated with the mercurial sulfhydryl reagent, $\text{p-}\text{chloromercuribenzene sulfonate}$, osmotic water permeability is suppressed and only diffusional water permeability remains (Macey, R.I. and Farmer, R.E.L. (1970) Biochim. Biophys. Acta 211, 104–106). It has been suggested that the route for the remaining water permeation is by diffusion through the membrane lipids. However, after making allowance for the relative lipid area of the membrane, the water diffusion coefficient through lipid bilayers which contain cholesterol is too small by a factor of two or more. We have measured the permeability coefficient of normal human red cells by proton $\text{T}{}_1$ NMR and obtained a value of 4.0 · 10^?3 cm · s^?1, in good agreement with published values. In order to study permeation-through red cell lipids we have perturbed extracted red cell lipids with the lipophilic anesthetic, halothane, and found that halothane increases water permeability. The same concentration of halothane has no effect on the water permeability of human red cells, after maximal pCMBS inhibition. In order to compare halothane mobility in extracted red cell membrane lipids with that in red cell ghost membranes, we have studied halothane quenching of $\text{N-}\text{phenyl}\text{-1-}\text{naphthylamine}$ by equilibrium fluorescence and fluorescence lifetime methods. Since halothane mobility is similar in these two preparations, we have concluded that the primary route of water diffusion in pCMBS-treated red cells is not through membrane lipids, but rather through a membrane protein channel.     

Mitochondrial phosphate transport and the N-ethylmaleimide binding proteins of the inner membrane

Mitochondria have been prepared from the flight muscles of mature blowflies (Sarcophaga bullata). Phosphate transport by these mitochondria, determined by rates of passive swelling in ammonium phosphate, is sensitive to inhibition by N-ethylmaleimide. 20 nmol of N-ethylmaleimide/nmol cytochrome a inhibit the swelling by 90%. When the mitochondria are inhibited by $\text{N-[}{}^3\text{H}\text{]}\text{ethylmaleimide}$, then solubilized in dodecyl sulfate/mercaptoethanol at 100°C and then electrophoresed on dodecyl sulfate-polyacrylamide gels, many labeled protein bands can be detected, including a large labeled peak that has the same mobility as the tracking dye, bromophenol blue. Sonic submitochondrial particles that are prepared from the $\text{N-[}{}^3\text{H}\text{]}\text{ethylmaleimidelabeled}$ mitochondria, solubilized, and electrophoresed on dodecyl sulfatepolyacrylamide gels, possess only seven major labeled protein bands with no radioactive peak at the tracking dye. These labeled proteins have molecular weights of 71, 68, 64, 45, 32, 30, and approx. 10 · 10³. The nmol $\text{N-[}{}^3\text{H}\text{]-}\text{ethylmaleimide}$ bound to each of these proteins per nmol cytochrome a are 0.15, 0.19, 0.35, 0.45, 0.87, 0.10, and 0.17, respectively, when the mitochondria are inhibited with 21.5 mol $\text{N-[}{}^3\text{H}\text{]}\text{ethylmaleimide/mol}$ cytochrome a at 10 μM cytochrome a. Coty and Pedersen ((1975) J. Biol. Chem. 250, 3515–3521) sensitized rat liver mitochondria to $\text{N-[}{}^3\text{H}\text{]}\text{ethylmaleimide}$ and identified five labeled proteins. Only the labeled 32 · 10³ dalton and the 45 · 10³ dalton proteins are common to both systems     

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.     

Compound 4880 is a selective and powerful inhibitor of calmodulin-regulated functions

Compound $\text{48}\text{80}$, a condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde, is composed of a family of cationic amphiphiles differing in the degree of polymerization. Compound $\text{48}\text{80}$ was found to be a potent inhibitor of the calmodulin-activated fraction of brain phosphodiesterase and red blood cell Ca²⁺-transport ATPase, with IC₅₀ values of 0.3 and 0.85 μg/ml, respectively. However, the basal activity of both enzymes is not at all suppressed by the drug at concentrations up to 300 μg/ml. Inhibition of Ca²⁺ transport into inside-out red blood cell vesicles by compound $\text{48}\text{80}$ follows a similar pattern in that basal, calmodulin-independent, transport is also not affected by the drug. Kinetic analysis revealed that the stimulation of Ca²⁺-transport ATPase induced by calmodulin is inhibited by compound $\text{48}\text{80}$ according to a competitive mechanism. It was demonstrated that the inhibitory constituents of compound $\text{48}\text{80}$ bind to calmodulin in a Ca²⁺-dependent fashion. Comparison of the specificity of several anti-calmodulin drugs showed that compound $\text{48}\text{80}$ is the most specific inhibitor of the calmodulin-dependent fraction of red blood cell Ca²⁺-transport ATPase that has been described hitherto. In addition, compound $\text{48}\text{80}$ was found to be a rather specific inhibitor of the calmodulin-induced activation of Ca²⁺-transport ATPase when compared with the stimulation induced by an anionic amphiphile or by limited proteolysis. Half-maximal inhibition of the activity stimulated by oleic acid or mild tryptic digestion required 8- and 32-times higher concentrations of compound $\text{48}\text{80}$, respectively, compared with the calmodulin-dependent fraction of the ATPase activity. Moreover, calmodulin-independent systems as rabbit skeletal muscle sarcoplasmic reticulum Ca²⁺-transport ATPase or calf cardiac sarcolemma (Na⁺ + K⁺)-transport ATPase are far less influenced by compound $\text{48}\text{80}$ as compared with trifluoperazine and calmidazolium. Because of its high specificity compound $\text{48}\text{80}$ is proposed to be a promising tool for studying calmodulin-dependent processes.     

Alkali-labile oligosaccharides from glycoproteins of different erythrocyte and milk fat globule membranes

Phenol extraction of horse, sheep, cow, pig and human erythrocyte membranes and human milk fat globule membranes gave glycoprotein fractions, all of which were shown by gas chromatography to contain the reduced disaccharide β-d-galactosyl $\text{(1?3)-N-}\text{acetyl}\text{-}\text{d}\text{-}\text{galactosaminital}$ after treatment with alkaline borohydride. Cow and pig erythrocyte membrane glycoproteins were found however to contain much lower amounts than the erythrocyte membrane glycoproteins of the other species tested. After gel filtration, a tetrasaccharide was isolated from horse and sheep glycoproteins containing the disaccharide plus two molecules of sialic acid. Periodate oxidation together with paper chromatography of alkaline degraded fragments showed these two molecules of sialic acid to be linked to positions C3 and C6 of the galactosyl and N-acetylgalactosamine residues respectively. Evidence was obtained for a similar structure from pig and cow erythrocyte glycoproteins and human milk fat globule membrane glycoproteins although the complete structure was not elucidated.In all native glycoprotein fractions, the unsubstituted disaccharide β-d-galactosyl $\text{(1?3)-N-}\text{acetyl}\text{-}\text{d}\text{-}\text{galactosamine}$ was found to be present to different extents.Haemagglutination inhibition tests against human anti-T serum, Arachis hypogoea and Vicia graminea by desialylated glycoproteins showed the presence of the T-antigen, confirming the chemical findings. Inhibition was found to be proportional to the chemically detected amounts of disaccharide in each fraction. Evidence for a second carbohydrate chain in horse, sheep and human erythrocyte glycoproteins with a sialic acid substituted N-acetylgalactosamine residue as the terminal sequence was obtained using the agglutinin from Helix pomatia.     

Specific detection of N-acetylglucosamine-containing oligosaccharide chains on ovine submaxillary asialomucin

Human milk β-N-acetylglucosaminide β1 → 4-galactosytransferase (EC 2.4.1.38) was used to galactosylate ovine submaxillary asialomucin to saturation. The major [¹⁴C]galactosylated product chain was obtained as a reduced oligosaccharide by β-elimination under reducing conditions. Analysis by Bio-Gel filtration and gas-liquid chromatography indicated that this compound was a tetrasaccharide composed of galactose, N-acetylglucosamine and reduced N-acetylgalactosamine in a molar ratio of 2:0.9:0.8. Periodate oxidation studies before and after mild acid hydrolysis in addition to thin-layer chromatography revealed that the most probable structure of the tetrasaccharide is $\text{Gal}\text{β1 → 3([}{}^{14}\text{C}\text{]}\text{Gal}\text{β1 → 4}\text{GlcNac}\text{β1 → 6)}\text{GalNAcol}$. Thus it appears that Galβ1 → 3(GlcNAcβ1 → 6)GalNAc units occur as minor chains on the asialomucin. The potential interference of these chains in the assay of α-N-acetylgalactosaminylprotein β1 → 3-galactosyltransferase activity using ovine submaxillary asialomucin as an receptor can be counteracted by the addition of N-acetylglucosamine.     

The effect of formate on cytochrome aa3 and on electron transport in the intact respiratory chain

1. Formate inhibits cytochrome $\text{c}$ oxidase activity both in intact mitochondria and submitochondrial particles, and in isolated cytochrome $\text{aa}{}_3$. The inhibition increases with decreasing pH, indicating that HCOOH may be the inhibitory species.2. Formate induces a blue shift in the absorption spectrum of oxidized cytochrome $\text{aa}{}_3\text{(a}{}^{\mathrm{3+}}\text{a}{}_3{}^{\mathrm{3+}}$) and in the half-reduced species ($\text{a}{}^{\mathrm{2+}}\text{a}{}_3{}^{\mathrm{3+}}$). Comparison with cyanide-induced spectral shifts, towards the red, indicates that formate and cyanide have opposite effects on the $\text{aa}{}_3$ spectrum, both in the fully oxidized and the half-reduced states. The formate spectra provide a new method of obtaining the difference spectrum of $\text{a}{}_3{}^{\mathrm{2+}}$ minus $\text{a}{}_3{}^{\mathrm{3+}}$, free of the difficulties with cyanide (which induces marked high → low spin spectral shifts in cytochrome $\text{a}{}_3{}^{\mathrm{3+}}$) and azide (which induces peak shifts of cytochrome $\text{a}{}^{\mathrm{2+}}$ towards the blue in both α- and Soret regions).3. The rate of formate dissociation from cytochrome $\text{a}{}^{\mathrm{2+}}\text{a}{}_3{}^{\mathrm{3+}}\text{-}\text{HCOOH}$ is faster than its rate of dissociation from $\text{a}{}^{\mathrm{3+}}\text{a}{}_3{}^{\mathrm{3+}}\text{-}\text{HCOOH}$, especially in the presence of cytochrome $\text{c}$. The $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ for formate inhibition of respiration is a function of the reduction state of the system, varying from 30 mM (100% reduction) to 1 mM (100% oxidation) at pH 7.4, 30 °C.4. Succinate-cytochrome $\text{c}$ reductase activity is also inhibited by formate, in a reaction competitive with succinate and dependent on [formate]².5. Formate inhibition of ascorbate plus $\text{N,N,N′,N′-}\text{tetramethyl}\text{-p-}\text{phenyl-enediamine}$ oxidation by intact rat liver mitochondria is partially released by uncoupler addition. Formate is permeable through the inner mitochondrial membrane and no differences in ‘on’ or ‘off’ inhibition rates were observed when intact mitochondria were compared with submitochondrial particles.6. NADH-cytochrome $\text{c}$ reductase activity is unaffected by formate in submitochondrial particles, but mitochondrial oxidation of glutamate plus malate is subject both to terminal inhibition at the cytochrome $\text{aa}{}_3$ level and to a slow extra inhibition by formate following uncoupler addition, indicating a third site of formate action in the intact mitochondrion.     

Chlorophyll-protein complexes of a Codium species,including a light-harvesting siphonaxanthin-Chlorophylla ab-protein complex,an evolutionary relic of some Chlorophyta

Eight chlorophyll-protein complexes were isolated from thylakoid membranes of a Codium species, a marine green alga, by mild SDS-polyacrylamide gel electrophoresis. CP 1a¹, CP 1a², CP 1a³ and CP 1a⁴ were partially dissociated Photosystem (PS) I complexes, which in addition to the core reaction centre complex, CP 1, possessed PS I light-harvesting complexes containing chlorophyll (Chl) a, Chl b and siphonaxanthin. LHCP¹ and LHCP³ are orange-brown green chlorophyll $\text{a}\text{b-}\text{proteins}$ ($\text{Chl}\text{a}\text{Chl}\text{b}$ ratios of 0.66) that contain siphonaxanthin and its esterified form, siphonein. CP a and CP 1, the core reaction centre complexes of PS II and PS I, respectively, had similar spectral properties to those isolated from other algae or higher plants. These P-680- or P-700-Chl a-proteins are universally distributed among algae and terrestrial plants; they appear to be highly conserved and have undergone little evolutionary adaptation. Siphonaxanthin and siphonein which are present in the Codium light-harvesting complexes of PS II and PS I are responsible for enhanced absorption in the green region (518 and 538 nm). Efficient energy transfer from both xanthophylls and Chl b to only Chl a in Codium light-harvesting complexes, which have identical fluorescence emission spectra at 77 K to those of the lutein-Chl $\text{a}\text{b-}\text{proteins}$ ($\text{Chl}\text{a}\text{Chl}\text{b}$ ratios of 1.2) of most green algae and all higher plants, proved that the molecular arrangement of these light-harvesting pigments was maintained in the isolated Codium complexes. The siphonaxanthin-Chl $\text{a}\text{b-}\text{proteins}$ allow enhanced absorption of blue-green and green light, the predominant light available in deep ocean waters or shaded subtidal marine habitats. Since there is a variable distribution of lutein, siphonaxanthin and siphonein in marine green algae and siphonaxanthin is found in very ancient algae, these novel siphonein-siphonaxanthin-Chl $\text{a}\text{b-}\text{proteins}$ may be ancient light-harvesting complexes which were evolved in deep water algae.     

Isolation and partial characterization of “galactoprotein a” (LETS) and “galactoprotein b” from hamster embryo fibroblasts

The cell surface glycoproteins of hamster NIL cells, labeled with galactose oxidase and NaB³H4, were selectively solubilized by sequential extraction with Tris buffer containing 1) sucrose-ATP-EDTA, 2) zwitterionic detergent (Empigen BB), and 3) 8 M urea. The previously reported “galactoprotein b” $\text{(Gap b)}$ and “galactoprotein a” ($\text{Gap a}$ or LETS) were isolated by affinity chromatography on insoluble Ricinus communis lectin colums (RCA column) from extracts 2) and 3), respectively. The affinity-purified $\text{Gap a}$ contained an actin-like protein, whereas the other affinity-purified galactoproteins did not contain the actin-like protein. The isolated $\text{Gap b}$ was heterogeneous, and an additional glycoprotein, specific for NILpy cells was copurified on RCA-column with $\text{Gap b}$.     

1H NMR spectroscopy of carbohydrates from the G glycoprotein of vesicular stomatitis virus grown in parental and Lec4 Chinese hamster ovary cells

Carbohydrate moieties derived from the G glycoprotein of Vesicular Stomatitis Virus (VSV) grown in parental Chinese hamster ovary (CHO) cells and the glycosylation mutant Lec4 have been analyzed by high-field ¹H NMR spectroscopy. The major glycopeptides of $\text{CHO}\text{VSV}$ and $\text{Lec4}\text{VSV}$ were purified by their ability to bind to concanavalin A-Sepharose. The carbohydrates in this fraction are of the biantennary, complex type with heterogeneity in the presence of α(2,3)-linked sialic acid and α(1,6)-linked fucose residues. A minor $\text{CHO}\text{VSV}$ glycopeptide fraction, which does not bind to concanavalin A-Sepharose but which binds to pea lectin-agarose, was also investigated by ¹H NMR spectroscopy. These carbohydrates are complex moieties which appear to contain N-acetylglucosamine in β(1,6) linkage. Their spectral properties are most similar to those of a triantennary complex oligosaccharide containing a 2,6-disubstituted mannose α(1,6) residue. Carbohydrates of this type are not found among the glycopeptides of VSV grown in the Lec4 CHO glycosylation mutant.     

Magnetic resonance study of 13C reductively methylated glycophorin and glycophorin glycopeptides

¹³C nuclear magnetic resonance (n.m.r.) spectral data for ¹³C reductively methylated N-terminal tryptic glycopeptides and for ¹³C reductively methylated N-terminal glyco-octapeptides derived from homozygous glycophorins A^M and A^N are presented. Their ¹³C chemical shift data are compared with the previously published ¹³C n.m.r. data for ¹³C reductively methylated homozygous glycophorins A^M and A^N in order to investigate the means of display of the MN blood determinants by these species. The pH dependence of the ¹³C resonances of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ leucine of glyco-octapeptide A^N and of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ serine of glyco-octapepti A^M indicated that only a slight structural perturbation occurs at the N-terminus when a large portion of the glycoprotein molecule is removed. However, one structural ‘state’ of ¹³C reductively methylated glycophorin A^M is lost when the glyco-octapeptide A^M is produced. The ¹³C resonance of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ leucine of glycooctapeptide A^N titrated with a $\text{p}\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ of 7.7 (Hill coefficient $\text{～ 1}$). The ¹³C resonance of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ serine, on the other hand, exhibited an unusual pH dependence, indicating the existence of some possible steric constraints or hydrogen bonding in this molecule. In comparison to the data obtained for ¹³C-labelled glycooctapeptide A^M molecule, the pH dependence of the chemical shift of the ¹³C resonance of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ serine of tripeptide tri-L-serine is also presented. Circular dichroism (c.d.) spectra indicated that the reductive methylation technique does not cause a large perturbation of the glycophorin A molecule.