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.     

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.     

Immunochemical studies on blood groups. 53. A study of various conditions of alkaline borohydride degradation on human and hog blood group substances and on known oligosaccharides

Blood group A, B, H, Le^a, Le^b, and I substances, their products of periodate oxidation and Smith degradation, and disaccharides containing 3-O-substituted reducing N-acetylhexosamines were treated with base-borohydride under three defined sets of conditions. Procedures for the assay and quantitation of the possible reduced base-degradation products, including hexenetetrol(s), 3-deoxygalactitol, galactitol, reduced chromogens, N-acetylglucosaminitol, and N-acetylgalactosaminitol are described. Extensive degradation occurred by two methods. 1 m NaBH₄ in 0.05 n NaOH at 50 ° cleaves the glycosidic linkage of the oligosaccharide chains from serine and threonine with reduction of the terminal-reducing N-acetylgalactosamine with minimal base degradation. The method is useful for isolation of complete reduced oligosaccharides from blood group substances; the structural implications of the free and oligosaccharide-bound N-acetylgalactosaminitol released are discussed.     

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.

     

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.     

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.     

The relationship between the N-acetylgalactosamine content and the blood group Sda activity of Tamm and Horsfall urinary glycoprotein

Substances with the human blood group Sd^a character occur on red cells and are also present in secretions. In urine Sd^a activity is associated with the Tamm and Horsfall (T-H) glycoprotein. About 4% of Caucasian individuals, whose red cells are Sd^a negative, have a T-H glycoprotein that is without Sd^a activity. The two immunologically distinct forms of T-H glycoprotein have almost identical qualitative and quantitative amino acid and carbohydrate compositions. The only exception is the $\text{N}$-acetylgalactosamine content which falls in the range of 1–2% for preparations from Sd(a+) individuals whereas the level is negligible in the Sd^a inactive preparations. These results strongly indicate that $\text{N}$-acetylgalactosamine makes an important contribution to the Sd^a determinant structure in the T-H glycoprotein.     

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.     

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.     

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.     

Purification and characterization of the major sialoglycoproteins of the rat erythrocyte membrane

The major sialoglycoproteins of the rat erythrocyte membrane were purified by hot phenol partitioning followed by cation-exchange chromatography on SP-Sephadex. Further purification was obtained by extraction with n-butanol and anion-exchange chromatography on DEAE-cellulose. The resulting sialoglycoprotein fraction was free of lipids and nonsialylated glycoproteins and gave rise to four major periodic acid-Schiff staining bands when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fastest migrating protein on these gels with an apparent molecular weight of 19,000 was purified to homogeneity by gel filtration. The amino acid and sugar compositions of these materials are reported. The protein moiety is rich in serine, threonine, and hydrophobic amino acids and the carbohydrate moiety is high in sialic acid and N-acetylgalactosamine. Most of the carbohydrate is linked O-glycosidically to serine and threonine residues, as shown by susceptibility to base-catalyzed β-elimination and concomitant reduction of serine and threonine to alanine and α-aminobutyric acid and of N-acetylgalactosamine to N-acetylgalactosaminitol in the presence of reducing agents. The significance of these data in light of the known role of the rat erythrocyte membrane sialoglycoproteins in erythropoiesis is discussed. The properties of the rat erythrocyte membrane sialoglycoproteins are compared to those of other species.     

Estimation of mutation rates in cultured mammalian cells

The factors that affect reliable estimations of mutation rates (μ) in cultured mammalian somatic cell populations by fluctuation analysis are studied experimentally and statistically. We analyze the differential effect of the final cell population size in each culture (N_t) and the number of parallel cultures (C) on the variation in the rate estimates ($\text{μ}$) inferred from the P₀ method. The analysis can be made after the derivation of the variance of $\text{μ}$, which is a measure of variation of $\text{μ}$ for a given combination of N_t and C in a number of repeat experiments. The variance of $\text{μ}$ is inversely proportional to C and to the square of N_t. N_t determines the probability of occurrence of mutation in a cell culture. By influencing the size of P₀, N_t also determines whether a rate estimate is obtainable from the experiment. Since P_o is estimated from the fraction of cultures containing no mutation in a set of C cultures, C becomes a determining factor for the accuracy of $\text{μ}$. The rate estimated from $\text{P}\text{?}{}_0$ is biased, but the bias is in general 2 orders of magnitude smaller than $\text{μ}$. By the selection of an appropriate combination of N_t and C for the experiment, this bias can be reduced even further.Based on the notion of comparing two proportions, we propose a test statistic and have applied it to experimental results for a test of equality of mutation rates in different cell lines. This development places the comparison of mutation rates on a statistical basis.     

Determination of zinc by flameless atomic absorption spectrophotometry

The flameless atomic absorption method described here is a simple, rapid, accurate microtechnique for determining zinc in aqueous solutions, serum, or urine. It requires no sample pretreatment, only 1.0 μl of sample per determination, no correction for viscosity differences between sample and standard solutions, and is not subject to ionic or organic interference. The average recovery of added zinc in serum is 97.5% and in urine is 97.6%. The values obtained for serum (mean ± SD: 94.6 ± 11.0 μAg/100 ml; N = 25) and urine (range: $\text{sol}\text{600–1000 μg}\text{24}\text{hr}$; N = 4) are comparable to the values reported in the literature. The coefficient of variation was less than 5.0% in all cases. The qualitative concentration limit was $\text{0.009}\text{μg}\text{100}\text{ml}$. The techniques and instrumentation described are also applicable to a number of trace minerals of common interest.     

Quantitation of the diastereoisomers of l-buthionine-(R,S)-sulfoximine in human plasma: a validated assay by capillary electrophoresis

An assay for the diastereoisomers of the biochemical modifier $\text{l}\text{-buthionine}\text{-(R,S)-}\text{sulfoximine}$ (BSO) in human plasma has been developed using capillary electrophoresis (CE). Separation of the diastereoisomers is achieved by the micellar electrokinetic chromatography (MEKC) mode of CE. Plasma is injected directly onto the separation capillary without any extraction step, and BSO is detected directly by ultraviolet absorbance measurements at 190 nm without prior derivatization. The whole assay, including capillary conditioning, takes approximately 30 min. Intra- and inter-day R.S.D. values are approximately 7% at sample concentrations around 25 μg ml⁻¹, and approximately 3% at sample concentrations around 500 μg ml⁻¹. The limit of detection in plasma is 3.9 μg ml⁻¹ (S/N = 2). The assay has been used to quantitate the diastereoisomers of BSO in patient samples in a pharmacokinetic study.     

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.     

Spectroscopic and electrochemical properties of chloro-N-methylprotoporphyrin IX dimethyl ester iron(II), a species related to the intermediate in the formation of N-alkylprotoporphyrins from the cytochrome P-450 prosthetic group

N-alkylporphyrins are formed when certain agents such as 3,5-diethoxycarbonyl-2,4,6-trimethyl-1,4-dihydropyridine or ethylene interact with cytochrome P-450 in rats. It is likely that the iron protoporphyrin complex in cytochrome P-450 is first alkylated and then demetallated to form the free base N-alkylprotoporphyrins that are observed. An iron complex of N-methylprotoporphyrin IX dimethyl ester, chloro-N-methylprotoporphyrin IX dimethyl ester iron(II), shows the following properties: a double Soret band (λ_max = 435 nm, with a shoulder at 390 nm) relatively facile reduction (E_{$\text{1}\text{2}$} for Fe(III)/Fe(II) of 0.385 V vs Ag/AgCl in acetonitrile) and facile demetallation by acid or good nucleophiles such as thiophenol. A knowledge of such properties should be useful in determining the mechanism of formation of N-alkylprotoporphyrins in vivo.     

Transfer of N, N'-diacetylchitobiose from dolichyl diphosphate into a gray matter membrane glycoprotein

Gray matter and white matter membranes catalyze the transfer of label from UDP-N-acetyl-[${}^{14}\text{C}$] glucosamine into N-acetyl[${}^{14}\text{C}$]glucosaminyl-pyrophosphoryl-dolichol, N,N′-diacetyl [${}^{14}\text{C}$]chitobiosyl-pyrophosphoryl-dolichol, and N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein. Gel filtration of the Pronase digests of gray matter N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein reveals two N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide fractions. One fraction (A) contains approximately eight glycose units. All of the radioactivity is at nonreducing termini and can be released by treatment with an exo-β-N-acetylglucosaminidase. A smaller N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide (B) is recovered in the elution volume expected for an asparaginyl disaccharide. Structural studies show that the labeled saccharide unit in glycopeptide B is N,N′-diacetyl[${}^{14}\text{C}$]chitobiose. The linkage between the ${}^{14}\text{C}$-labeled disaccharide and the polypeptide has the properties of an N-glycosidic attachment to asparagine. Only the larger N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide (A) is found in Pronase digests of white matter membrane N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein after incubation with UDP-N-acetyl[${}^{14}\text{C}$]glucosamine. When gray matter membranes are incubated with UDP-N-acetyl[${}^{14}\text{C}$]glucosamine in the presence of tunicamycin or UMP, the labeling of glycolipid and the asparaginyl disaccharide is inhibited. UMP and tunicamycin have no effect on the transfer of N-acetyl[${}^{14}\text{C}$]glucosamine to external acceptor sites of the larger glycopeptide (A). The transfer of N,N′-diacetyl[${}^{14}\text{C}$]-chitobiose from carrier lipid to protein is observed when extensively washed membranes containing endogenous, prelabeled ${}^{14}\text{C}$-labeled glycolipids are incubated in the presence or absence of unlabeled GDP-mannose. UMP treatment of the prelabeled membranes selectively discharged over 80% of the label from N-acetyl[${}^{14}\text{C}$]glucosaminyl-pyrophosphoryl-dolichol, but had no effect on the transfer of the ${}^{14}\text{C}$-labeled disaccharide to protein. All of these results are concordant with transfer of N,N′-diacetylchitobiose from dolichyl diphosphate to gray matter glycoprotein. The major membrane glycoprotein labeled by the lipid-mediated [${}^{14}\text{C}$]disaccharide transfer reaction has an apparent molecular weight of 24,000. Tunicamycin prevents the enzymatic labeling of the gray matter glycoprotein having an apparent molecular weight of 24,000.     

Studies on lectins: XXXIX. S-glycosyl polyacrylamide gels for affinity chromatography of lectins

Hydrophilic water-insoluble gels suitable for affinity chromatography of lectins have been prepared by copolymerization of acrylamide, N,N′-methylene bisacrylamide and alkenyl 1-thioglycosides. Water-soluble copolymers of analogous type have been obtained by omitting the cross-linking agent, N,N′-methylene bisacrylamide.In affinity chromatography of the Ricinus communis lectin it could be shown that the capacity for the lectin of the water insoluble copolymers was more than four times higher in copolymers having the $\text{S-β-}\text{D}\text{-}\text{galactosyl}$ ligand attached through a methylene bridge than in derivatives with a nonamethylene spacer.None of the insoluble $\text{S-β-}\text{D}\text{-}\text{glycosyl}$ copolymers prepared could be shown usable as affinity adsorbent for glycosidases though the corresponding soluble copolymers inhibited the activity of the enzymes.     

Control of pattern duplication in the retinotectal system of Xenopus. Induction of duplication in eye fragments by secondary cuts.

Following surgical ablation of the temporal (posterior) region of the eye-bud in stage 32 Xenopus frog embryos, the surviving nasal (anterior) fragment gradually rounds up to form a functional eye and orderly retinotectal map. Large nasal fragments ($\text{N}\text{-}\text{2}\text{3}$) assemble topographically normal maps, as does the majority of nasal “half-eye” fragments; small nasal fragments ($\text{N}\text{-}\text{1}\text{3}$), and a minority of nasal half-eye fragments, give a characteristic, mirror-symmetrical duplication map, similar (but not identical) to the “double-nasal” maps which develop when two nasal half-eyes are fused to form a frank NN double-eye. Ventral fragments and temporal fragments show similar size-dependent behavior, although their characteristic duplicate maps are topographically different from those of nasal fragments and more similar to the “double-ventral” and “double-temporal” maps of VV and TT recombinant eyes. Here we show that a simple surgical transection, applied either dorsally or ventrally to large nasal ($\text{N}\text{-}\text{2}\text{3}$) fragments so as to isolate a subregion of the tissue at the dorsum or venturm of the fragment, induces full or partial duplication of the nasal type in the majority of cases. The results refute the hypothesis that special properties at the eye-bud center, by their presence or absence in the fragment, control pattern duplication, and point instead toward interactions around the circumference of the eye-bud as a crucial parameter in determining positional information in the retina.     

Proton and carbon magnetic resonance studies of the synthetic polypentapeptide of elastin.

Proton and ¹³C magnetic resonance studies are reported on the synthetic polypentapeptide of elastin, HCO-(Val₍₁₎-Pro₍₂₎-Gly₍₃₎-Val₍₄₎-Gly₍₅₎)_n-Val-OMe, where n ∼- 18. Temperature and solvent dependence of peptide N$\text{H}$ chemical shift and solvent dependence of peptide carbonyl chemical shift were used to delineate these moieties preliminary to identification of secondary structure.Based on these studies it is proposed, for the organic solvents of dimethyl sulfoxide, methanol, and low-temperature trifluoroethanol, that dynamic hydrogen bonds form in order of decreasing frequency of occurrence between the Val₍₁₎$\text{C}$O and the Val₍₄₎ N$\text{H}$ (a β-turn), between the Gly₍₃₎ N$\text{H}$ and the Gly₍₅₎$\text{C}$O (an 11-atom, hydrogen-bonded ring), and a more limited interaction between the Gly₍₃₎$\text{C}$O and the Gly₍₅₎ N$\text{H}$ (a γ-turn).Arguments are presented that relate the conformational features proposed above to the coacervate, which is a filamentous state.