Distribution of C22-, C24- and C26-α-Unit-Containing Mycolic Acid Homologues in Mycobacteria

There are three mycolic acid homologues with C₂₂-, C₂₄- and C₂₆-α-units in Mycobacterium. In order to reveal the composition and distribution of these homologues in each subclass and molecular species of mycolic acids and to compare them with the composition of constitutive non-polar fatty acids (free and bound forms), we have separated non-polar fatty acids and each subclass of mycolic acids from 21 mycobacterial species by thin-layer chromatography, and analyzed non-polar fatty acid methyl esters by gas chromatography (GC) and the cleavage products of methyl mycolate by pyrolysis GC. We further performed mass chromatographic analysis of trimethylsilyl (TMS) ether derivatives of mycolic acid methyl esters by monitoring [B-29]⁺ ions (loss of CHO from the α-branched-chain structure of mycolic acids) of m/z 426, 454 and 482 which are attributed to C₂₂-, C₂₄- and C₂₆-α-units of TMS ether derivatives of methyl mycolates, respectively, (Kaneda, K. et al, J. Clin. Microbiol. 24: 1060-1070, 1986). By pyrolysis GC, C_22:0, C_24:0 and C_26:0 fatty acid methyl esters generated by the C₂-C₃ cleavage of C₂₂-, C₂₄- and C₂₆-α-unit-containing mycolic acid methyl esters, respectively, were detected. Their proportion was almost the same among subclasses of mycolic acids in every Mycobacterium and also similar to the proportion of constitutive non-polar C_22:0, C_24:0 and C_26:0 fatty acids. By mass chromatography, the composition and distribution of C₂₂- and C₂₄-α-unit-containing homologues were revealed to be similar between α- and α'-mycolic acids in every Mycobacterium. We further analyzed in detail M. vaccae and demonstrated that the mass chromatogram of C₂₂-α-unit-containing homologue was analogous in shape to that of the C₂₄-α-unit-containing one, with the latter mass chromatogram being up-shifted from the former by two carbon numbers, in every subclass of α-, α'-, keto and dicarboxy mycolic acids. The present study suggests that the compositions of three homologues of both mycolic acids and constitutive non-polar fatty acids, which are characteristic to each mycobacterial species, may reflect the proportion of the amount of free C_22:0, C_24:0 and C_26:0 fatty acids synthesized in the cell. It is further demonstrated that intermolecular condensation of two fatty acids which become α- and β-units of mycolic acids will occur independently of the carbon chain length or kinds of polar moieties of fatty acids.     

Synthesis of β-hydroxy fatty acids and β-amino fatty acids by the strains of Bacillus subtilis producing iturinic antibiotics

The iturinic antibiotics, which contain long chain β-amino acids, are produced by Bacillus subtilis. Screening these strains for the presence of a possible precursor of the iturinic antibiotics, we isolated a lipopeptide containing β-hydroxy fatty acids. The structure of this compound was studied and it appears to be identical or structurally very similar to surfactin. The carbon chain of its β-hydroxy fatty acids was n C₁₆, iso C₁₆, iso C₁₅ or anteiso C₁₅. The percentages of each β-hydroxy fatty acids varied according to the strain producing iturinic antibiotics and were influenced by addition of branched-chain α-amino acids to the culture medium. These results demonstrate for the first time that iso C₁₄ β-hydroxy fatty acid is a constituent present in such a surfactin like lipopeptide. Besides, the presence of radioactive β-hydroxy fatty acids in the phospholipids when the strains were grown in the presence of sodium [¹⁴C]acetate seems also characterize the different strains producing iturinic antibiotics.     

Synthesis of anomeric sulfonamides and their behaviour under radical-mediated bromination conditions

O-Peracetylated methyl 3-(d-glycopyranosylthio)propanoates of β-d-gluco, and α- and β-d-galacto configurations were oxidized to the corresponding S,S-dioxides (sulfones) by Oxone^® or MCPBA. Oxidation of the β-d-gluco derivative with H₂O₂/Na₂WO₄ gave the corresponding S-oxide (sulfoxide). DBU-induced elimination of methyl acrylate from the β-d-gluco and β-d-galacto configured S,S-dioxides (sulfones) gave O-peracetylated β-d-glycopyranosyl-1-C-sulfinates which, on treatment with H₂NOSO₃H, furnished the corresponding β-d-glycopyranosyl-1-C-sulfonamides. Radical-mediated bromination of the protected methyl 3-(β-d-glycopyranosylthio)propanoate S,S-dioxides gave mixtures of 1-C- and 5-C-bromoglycosyl compounds. Similar brominations of the O-peracetylated β-d-glycopyranosyl-1-C-sulfonamides resulted in the formation of α-d-glycopyranosyl bromides and 1-C- and 5-C-bromoglycosyl sulfonamides. A rationale for these observations was proposed. Methyl 3-(β-d-glucopyranosylthio)propanoate, its S,S-dioxide, and β-d-glucopyranosyl-1-C-sulfonamide proved inefficient when tested as inhibitors of rabbit muscle glycogen phosphorylase b.     

A technique for the isolation of yeast alcohol dehydrogenase mutants with altered substrate specificity.

α-Amylases have been found to convert starch and glycogen, in part, to products other than hemiacetal-bearing entities (maltose, maltodextrins, etc.)—hitherto, the only products obtained from natural α-glucans by α-amylolysis. Glycosides of maltosaccharides were synthesized by purified α-amylases acting on starch or bacterial glycogen in the presence of p-nitrophenyl α- or β-d-glucoside. From a digest with crystallized B. subtilis var. amyloliquefaciens α-amylase, containing 4 mg/ml of [¹⁴C]glycogen and 40 mmp-NP β-d-glucoside, three pairs of correspondingly labeled glycosides and sugars were recovered: p-NP α-d-[¹⁴C]glucopyranosyl (1 → 4) β-d-glucopyranoside, and [¹⁴C]glucose; p-NP α-[¹⁴C]maltosyl (1 → 4) β-d-glucopyranoside, and [¹⁴C]maltose; p-NP α-[¹⁴C]maltotriosyl (1 → 4) β-d-glucopyranoside, and [¹⁴C]maltotriose. The three glycosides accounted for 11.4% of the [¹⁴C]glycogen donor substrate; the three comparable sugars, for 30.4%; higher maltodextrins, for 58.2%. Calculations based on the molar yields of all reaction products show that [¹⁴C]glycosyl moieties were transferred from donor to p-NP β-d-glucoside with a frequency of 0.234 relative to all transfers to water. This is a very high value considering the minute molar ratio (0.0007) of β-d-glucoside-to-water concentration. Less striking but similar findings were obtained with cryst. hog pancreatic and Aspergillus oryzae α-amylases. The results extend earlier findings (Hehre et al., Advan. Chem. Ser. (1973) 117, 309) in showing that α-amylases have a substantial capacity to utilize the C₄-carbinols of certain d-glucosyl compounds as acceptor sites.     

Blood Group Substance-degrading Enzymes Obtained from Streptomyces sp.

The blood group B substance-degrading activity of Streptomyces 9917S₂ is induced by galactosides as α-galactosidase activity is. Purification of the α-galactosidase was attempted by chromatography on DEAE-Sephadex and Sephadex. The purified preparation was shown to be free from α- and β-glucosidases, β-galactosidase, α- and β-glucosaminidases, and α- and β-galactosaminidases activities. The blood group B substance-degrading activity was present only in this fraction. This enzyme preparation cleaves α-(1→3)- and α-(1→6)-galactosidic linkages. The activity is inhibited by d-galactose, melibiose, and raffinose and also by l-arabinose and d-xylose.     

Characterization of mycolic acids from the pathogen Rhodococcus equi by tandem mass spectrometry with electrospray ionization

We describe a simple tandem mass spectrometric approach toward structural characterization of mycolic acids, the long-chain α-alkyl-β-hydroxy fatty acids unique to mycobacteria and related taxa. On collisionally activated dissociation in a linear ion trap or tandem quadrupole mass spectrometer, the [M−H]⁻ ions of mycolic acid generated by electrospray ionization undergo dissociation to eliminate the meroaldehyde residue, leading to formation of carboxylate anions containing α-alkyl chains. The structural information from these fragment ions affords structural assignment of the mycolic acids, including the lengths of the meromycolate chain and the α-branch. This study revealed that the mycolic acids isolated from pathogenic Rhodococcus equi 103 contained a series of homologous ions having C₃₀ to C₅₀ chain with 0–2 double bonds. The α-branch ranged from C₁₀ to C₁₈ with 0 to 1 double bond, in which 16:0 and 14:0 are the most prominent, whereas the meromycolate chain ranged from C₁₄ to C₃₄ with 0 to 2 double bonds. The major molecular species consisted of more than 3 isomers that differ by the lengths of the α-branch or meromycolate chain, and up to 10 isobaric isomers were identified for some minor ions. We also employed tandem quadrupole mass spectrometry with precursor ion and neutral loss scans for profiling mycolic acid with specific structure in mixtures. The tandem spectra obtained from precursor ion scans of m/z 255 (16:0-carboxylate anion) and m/z 227 (14:0-carboxylate anion) may provide a simple specific means for classification of Rhodococci species, whereas tandem spectra from neutral loss of meroaldehyde residue scans provided a simple approach to reveal the mycolic acid molecules with specific meromycolate chain in mixtures.     

Product-identification and substrate-specificity studies of the GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (fuc→asn-linked GlcNAc) 6-α-l-fucosyltransferase in a golgi-rich fraction from porcine liver

Golgi-rich membranes from porcine liver have been shown to contain an enzyme that transfers l-fucose in α-(1→6) linkage from GDP-l-fucose to the asparagine-linked 2-acetamido-2-deoxy-d-glucose r residue of a glycopeptide derived from human α₁-acid glycoprotein. Product identification was performed by high-resolution, ¹H-n.m.r. spectroscopy at 360 MHz and by permethylation analysis. The enzyme has been named GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (Fuc→Asn-linked GlcNAc) 6-α-l-fucosyltransferase, because the substrate requires a terminal β-(1→2)-linked GlcNAc residue on the α-Man (1→3) arm of the core. Glycopeptides with this residue were shown to be acceptors whether they contained 3 or 5 Man residues. Substrate-specificity studies have shown that diantennary glycopeptides with two terminal β-(1→2)-linked GlcNAc residues and glycopeptides with more than two terminal GlcNAc residues are also excellent acceptors for the fucosyltransferase. An examination of four pairs of glycopeptides differing only by the absence or presence of a bisecting GlcNAc residue in β-(1→4) linkage to the β-linked Man residue of the core showed that the bisecting GlcNAc prevented 6-α-l-fucosyltransferase action. These findings probably explain why the oligosaccharides with a high content of mannose and the hybrid oligosaccharides with a bisecting GlcNAc residue that have been isolated to date do not contain a core l-fucosyl residue.     

The crystal structure of methyl 3,4-O-isopropylidene-2,6-di-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-α-d-galactopyranoside at 97 K

The crystal structure of methyl 3,4-O-isopropylidene-2,6-di-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-α-d-galactopyranoside (1), C₃₈H₅₄O₂₄ · (C₄H₈O₂)_0.32 was determined by X-ray diffraction;1 crystallises in space group P2₁ with a = 12.480(3), b = 8.821(3), c = 21.182(4)Å, β = 98.46(3)°, and Z = 2. The structure was solved by Patterson-search and Fourier-recycling procedures and refined to R_w(R) = 0.048(0.063), using 4348 [3112 with I> 2σ(I)] independent reflections. The β-d-galactosyl rings are slightly distorted and, due to the isopropylidene group, the α-d-galactoside ring is severely distorted. The conformation near the β-(1→6) and β-(1→2) linkages between the pyranoid rings is not significantly affected by the acetyl groups, but the anomeric C-O-C bridge angles have unusual values. The C-6O-6 bond in the β-d-galactosyl group (1→2)-linked to the α-d-galactoside residue has an unusual gauche—trans conformation with respect to C-4 and O-5. The CH₃-(C = O)-O-C moieties are planar within 0.01Å, and 32.6% of all unit cells contain a molecule of ethyl acetate.     

Suppression of temperature-sensitive assembly mutants of heat-labile enterotoxin B subunits

Deletions or substitutions of amino acids at the carboxyl-terminus of the heat-labile enterotoxin B sub-unit (EtxB) affect its assembly into pentamers in a temperature-dependent manner. At 42°C, the mutations prevent the B subunits from achieving their final pentameric structure resulting in membrane association of the monomers. However, mutant B subunits produced at 30°C assemble, in the periplasm, into pentamers that remain stable when transferred to 42°C, indicating that the mutant pentamers are stable under conditions where their formation is inhibited. The mutant pentamers are, similarly to wild-type pentamers, SDS-resistant and stable, in vitro, at temperatures up to 65°C. This suggests that although the C-terminal amino acids are part of the subunit interface, they appear not to contribute significantly to the stability of the final pentameric complex, but are instead essential for the formation or stabilization of an assembly intermediate in the pentamerization process. Single second site mutations suppress the assembly defect of mutant EtxB191.5, which carries substitutions at its C-terminus. The Thr→IIe replacement at position 75 in the α₂-helix probably restores the van der Waals contact between residues 75 and 101, which had been greatly reduced by the Met→Leu substitution at position 101 in the β₆-strand of EtxB191.5. Interaction between the α₂-helix and β₆-strand which contains the c-terminus probably stabilizes a conformation essential for assembly and is therefore required for the formation of pentamers.     

Structure Determination of Fatty Acids from Tunicamycin Complex

The structures of ten fatty acids, which were obtained by the hydrolysis of tunicamycin complex, were determined. GLC-mass, ¹H NMR and IR spectra showed that the major acids were trans-α, β-unsaturated iso acids with the formula C₁₄H₂₈O₂, C₁₆H₂₈O₂, C₁₆H₃₀O₂ and C₁₇H₃₂O₂. The minor acids were α, β-unsaturated normal acids and saturated normal and iso acids.     

The crystal and molecular structure of O-α-D-mannopyranosyl-(1→3)-O-β-D-mannopyranosyl-(1→4)-2-acetamido-2-deoxy-α-D-glucopyranose

The crystal structure of α-D-Manp-(1→3)-β-D-Manp-(1→4)-α-D-GlcNAcp has been determined by the direct method using the multi-solution, tangent formula, and “magic integer” procedures. The space group is P2₂, and 2 molecules are in the unit cell with a  9.894 (5), b  10.372 (6), c  11.816 (6) Å, and β  95.03° (6). The structure was refined to R 0.059 for 2099 reflections measured with Mo Kα radiation. Difference synthesis showed all the hydrogen atoms, and indicated a partial (～30%) substitution of the α-anomer molecules by the β-anomer molecules. The D-mannopyranose and the D-glucopyranose have the normal ⁴C₁ conformation; an intramolecular hydrogen-bond O-3″-H.....O-5′ (2.703 Å) stabilises the GlcNAc in relation to β-D-mannopyranose.     

A glucomannan from the tubers of Arum orientale

Two glucomannans (A and B), and an attendant polysaccharide (C) have been isolated from the tubers of Arum orientale (L.) by gel chromatography. Glucomannan B was found to be composed of d-glucose and d-mannose in a molar ratio of 2:3·1, and traces of uronic acid. IR spectra, enzymatic hydrolysis with α- and β-amylase, and periodate oxidation followed by reduction, showed the presence of a β-(1→4) glucoside linkage in this glucomannan, and of hexose residues in a β-pyranose form.     

FATTY ACIDS AND HYDROXY FATTY ACIDS IN THREE SPECIES OF FRESHWATER EUSTIGMATOPHYTES

The monocarboxylic fatty acids and hydroxy fatty acids of three species of freshwater microalgae—Vischeria punctata Vischer, Vischeria helvetica (Vischer et Pascher) Taylor, and Eustigmatos vischeri (Hulbert) Taylor, all from the class Eustigmatophyceae— were examined. Each species displayed a very similar distribution of fatty acids, the most abundant of which were 20:5n-3, 16:0, and 16:1n-7; C₁₈ polyunsaturated fatty acids were minor components. These fatty acid distributions closely resemble those found in marine eustigmatophytes but are quite distinct from those found in most other algal classes. These microalgae also contain long-chain saturated and unsaturated monohydroxy fatty acids. Two distinct types of hydroxy fatty acids were found: a series of saturated α-hydroxy acids ranging from C₂₄ to C₃₀ with a shorter series of monounsaturated α-hydroxy acids ranging from C₂₆ to C₃₀ together with a series of saturated β-hydroxy acids ranging from C₂₆ to C₃₀. The latter have not previously been reported in either marine or freshwater microalgae, although C₃₀ to C₃₄ midchain (ω-18)-hydroxy fatty acids have been identified in hydrolyzed extracts from marine eustigmatophytes of the genus Nannochloropsis, and C₂₂ to C₂₆ saturated and monounsaturated α-hydroxy fatty acids have been found in three marine chlorophytes. These findings have provided a more complete picture of the lipid distributions within this little studied group of microalgae as well as a range of unusual compounds that might prove useful chemotaxonomic markers. The functions of the hydroxy fatty acids are not known, but a link to the formation of the lipid precursors of highly aliphatic biopolymers is suggested.     

Characterization of spinach leaf α-l-arabinofuranosidases and β-galactosidases and their synergistic action on an endogenous arabinogalactan-protein

Two β-galaclosidases (β-Galase-I and -II, EC 3.2.1.23) and two α-l -arabinofuranosidases (α-l -Arafase-I and -II. EC 3.2.1.55). were purified from mesophyll tissues of spinach (Spinacia oleracea L.), using chromatography on DEAE-cellulose, lactose-conjugated Sepharose CL-4B, and Sephadex G-100, or on hydroxylapatite and Sephadex G-150. The apparent molecular mass (M_r) of β-Galase-I and -II, respectively, were estimated to be 38 000 and 58 000 on SDS-PAGE and 64 000 and 60 000 on gel-permeation chromatography, indicating that the former was a dimeric protein. The isoelectric points of β-Galase-I and -II were 6.9 and 5.2, respectively. Both enzymes hydrolyzed maximally p-nitrophenyl (PNP) β-galactoside at pH 4.3, and were activated about 2-fold in the presence of BSA (100 μg ml^?1). The activity of both enzymes was inhibited strongly by heavy metal ions and p-chloromercuriberszoate (p-CMB). d -Galactono-(1→4)-lactone and d -galactal served as potent competitive inhibitors for the enzymes. β-Galase-I and -II could be distinguished from each other in their relative rates and kinetic properties in the hydrolysis of aryl β-galactosides as well as of lactose and galacto-oligosaccharides. In particular. β-Galase-I exhibited a preferential exowise cleavage of β-1,6-galactotriose and β-1.3-galactan. α-l -Arafase-l (M_r 118000) and -II (M, 68 000) were optimally active on PNP α-l -arabinofuranoside at pH 4.8 and gave K_m values of 1.2 and 2.2 mM. respectively. l -Arabino-(1 → 4)-lactone. Ag⁺, and SDS acted as inhibitors for the isozymes. α-l Arafase-I was characterized by its activity to hydrolyze PNP β-d -xylopyranoside besides PNP α-l -arabinofuranoside. inhibition by d -xylose and d -glucono-(1 → 5)-lactone. and less sensitivity to Hg²⁺. Cu²⁺, and p-CMB. Sugar beet arabinan was hydrolyzed rapidly by α-l Arafase-II at one-half the rate for PNP α-l arabinofuranoside, while the polysaccharide was less susceptible to α-l Arafase-I. A spinach leaf arabinogalactan-protein was practically resistant to the action of β-Galases, but its susceptibility to the enzymes increased remarkably after prior hydrolysis with α-l Arafase-Il.     

Étude complémentaire de la structure de trois galactoxyloglucanes (amyloïdes) de graines

D-Galacto-D-xylo-D-glucans (amyloids) from Balsamina, Tropaeolum, and Tamarindus seeds behave in a similar manner in the presence of various glycosidase preparations: slow depolymerization by enzymes from several germinated or non-germinated seeds, and hydrolysis into monosaccharides and oligosaccharides by commercial cellulase and hemicellulase preparations from fungi. A purified cellulase from Penicillium notatum gave a dialyzable fraction almost exclusively composed of α-D-xylopyranosyl-(1→6)-D-glucose residues and a nondialyzable fraction composed of chains of β-D-(1→4)[withsome (1→3)]-glucopyranosyl residues; β-D-galacto-pyranosyl-(1→2)-α-D-xylosyl groups are linked to some of the β-D-glucosyl residues at 0-6. The presence of (1→3)-linkages in the D-glucan chain of the Balsamina was verified by methylation and sequential periodate oxidation-borohydride reduction; the distribution of the substituents on the D-glucan chain is not regular. The main D-glucan backbone, where the β-D-glucosyl residues are partly linked at 0-6 to β-D-galactosyl-(1→2)-D-xylosyl groups, is linked to D-glucan chains where almost all the D-glucose units are linked at 0-6 by one α-D-xylosyl group. The presence of 3,6-di-O-methyl-D-glucose after permethylation and hydrolysis suggests that the xyloglucan chains are linked to 0-2 of the D-glucosyl units of the galactoxyloglucan backbone.     

Attribution des signaux de résonance magnétique nucléaire-13c de disaccharides peracétylés dans la série du D-glucose

Selective, double irradiation allows the assignment of most ¹³C-n.m.r. signals in a series of per-O-acetyl disaccharides composed of two D-glucose residues linked α-(1→3), β-(1→3), α-(1→4), β-(1→4), α-(1→6), β-(1→6), and α,α-(1→1). The main influences that affect the chemical shifts are discussed and the spectra of β-cellobiose octaacetate and β-maltose octaacetate are compared to those of cellulose and amylose triacetate, respectively, to show the possibilities and limitations of a disaccharide model for the interpretation of the ¹³C-spectrum of a polymer.     

Trans-α-xylosidase,a widespread enzyme activity in plants,introduces (1→4)-α-d-xylobiose side-chains into xyloglucan structures

Angiosperms possess a retaining trans-α-xylosidase activity that catalyses the inter-molecular transfer of xylose residues between xyloglucan structures. To identify the linkage of the newly transferred α-xylose residue, we used [Xyl-³H]XXXG (xyloglucan heptasaccharide) as donor substrate and reductively-aminated xyloglucan oligosaccharides (XGO–NH₂) as acceptor. Asparagus officinalis enzyme extracts generated cationic radioactive products ([³H]Xyl·XGO–NH₂) that were Driselase-digestible to a neutral trisaccharide containing an α-[³H]xylose residue. After borohydride reduction, the trimer exhibited high molybdate-affinity, indicating xylobiosyl-(1→6)-glucitol rather than a di-xylosylated glucitol. Thus the trans-α-xylosidase had grafted an additional α-[³H]xylose residue onto the xylose of an isoprimeverose unit. The trisaccharide was rapidly acetolysed to an α-[³H]xylobiose, confirming the presence of an acetolysis-labile (1→6)-bond. The α-[³H]xylobiitol formed by reduction of this α-[³H]xylobiose had low molybdate-affinity, indicating a (1→2) or (1→4) linkage. In NaOH, the α-[³H]xylobiose underwent alkaline peeling at the moderate rate characteristic of a (1→4)-disaccharide. Finally, we synthesised eight non-radioactive xylobioses [α and β; (1↔1), (1→2), (1→3) and (1→4)] and found that the [³H]xylobiose co-chromatographed only with (1→4)-α-xylobiose. We conclude that Asparagus trans-α-xylosidase activity generates a novel xyloglucan building block, α-d-Xylp-(1→4)-α-d-Xylp-(1→6)-d-Glc (abbreviation: ‘V’). Modifying xyloglucan structures in this way may alter oligosaccharin activities, or change their suitability as acceptor substrates for xyloglucan endotransglucosylase (XET) activity.     

The chemistry of thujone: I. Synthesis of insect juvenile hormone analogs via wittig coupling

Syntheses of the C₈ and C₁₀ olefinic units cis- and trans-5-ethyl-1-iodo-hex-4-enes and cis- and trans-7-ethyl-3-iodo-oct-6-enes are described. The Wittig coupling of such units with derivatives of α- and β-thujaketonic acids to give analogs of insect juvenile hormones is discussed.     

The presence of -mannosidic linkage in acidic glycopeptide from porcine thyroglobulin

The mannose residue in (Man)₁ (GlcNAc)₂-Asn obtained by a Smith degradation of the acidic glycopeptide from porcine thyroglobulin was found to be insusceptible to α-mannosidase. This residue was hydrolyzed, however, by purified β-mannosidase. After β-mannosidase treatment, the resulting (GlcNAc)₂-Asn was compared with synthetic glycosyl-asparagine derivatives. From these experiments, the core structure of the acidic glycopeptide was proposed to be β-Man-(1 → 3 or 4)-β-GlcNAc-(1 → 4)-GlcNAc-Asn.     

Glycogen in the Marine Protozoon Parauronema acutum*

SYNOPSIS. The carbohydrate which accumulates in the cytoplasm of the marine protozoon, Parauronema acutum, during normal growth was isolated, purified and characterized chemically. The highly purified material yielded only glucose residues following hydrolysis in 0.6 N HCl for 3 h at 100 C; measurement of total carbohydrate by the phenol-sulfuric acid method and by treatment with amylo-glucosidase and glucose oxidase gave similar values. Aqueous solutions of the purified material reacted with iodine to form a complex which exhibited an absorption peak at 456 nm with a shift to 484 nm in the presence of 50% saturated (NH₄)₂SO₄. Digestion with α-amylase, β-amylase, and isoamylase yielded 71%, 45% and 8.3% hydrolysis, respectively. Treatment sequentially with both isoamylase and β-amylase gave complete hydrolysis of the polymer. The average chain length (CL) determined by the isoamylase procedure was 12. These observations are consistent with the view that the carbohydrate isolated from the protozoan is a polymer consisting of α-D-glucose residues arranged in chains containing α-(1→4) linkages with branch points containing α-(1→6) linkages occurring once on the average of ～ 12 glucose residues and, as such, is indistinguishable from glycogen isolated from mammalian sources.