Human umbilical cord hyaluronate neutral sugar content and carbohydrate-protein linkage studies

Paper chromatography of neural sugars and gas chromatography of their aldononitrile acetates indicated the presence of fucose, arabinose and a small amount of glucose in purified human umbilical cord hyaluronate. The molar ratios of serine, threonine and aspartic acid to neural sugars were not unity, suggesting the non-involvement of the neutral sugars and the amino acids in a carbohydrate-protein linkage. The same was indicated by an increase in the percentage of the aforementioned amino acids and by the absence of sugar alditols in umbilical cord hyaluronate reduced with NaBH₄-PdCl₂, after alkali treatment. This reduction caused a decrease in the intrinsic viscosity and molecular weight to about one-half and an appreciable decrease in the specific rotation of hyaluronate, suggesting a separation of the two antiparallel chains of the double helical hyaluronate. The umbilical cord hyaluronate contained bound silicon and it is possible that this bound silicon may cross-link the two chains at interspersed intervals through the uronic acid moiety and/or through neutral sugars.     

On the carbohydrate—protein linkage group in vitreous humor hyaluronate

Fractional molar ratios of serine, threonine and aspartic acid to neutral sugars in the purified bovine vitreous humor hyaluronate, and a 4–5-fold increase in the percentage of these amino acids and the absence of sugar alditols in hyaluronate reduced with NaBH₄---PdCl₂ after alkali treatment indicated the absence of a carbohydrate—protein linkage. Gel filtration behavior, a decrease in intrinsic viscosity of reduced hyaluronate to about one-half and a significant decrease in its specific rotation suggested that the two antiparallel chains of the hyaluronate double helix may come apart upo reduction. The vitreous humor hyaluronate contained 109.2 ppm of “bound” silicon. It is suggested that the bound silicon may bridge the two antiparallel chains through the neutral sugars and/or through the hydroxyl group of the uronic acid moiety.     

Crystallization and partial characterization of Staphylococcus aureus hyaluronate lyase

Staphylococcus aureus, strain 1801, hyaluronate lyase was purified and crystallized to homogeneity as ascertained by chromatography and disc-gel electrophoresis. Purification procedures included sequential ammonium sulfate fractionation, acetone precipitation, Sephadex gel filtration, and ion exchange chromatography. During its passage through the cation exchange column, the hyaluronate lyase was resolved into two minor and one major fraction. The major peak, which was found to be cationic, was further characterized and designated as Fraction III. Carbohydrate analysis showed the presence of neutral sugars galactose, glucose, and mannose in the ratio of 1:3:6. Amino sugars galactosamine and glucosamine (or mannosamine) were present in a ratio of 1:1. Quantitative amino acid analysis of the Fraction III showed a relative abundance of the basic amino acids lysine and histidine.     

Purification and characterisation of a minor low-sulphated dermatan sulphate-proteoglycan from ray skin.

A minor low-sulphated dermatan sulphate proteoglycan was isolated from ray skin by extraction with 2% sodium dodecyl sulphate, followed with ion-exchange chromatography, gel chromatography and density gradient centrifugation. The proteoglycan with a relative molecular mass (Mr) ranging from 70 to 120 kDa is composed of about two dermatan sulphate chains (Mr 33 kDa) bound on a protein core of Mr 27 kDa, and oligosaccharides consisting of uronic acids, hexosamines and neutral sugars. The major amino acids of the protein core were glycine (corresponding to about one-fourth of the total amino acids), serine, threonine, glutamic acid/glutamine, leucine and cysteine, together amounting to 56% of the total. The isolated proteoglycan does not interact with hyaluronic acid and does not form self-aggregates. Dermatan sulphate was rich in iduronic acid (62% of total uronic acid) and composed of non-sulphated (44%), and mono-sulphated disaccharides bearing esterified sulphate groups at positions C-4 (53%) or C-6 (3%) of the N-acetyl galactosamine. HPLC analysis of a pure preparation of dermatan sulphate, showed the presence of galactose and glucose possibly as branches on the dermatan sulphate chain.     

The composition of β-lactamase I and β-lactamase II from Bacillus cereus 569/H

1. Crystalline beta-lactamase I from Bacillus cereus 569/H yielded only amino acids on acid hydrolysis, but crystalline beta-lactamase II from the same organism yielded also substantial quantities of neutral sugars and amino sugars. 2. Analysis with an amino acid analyser indicated that the two enzymes were similar though not identical in overall amino acid composition. Analysis of neutral and amino sugars as their silyl derivatives by gas-liquid chromatography showed that the carbohydrate moiety of beta-lactamase II contained residues of glucose, galactose, mannose, fucose, glucosamine and galactosamine. 3. After oxidation and hydrolysis both beta-lactamases gave small amounts of cysteic acid. After treatment of inactive Zn(2+)-free beta-lactamase II with N-ethylmaleimide or iodoacetate enzymic activity was not restored by the addition of Zn(2+).     

Amino sugars in the glycoprotein toxin from Bacillus thuringiensis subsp. israelensis.

The carbohydrate content of purified Bacillus thuriniensis subsp. israelensis crystal toxin was determined by six biochemical tests, column chromatography on an amino acid analyzer, and the binding of 11 fluorescent lectins. The crystals contained approximately 1.0% neutral sugars and 1.7% amino sugars. The amino sugars consisted of 70% glucosamine and 30% galactosamine. No N-acetylneuraminic acid (sialic acid) was detected. The presence of amino sugars was confirmed by the strong binding of fluorescent wheat germ agglutinin and the weak binding of fluorescent soybean agglutinin. These lectins recognize N-acetyl-D-glucosamine and N-acetyl-D-galactosamine, respectively. The lectin-binding sites appeared evenly distributed among the protein subunits of the crystal. The sugars were covalently attached to the crystal toxin because wheat germ agglutinin still bound alkali-solubilized toxin which had been boiled in sodium dodecyl sulfate, separate by polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. This study demonstrates the covalent attachment of amino sugars and indicates that the B. thuringiensis subsp. israelensis protein toxins should be viewed as glycoprotein toxins. The crystals used in the present study were purified on sodium bromide density gradients. Studies employing crystals purified on Renografin density gradients can give artificially high values for the anthrone test for neutral sugars.     

Rapid separation of the alpha, beta, G gamma and A gamma globin chains by reversed-phase high pressure liquid chromatography.

The main human globin chains present in cord blood hemoglobins, α,β,^Gγ and ^Aγ, can be separated in 45 minutes by reversedphase high pressure liquid chromatography. The chains were identified by carboxymethyl cellulose chromatography and partial amino acid analysis of the cyanogen bromide fragments of the two γ chains. The purification of cyanogen bromide fragments and the separation and quantitation of their dansylated amino acids were accomplished using a similar system to that used for the separation of the globin chains. These results show the potential of this type of chromatography for the analytical and semi-preparative analysis of globin chains and the large advantages over conventional chromatographic techniques.     

Reciprocal inhibition of umbilical uptake within groups of amino acids

Eight pregnant sheep were infused with two amino acid mixtures of different composition: essential amino acids only and the essentials plus some of the nonessentials. Uterine and umbilical uptakes of amino acids were measured before and during infusion. For most of the amino acids, the infusion increased both maternal plasma concentration and umbilical uptake. However, depending on the infusate composition, the increase in maternal concentration of some amino acids was associated with no change or a significant reduction in umbilical uptake. Data were pooled from this and other, similar studies to test the hypothesis that umbilical uptake of several amino acids can be inhibited by coinfused amino acids. The test consisted of fitting the data, by means of multiple regression analysis, to the linear transformation of a saturation kinetics equation in which uptake is assumed to depend on maternal arterial concentrations. The analysis showed significant inhibitory effects within the neutral essential amino acids group and within the lysine-arginine group, with no demonstrable interaction between the two groups. Uterine uptakes did not show clear evidence of saturability and inhibitory interactions, suggesting a large transport capacity and low transporter affinity on the maternal surface of the trophoblast. We conclude that the transport of any given amino acid from placenta to fetus is a function of both its own maternal concentration and the maternal concentration of inhibitory amino acids.     

MUCOPOLYSACCHARIDES PRODUCED BY A STRAIN OF CLOSTRIDIUM PERFRINGENS.

Izumi, Kunihiko (Kanazawa University, Kanazawa, Japan). Mucopolysaccharides produced by a strain of Clostridium perfringens. J. Bacteriol. 83:956-959. 1962.-A new series of mucopolysaccharides was isolated from the culture medium of Clostridium perfringens and partially purified by the use of a column of anion-exchange resin. A large part of the substance was composed of neutral sugars, amino sugars, uronic acids, and oligopeptides, suggesting a structure analogous to that of bacterial cell walls. Acidic amino acids, especially aspartic acid, were the main constituents of the oligopeptides. The substance exhibited high viscosity when dissolved in water. The degree of viscosity in each fraction seemed to depend on the content of amino sugars and the chain length of the oligopeptides.     

Fractionation of plant extracts by thin-layer electrophoretic and chromatographic procedures

Methods are described for separating plant tissue extracts into amino acid, organic acid, and neutral fractions by means of thin-layer electrophoresis. The electrophoresis also provides the first-dimensional oeparation for both amino acid and organic acid fractions. Separation of amino acids and organic acids in the second dimension, and of sugars in both dimensions, is achieved by thin-layer chromatography. Suitable procedures for including the study of phospholipids and phosphate esters are suggested. Extracts from 20 mg tissue can be studied easily in this way.     

Composition of Fatty Acids and Carbohydrates in Leptospira

The fatty acid and monosaccharide composition of four pathogenic and two saprophytic strains of Leptospira was analyzed by gas chromatography (GC) and GC-mass spectrometry. Among the fatty acids, palmitic acid was most abundant and constituted 30 to 50% of the total fatty acids. Even-numbered unsaturated acids including octadecenoic, hexadecenoic, octadecadienoic, and tetradecadienoic acids comprised 40 to 60% of the total fatty acids. Tetradecanoic acid was about 5% in saprophytic strains, but 1% or less in pathogenic strains. The amount of chloroform-methanol extract of L. biflexa strain Ancona was 14 to 20% of the dry weight of the cell. Tetradecadienoic acid was found in the chloroform-methanol insoluble fraction, suggesting the presence of the acid in a bound form. GC analysis of monosaccharides revealed the existence of arabinose, xylose, rhamnose, mannose, galactose, glucose, glucosamine, and muramic acid in the cells. Among the neutral sugars, glucose was a minor component and was especially low in pathogenic strains. Total pentose content was about two to three times greater than total hexose.     

Human liver alanine aminopeptidase. Inhibition by amino acids.

Human liver alanine aminopeptidase is inhibited by L-amino acids having hydrophobic side chains such as Phe, Tyr, Trp, Met, and Leu. Blocking of the amino group or the carboxyl group greatly reduces the inhibitory capacity of the amino acid. Kinetic studies demonstrate that inhibition of hydrolysis of the substrate L-Ala-beta-naphthylamide is of the noncompetitive type. Inhibition of the substrate L-Leu-L-Leu is of the mixed type. Inhibition of the substrate L-Ala-L-Ala-L-Ala is of the competitive type. These changes in the mechanism of inhibition are thought to be the result of the binding of the amino acid to the third residue binding site on the enzyme. This is the part of the active center to which the third residue from the amino end of a peptide substrate is normally bound. The inhibitor constants of several alanine oligopeptides are shown to decrease with increasing length through L-Ala-L-Ala-L-Ala-L-Ala, demonstrating that alanine aminopeptidase is a multisited enzyme with three and possibly four residue sites per active center. The inhibitor constant for Gly-Gly--Phe suggesting that indeed the third residue site preferentially binds large hydrophobic residues.     

Nutritional studies of some members of mucorales IV. 1. Sugars,amino- and organic acids of the mycelium

Summary and Conclusions The sugars and amino acids of the fungal mycelium of six species of the order Mucorales were determined with the use of two-dimensional as well as circular paper chromatography. A critical survey of the above conclusions revealed that aspartic acid, glutamic acid, glycine and serine and -alanine were found in either free and bound form. Glutamine and arginine were absent in bound form. Threonine, histidine, isoleucine and tryptophane were absent in the ethanol soluble fraction. The presence and absence of certain amino acids in these species are good subsidiary characters for distinguishing one of them from the other.In the majority of the species three sugars viz., glucose, fructose and sucrose were found. InM. indica andC. bertholletiae sucrose was absent. Amongst the organic acid only tartaric and malic acids were detected in all the organisms.     

Analysis of carbohydrates and amino acids in vegetable waste waters by ion chromatography

High-performance anion exchange chromatography coupled with pulsed amperometric detection was used for the quantitative determination of total and free sugars in olive oil mill waste waters (OMWW). Automated amino acid ion chromatography was employed to analyse total and free amino acids in the same OMWW. Sugars were analysed in samples pre-purified by means of a three-step purification procedure involving: (i) methanol precipitation of OMWW; (ii) dialysis of the obtained solid and liquid fractions; and (iii) chromatographic purification on RP18 phase followed by Amberlite resin. The amino acids were determined directly in samples obtained from the first two steps performed for sugar analysis. The analysis carried out with the reported methodologies allowed the quantitative determination of total sugars and amino acids and the differentiation between their free and bound forms. The sugars determined were arabinose, fructose, galactose, glucose, rhamnose, xylose, galacturonic and glucuronic acids, and the amino acids were Asp, Glu, Thr, Ser, Pro, Gly, Ala, Val, Met, Ile, Leu, Tyr, Phe, Lys, His, Arg and Cys. Asn, Gin, and Trp were not detected. The technological, biotechnological and environmental advantages arising from this analytical methodology applied to OMWW are briefly discussed.     

On arabinose as a component of brain hyaluronate Confirmation by chromatographic,enzymatic and chemical ionization-mass spectrometric analyses

The controversy about the presence of the pentose arabinose in brain hyaluronate was reinvestigated using modern analytical technics. The purified bonive brain hyaluronate contained the neutral sugars: arabinose, 0.18%; glucose; 0.05%; and fucose, 0.22%. The confirmation of the presence of arabinose was obtained by paper and thin layer chromatography of the neutral sugars in deionized hyaluronate hydrolysate. Gas-liquid chromatography of the aldononitrile peracetate of the pentose isolated by preparative paper chromatography gave a single distinct peak, corresponding to standard arabinose on three columns packed with three different phases. Chemical ionization data and mass spectrum of the aldononitrile peracetate drivative agreed with those of the authentic arabinonitrile tetracetate. Analysis of the isolated pentose with the help of the enzymes l-arabinose isomerase and l-ribulose kinase, which are specific for their substrates, further established its identity as l-arabinose.     

Studies on the influence of foliar nutrient sprays on the root exudation pattern in four crop plants

Summary Studies conducted to examine the exudation pattern of amino acids and sugars in four crop plants,viz sorghum, sunnhemp, ragi, and tomato indicated that in all, 17 known amino acids and 4 sugars were exuded and that the number and nature of the exuded amino acids and sugars differed with the plant species and with the age of plant. Glutamic and aspartic acids were found to be present in the exudates of all the plant species at all stages of plant growth examined. The quantities of amino acids and sugars differed with plant species and the maximum quantity of the chemicals was exuded during the early stages of plant growth. Glutamic acid among amino acids, and glucose among sugars, were always present in higher concentrations than the others, in the exudates in all the four crop plants.Foliar application of nitrogen in the form of NaNO₃ and phosphorus as Na₂HPO₄, was found to alter the exudation pattern of amino acids and sugars and such influence differed in different plant species. There was a general increase in the total concentration of amino acids and a decrease in sugar content in the exudates after treatment of the foliage with N, while a decrease in the amino acid content and increase in total sugars with P-treatment was observed.     

Nucleotide sequence analysis of the gene encoding the Deinococcus radiodurans surface protein, derived amino acid sequence, and complementary protein chemical studies.

The complete nucleotide sequence of the gene encoding the surface (hexagonally packed intermediate [HPI])-layer polypeptide of Deinococcus radiodurans Sark was determined and found to encode a polypeptide of 1,036 amino acids. Amino acid sequence analysis of about 30% of the residues revealed that the mature polypeptide consists of at least 978 amino acids. The N terminus was blocked to Edman degradation. The results of proteolytic modification of the HPI layer in situ and Mr estimations of the HPI polypeptide expressed in Escherichia coli indicated that there is a leader sequence. The N-terminal region contained a very high percentage (29%) of threonine and serine, including a cluster of nine consecutive serine or threonine residues, whereas a stretch near the C terminus was extremely rich in aromatic amino acids (29%). The protein contained at least two disulfide bridges, as well as tightly bound reducing sugars and fatty acids.     

Sheath and outer membrane components from the cyanobacterium Fischerella sp. PCC 7414

A purified sheath fraction and an outer membrane fraction were obtained from the cyanobacterium Fischerella sp. PCC 7414. The sheath had a fine structure with osmiophilic fibers running in parallel to the cell surface in two distinct layers. The sheath fraction contained mainly neutral sugars (Glc, Man, Gal, Xyl, Fuc, 2-O-methylhexose), GlcN, uronic acids, and minor components such as amino acids, sulfate, phosphate, and fatty acids. The protein moiety was removable from the sheath fraction by treatment with boiling sodium dodecyl sulfate. The presence of three different 3-hydroxy fatty acids (3-OH-14:0, 3-OH-16:0, 3-OH-18:0) in addition to GlcN indicated the presence of lipopolysaccharide in the outer membrane. One major (M_r 50,000) and two minor (M_r 54,000 and 65,000) proteins were detected as constituents of the outer membrane.Abbreviations A₂pm diaminopimelic acid - GLC gas-liquid chromatography - GlcN glucosamine - Ino inositol - MurN muramic acid - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Isolation and a partial amino acid sequence of insulin from the islet tissue of cod (Gadus callarias)

1. Insulin has been isolated by gel filtration and ion-exchange chromatography from extracts of the discrete islet tissue of cod. The final preparation yielded a single band on electrophoresis at two pH values. The biological potency was 11.5 international units/mg. in mouse-convulsion and other assay procedures. 2. Glycine and methionine were shown to be the N-terminal amino acids of the A and B chains respectively. An estimate of the molecular weight together with amino acid analyses indicated that cod insulin, like the bovine hormone, consists of 51 amino acid residues. In contrast, the amino acid composition differs markedly from bovine insulin. 3. Oxidation of insulin with performic acid yielded the A and B peptide chains, which were separated by ion-exchange chromatography. Sequence studies on smaller peptides isolated from enzymic digests or from dilute acetic acid hydrolysates of the two chains have established the sequential order of 14 of the 21 amino acid residues of the A chain and 25 of the 30 amino acid residues of the B chain.     

Modelling enteric bacteria survival in aquatic systems

We measured the uptake of carbon and inorganic nitrogen in nutrient-enriched water samples during 15-days incubation in summer in Lake Nakanuma, Japan. We calculated daily variations in neutral sugars and amino acids of the phytoplankton and estimated the efficiency of the increases in sugars and amino acids relative to photosynthetic activities. Only a small portion of carbon incorporated via photosynthesis was used for synthesis of sugars and amino acids during the incubation periods. The percentage increase in neutral sugars plus amino acids compared to photosynthetic rates ranged from 3.7 to 26.9% with an average of 12.8%. These findings suggest that large amounts of photosynthates were not used for the synthesis of cell components of phytoplankton and were lost through processes such as excretion and respiration.