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S Hase

Publications and source records attributed to S Hase.

At least 145 records · Page 8Linked to original sources
PubMed1987

Identification method for twelve oligomannose-type sugar chains thought to be processing intermediates of glycoproteins.

A mixture of the pyridylamino (PA) derivatives of 12 oligomannose-type sugar chains was fractionated into five fractions (mannose5N-acetylglucosamine2-PA approximately mannose9N-acetylglucosamine2-PA) by size-fractionation HPLC with a MicroPak AX-5 column. Each fraction thus obtained was then analyzed by reversed-phase HPLC with a Cosmosil 5C18-P column. In this way, the 12 PA-oligomannose-type sugar chains were completely separated from each other. The method was used to identify the structures of oligomannose-type sugar chains of human C3, the third component of human complement.

Carbohydrate Sequence↗
PubMed1987

Possible pathway for the processing of sugar chains containing xylose in plant glycoproteins deduced on structural analyses of sugar chains from Ricinus communis lectins.

The structures of sugar chains from two lectins in seeds of the castor bean (Ricinus communis) were identified. The sugar chains were liberated from the lectins by hydrazinolysis. After N-acetylation, the reducing-end residues of the sugar chains were coupled with 2-aminopyridine. The pyridylamino derivatives thus obtained were purified by gel filtration and HPLC. The structures of the purified derivatives were identified by component sugar analysis, stepwise exoglycosidase digestion, partial acetolysis, and 500 MHz 1H-NMR spectroscopy. A new processing pathway for sugar chains in plant glycoproteins was proposed on the basis of the structures of the sugar chains.

Carbohydrate Sequence↗
PubMed1986

1H-NMR analysis of the sugar structures of glycoproteins as their pyridylamino derivatives.

The 1H-NMR spectra of a series of pyridylamino (PA-) derivatives of oligosaccharides were obtained and compared with those of the corresponding asparagine-linked sugar chains in order to elucidate the effect of the PA-group on the chemical shifts of structural-reporter signals. The effects were found to be localized within the two residues from the end group. Thus, the data for asparagine-linked chains in the literature are applicable to PA-derivatives, so the combination of pyridylamination and NMR measurements greatly reduces the time required for structure analysis of sugar chains of glycoproteins, because the isolation and purification of the chains as PA-derivatives are easy and efficient.

Asparagine↗
PubMed1986

Structure of a sugar chain of a protease inhibitor isolated from barbados pride (Caesalpinia pulcherrima Sw.) seeds.

An asparagine-linked sugar chain of a protease inhibitor from barbados pride (Caesalpinia pulcherrima Sw.) was liberated by hydrazinolysis. After N-acetylation, the reducing end residue of this carbohydrate unit was coupled with 2-aminopyridine and the pyridylamino (PA-) derivative was purified by gel-filtration and reversed-phase HPLC. The structure of the resulting PA-sugar chain was determined mainly by stepwise exoglycosidase digestions and 500 MHz 1H-NMR spectroscopy and proved to be as follows: (formula; see text).

Carbohydrate Conformation↗
PubMed1986

The structure of sugar chains of Japanese quail ovomucoid. The occurrence of oligosaccharides not expected from the classical biosynthetic pathway for N-glycans; a method for the assessment of the structure of glycans present in picomolar amounts.

While the structure of the major oligosaccharide of Japanese quail ovomucoid was reported earlier (Hase, S. et al. (1982) J. Biochem. 91, 735-737), the structures of the minor oligosaccharide units were investigated for the first time in the present studies. For this purpose, the glycans of the protein were liberated from the polypeptide chain by hydrazinolysis. After N-acetylation, the reducing ends of the oligosaccharides obtained were coupled with 2-aminopyridine, and then the resulting fluorescent derivatives were purified by Bio-Gel P-2 column chromatography and reversed-phase HPLC. The chemical structures of two minor oligosaccharide units were determined with the aid of exoglycosidases, and by methylation analysis and Smith degradation. The results demonstrated that the ovomucoid contains the following two monoantennary glycans: Man alpha 1-6(Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc and Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc. The latter structure was not predicted by the classical metabolic pathway for the N-glycans to be formed. The structures of three additional minor heterosaccharides were deduced from their elution positions on HPLC together with the results of determination of their molecular sizes and the HPLC elution positions of their enzymatic degradation products. It is noteworthy that for the latter procedure for the estimation of the structures of oligosaccharides only minute quantities of glycans (several hundreds pmol) are required.

Amino Acids↗
PubMed1985

Microquantitative analysis of neutral and amino sugars as fluorescent pyridylamino derivatives by high-performance liquid chromatography.

A method for the quantitation of picomole amounts of neutral and amino sugars in glycoconjugates was developed. Glycoconjugates were hydrolyzed with a mixture of equal amounts of 4 M trifluoroacetic acid and 4 M hydrochloric acid, and the free amino groups were acetylated. Sugars were coupled with 2-aminopyridine. After the excess reagents were removed by gel-permeation high-performance liquid chromatography, the fluorescent pyridylamino derivatives of sugars were separated and quantified by high-performance liquid chromatography on a reversed-phase column. This method allowed the determination of 0.01-10 nmol of sugars. About 100 pmol of several glycoconjugates were analyzed by the present method, with satisfactory results.

Amino Sugars↗
PubMed1985

Comparative study on amino acid sequences of Kunitz-type soybean trypsin inhibitors, Tia, Tib, and Tic.

The amino acid sequences of three variants of the Kunitz-type trypsin inhibitors, Tia, Tib, and Tic, obtained from some cultivars of soybean were determined by conventional methods. All three inhibitors consisted of 181 amino acid residues. The differences in the amino acid sequences are as follows: Tia E12 G55 Y62 H71 S74 M114 L120 P137 L176; Tib S F N R V I T V; Tic E. The amino acid sequences of Pro(60)-Ser(61) and Asp(154)-Asp(155)-Gly(156)-His(157) of Tia reported previously (Koide & Ikenaka (1973) Eur. J. Biochem. 32, 417-431) were amended to Ser(60)-Pro(61) and His(154)-Asp-Asp-Gly(157), respectively.

Amino Acid Sequence↗
PubMed1985

Structures of sugar chains of the third component of human complement.

Human C3, the third component of human complement, contained mannose and N-acetylglucosamine as sugar components. The sugar chains were liberated from the polypeptide chains by hydrazinolysis, and the free amino groups were N-acetylated. The reducing end residues of the sugar chains thus obtained were tagged with 2-aminopyridine, and the pyridylamino (PA-) derivatives of sugar chains were separated by high-performance liquid chromatography. The structures of purified PA-sugar chains were analyzed by a combination of stepwise exoglycosidase digestions, size determination by paper electrophoresis, methylation analysis, Smith degradation, and partial acetolysis. These results showed that C3 contained two high-mannose type sugar chains ranging from Man5GlcNAc2 to Man9GlcNAc2. Analyses of the sugar chains of alpha- and beta-chains of C3 indicated that the alpha-chain contained mainly Man8GlcNAc2 and Man9GlcNAc2, while the beta-chain contained mainly Man5GlcNAc2 and Man6GlcNAc2.

Acetates↗
PubMed1984

Reexamination of the pyridylamination used for fluorescence labeling of oligosaccharides and its application to glycoproteins.

The pyridylamination reaction of sugar chains from glycoproteins was re-investigated to raise the total yield of pyridylamino sugars. Sugar chains of glycoproteins were released by hydrazinolysis, and free amino groups were N-acetylated. The sugar chains were coupled with 2-aminopyridine, and the pyridylamino derivatives thus obtained were purified by Sephadex G-15 column chromatography and analyzed by high performance liquid chromatography. In this study, conditions for the coupling reaction (temperature, time, pH, concentration of 2-aminopyridine, and amount of the reducing reagent) were re-investigated. Under the conditions established in the present study, the total recovery of pyridylamino derivatives of sugar chains of glycoproteins was about 70%, and 0.15 nmol of a glycoprotein was enough to detect the pyridylamino derivatives of sugar chains. The stability of sialyl and fucosyl linkages under the present conditions was also studied.

Acetylation↗
PubMed1983

The effect of cellular energy reserves and internal calcium ions on the potassium conductance in skeletal muscle of the frog.

The increase in K+ conductance induced by repetitive stimulation in metabolically poisoned sartorius muscle fibres of the frog was investigated, using a two-micro-electrode voltage-clamp technique. After the inhibition of creatine kinase by 0.4 mM-1-fluoro-2,4-dinitrobenzene (FDNB) and a complete and irreversible exhaustion of contractility, a nearly linear current-voltage relation was measured between -100 and 0 mV. In the presence of CN- (4 mM) an 'intermediate state' could be established by repetitive stimulation towards complete mechanical exhaustion. In this labile state, the high and potential-independent K+ conductance could be induced by repetitive voltage-clamp pulses (100 ms duration) from -85 to 0 mV membrane potential. After the pulses had ceased, fibres regained their original membrane conductance within several minutes. After the electrophoretic injection of the Ca2+-chelating agent H2EGTA2- into fibres in the intermediate state, an increase in membrane conductance by repetitive voltage-clamp pulses could no longer be induced. Fibres in the intermediate state into which H2EGTA2- -buffered Ca2+ (free Ca2+ approximately 10(-5) M) was injected, or to which external caffeine (1.5 mM) was applied, showed a spontaneous and reversible increase in membrane conductance. In metabolically poisoned and mechanically exhausted sartorius muscles the concentrations of creatine phosphate (CP) and ATP were estimated using biochemical standard methods. The concentration of CP remained basically unchanged after FDNB poisoning. In solutions containing CN- plus iodoacetate CP fell below the detectable concentration of about 0.5-1% of the normal value. ATP decreased to slightly less than 20% under both conditions. It is concluded that internal free Ca2+ promotes the activation of the K+ conductance in exhausted muscle fibres, and that a shortage of energy reserves increases the 'sensitivity' of K+ channels to Ca2+ ions.

Adenosine Triphosphate↗
PubMed1982

Identification of the trimannosyl-chitobiose structure in sugar moieties of Japanese quail ovomucoid.

Ovomucoid isolated from egg white of japanese quail contained mannose, galactose, glucosamine, and sialic acid (8.6 : 1.3 : 6.6 : 0.5 mol/mol) as sugar components. Sugar chains were liberated from the polypeptide portion by hydrazinolysis, N-acetylated and fractionated on Bio-Gel P-2. The reducing ends of the thus obtained sugar moieties were pyridylaminated. Chemical and exoglycosidase digestion studies of the fluorescent pyridylamino derivative of a major sugar fraction indicated the following structure for one of the major sugar chains of japanese quail ovomucoid; Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc.

Animals↗
PubMed1982

Studies on heterogeneity of Taka-amylase A: isolation of an amylase having one N-acetylglucosamine residue as the sugar chain.

Crystalline Taka-amylase A, prepared from Takadiastase, was fractionated into four fractions by DEAE-Sephacel and Concanavalin A-Sepharose column chromatography. The relative weight ratio of the fractions was 90 : 4 : 4 : 2. They had similar molecular weights (51,000), amino acid compositions, and hydrolytic activity against soluble starch, but different phenyl maltosidase activities and electrophoretic mobilities on polyacrylamide gel electrophoresis. Three of the fractions mainly had the high mannose type sugar chain with the sugar composition of Man5-GlcNAc2, but the other fraction had only one N-acetylglucosamine residue as the sugar chain. These results suggested that Taka-amylase A was heterogeneous both in the sugar portions and in the polypeptide portions.

Acetylglucosamine↗
PubMed1981

A highly sensitive method for analyses of sugar moieties of glycoproteins by fluorescence labeling.

The sensitivity of a fluorescence labeling method ((1979). J. Biochem. 85, 989--994; 995--1002) for structure analyses of asparagine-linked sugar moieties of glycoproteins was increased by using HPLC with a fluorescence detector. Sugar moieties were separated from polypeptide portions by hydrazinolysis. Free amino groups thus exposed were acetylated and the reducing ends of sugar chains were reductively aminated with a fluorescent reagent, 2-aminopyridine, by the use of sodium cyanoborohydride. The pyridylamino derivatives were purified on a Dowex 1 column to eliminate undesired substances. The separation and identification of the pyridylamino derivatives were carried out by HPLC with a column of C18 reversed phase or gel permeation phase. As little as 0.1 pmol of pyridylamino derivatives can be detected. Ten microgram of Taka-amylase A was easily detected by this system. The method was also applied to some other glycoproteins.

Aminopyridines↗
PubMed1981

A highly sensitive method for determination of molecular weights of neutral oligosaccharides by fluorescence labeling.

The electrophoretic mobilities of pyridylamino derivatives of oligosaccharides in paper electrophoresis at pH 5 ((1979) J. Biochem. 85, 989--994) were independent of linkage positions, anomeric configurations, and acetamido groups up to a molecular weight of 1,700 and dependent on only their molecular weights. To increase the accuracy and sensitivity of the method, the pyridylamino derivative of a sample (10--50 pmol) was mixed with small amounts of pyridylamino derivatives of internal standard oligosaccharides of different molecular weights. The mixture was electrophoresed on a paper. Sections of paper corresponding to the standard oligosaccharides were extracted with water and then each extract was analyzed by high performance liquid chromatography with a fluorescence spectrophotometer. The molecular weight of the pyridylamino derivative of the sample was calculated by comparing its mobility with those of the pyridylamino derivatives of the internal standard oligosaccharides which were detected together with the sample on the chromatogram.

Chromatography, High Pressure Liquid↗