Variability in the 11S globulin fraction of seed storage proteins ofHelianthus (Asteraceae)

The seed storage globulins from sixHelianthus and four hybrids were studied using mono and bidimensional gel SDS electrophoresis (+ 2 mercaptoethanol). The polypeptide composition of each subunit was determined. Different pairs are specifically expressed according to the species studied. Three typical patterns were discriminated. All the studied species exhibit five subunits: two of them are expressed in all the species (₁₁ and ₂₂). The subunit corresponding to the ₁₁ pair is present inH. petiolaris and in the three populations ofH. annuus studied. The _2b2 pair is common toH. annuus andH. argophyllus. H. petiolaris presents two specific _2a2 and ₄₄ pairs andH. annuus a specific ₃₃ pair. InH. argophyllus _{11 33} or ₄₄ are never observed but are replaced by ₁₃ and ₃₁ pairs. Some globulins, poorly represented, are of forms but present chains of higher molecular weights (in the range 54–56 kDa). Expressing variations in the banding patterns between these species by the use of a similarity index reveals complete identity between the three populations ofH. annuus. Identity between the twoH. petiolaris studied is also observed.H. annuus andH. argophyllus appear to be closer to each other thanH. petiolaris concerning the seed storage globulins.     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

The glycolipid specificity ofErythrina cristagalli agglutinin

The interaction of¹²⁵I-labeledErythrina cristagalli agglutinin (ECA) with neutral glycosphingolipids on thin layer chromatograms was examined by the overlay technique followed by radioautography. The lectin bound topara-globoside with a sensitivity about 10 times higher than to lactosylceramide or globoside, in agreement with the specificity of the lectin forN-acetyllactosamine. The lower limit of detection ofpara-globoside was about 0.66 nmol. The specific binding of ECA to this glycolipid was confirmed by a highly sensitive enzyme-linked lectin assay (ELLA), utilizing the horseradish peroxidase-avidin-biotin system for detection of bound lectin. Overlays of neutral glycosphingolipid extracts from human erythrocyte membranes and from human granulocytes with ECA demonstrated that the lectin can be employed for the detection of small amounts ofpara-globoside in biological materials also in the presence of excess globoside. No staining was obtained when thin layer chromatograms of neutral glycosphingolipid extracts from rabbit erythrocyte membranes were overlayed with¹²⁵I-ECA. Afterin situ treatment of the chromatograms with -galactosidase, the lectin bound to several components, one of which had a mobility corresponding to that of the pentahexosylceramide Gal3Gal4GlcNAc3Gal4Glc1Cer, the major neutral glycosphingolipid of rabbit erythrocytes, thus providing further evidence for the specificity of ECA forpara-globoside.Abbreviations GSL glycosphingolipid(s) - CDH lactosylceramide, Gal4Glc1Cer - CTH trihexosylceramide, Gal4Gal4Glc1Cer - GLOB globoside, GalNac3Gal4Gal4Glc1Cer - PG para-globoside, Gal4GlcNAc3Gal4Glc1Cer - AsG_M1 asialo-G_M1, Gal3GalNAc4Gal4Glc1Cer - FORS Forsmann antigen, GalNAc3GalNAc3Gal4Gal4Glc1Cer - CPH pentahexosylceramide, Gal3Gal4GlcNAc3Gal4Glc1Cer - ECA Erythrina cristagalli agglutinin - SBA soybean agglutinin - PBS phosphate-buffered saline - PVP-40 polyvinylpyrrolidone M.W. 40000 - BSA bovine serum albumin - HRP-avidin horseradish peroxidase conjugated to avidin - ELLA enzyme-linked lectin assay - ELISA enzyme-linked immunosorbent assay - PMNL polymorphonuclear leukocytes - HPTLC high performance thin layer chromatography     

Detection and partial characterization of two antigenically distinct β-amylases in developing kernels of wheat

A -amylase (EC 3.2.1.2) was identified in the outer pericarp (P) of developing seeds of wheat (Triticum aestivum L.) and compared with the well known -amylase which is synthesized during seed development in the starchy endosperm (E). The enzyme P already exists in the tissues before anthesis and vanishes at the time when E starts to accumulate. The isoelectric-focusing patterns of P and E are very similar. The relative molecular weight (M_r) of P is slightly higher than that of E (66 and 64.5 kDa, respectively). Both P and E exhibit common epitopes in addition to epitopes specific for each of them. The two enzymes were identified in small amounts in the green tissues of the developing seeds (inner pericarp and testa). No antigenic difference was detected between P and the -amylases of roots and leaves.Abbreviations P pericarp -amylase - E endosperm -amylase - IS1 anti--amylase immune serum - IS2 anti- and anti- amylase immune serum - IS3 anti- amylase immune serum - IEF isoelectric focusing - IgG immunoglobulin G The authors thank Dr. P. Ziegler (Universität Bayreuth, FRG) for stimulating discussion and for useful suggestions during the writing of the text. The authors thank Miss C. Mayer for her skillful technical assistance.     

Polymorphism of human liver alcohol dehydrogenase: Identification of ADH2 2-1 and ADH2 2-2 phenotypes in the Japanese by isoelectric focusing

Liver homogenate-supernatants from most Japanese exhibit an atypical pH optimum for ethanol oxidation at pH 8.8 instead of 10.5, the typical pH-activity optimum. It has been proposed that atypical livers contain alcohol dehydrogenase isozymes with ₂ subunits while typical livers contain isozymes with ₁ subunits, both produced by the ADH ₂ gene. Because it is difficult to differentiate the atypical ADH₂ 2-2 phenotype from the ADH₂ 2-1 phenotype by starch gel electrophoresis, an agarose isoelectric focusing procedure was developed that clearly separated the atypical Japanese livers into two groups, A1 and A2. The isozymes in A1 and A2 livers were purified. Type A1 livers contained a single isozyme with an atypical pH-rate profile; it was designated ₂₂. Three isozymes were isolated from A2 livers, two of which corresponded to ₁₁ and ₂₂. A third, absent from the typical and the atypical A1 livers, had an intermediate mobility; it was designated ₂₁. Type A1 livers are, therefore, the homozygous ADH₂ 2-2 phenotype, and type A2 livers, the heterozygous ADH₂ 2-1 phenotype. The ADH₂ 2-2 phenotype was found in 53% of 194 Japanese livers, and the ADH₂ 2-1 phenotype, in 31%. Accordingly, the frequency of ADH ₂ ² was 0.68.This study was supported by U.S. Public Health Service Grant AA 02342.     

β-Carotene production by Flavobacterium multivorum in the presence of inorganic salts and urea

Flavobacterium multivorum, a non-fermenting Gram-negative bacteria, normally produces zeaxanthin (3R, 3 R-, -carotene-3, 3 diol) as its main carotenoid. The effect of supplementation of various inorganic salts and urea on the growth, total carotenoid production, and proportion of -carotene (, -carotene), -cryptoxanthin (, -caroten-3-ol), and zeaxanthin produced by F. multivorum was investigated. Urea and several salts, such as calcium chloride, ammonium chloride, lithium chloride, and sodium carbonate, improved total carotenoid production by 1.5- to 2.0-fold. Urea and sodium carbonate had an unexpectedly strong positive effect on -carotene production at the expense of zeaxanthin formation. The effect was found to be independent of incubation time, and -carotene represented 70% (w/w) of the total carotenoid content. The cumulative effect of urea and sodium carbonate was further studied using response surface methodology. An optimum medium was found to contain 4,000 and 4,070 mg l^–1 urea and sodium carbonate, respectively. The maximum -carotene level was 7.85 g ml^–1 culture broth, which represented 80% (w/w) of the total carotenoid produced. Optimization resulted in 77- and 88-fold improvements in the volumetric and specific -carotene levels, respectively, accompanied by a simultaneous decrease in the zeaxanthin level as compared to the control medium. The carotenoid production profile in the optimized medium indicated that -carotene was produced maximally during the late exponential phase at 0.41 g ml^–1 h^–1. It is possible that this organism could be an excellent commercial source of either -carotene or zeaxanthin, depending on initial culture conditions.     

Purification and characterization of two intracellular β-glucosidases from the Neurospora crassa mutant cell-1

Two intracellular -glucosidases (E.C. 3.2.1.21) were purified from the filamentous fungus Neurospora crassa, mutant cell-1 (FGSC no. 4335) and characterized. The extent of purification were 2.55- and 28.89-fold for -glucosidase A and -glucosidase B, respectively. -Glucosidase A was a dimeric protein, and B a monomeric protein, with molecular masses of 178 and 106 kDa, respectively. Both isoenzymes were glycoproteins with relatively high carbohydrate contents (-glucosidase A, 29.2%; -glucosidase B, 34.2%). The isoelectric points determined by IEF were 6.27 and 4.72, respectively. pH optima for activity were determined to be 5.0 and 5.5, and temperature optima to be 55 and 60 °C, for -glucosidases A and B, respectively. Both purified -glucosidases. especially -glucosidase B, showed relatively high stability against pH and temperature. Both enzymes were stable in the pH range of 5.0–9.0. The activities were completely retained up to 48 h at temperatures below 40 °C. At higher temperatures, enzymes were relatively unstable and lost their activities at 60 °C after 24 h. Both -glucosidases were highly activated by CuCl₂, and inhibited by SnCl₂ and KMnO₄. Hg²⁺ and Ag⁺ also inhibited severely -glucosidase B. The K _m and V _max values of the isoenzymes against cellobiose as substrate were 1.50 mM and 12.2mol min^–1 mg^–1 for -glucosidase A and 2.76 mM and 143.5 mol min^–1 mg^–1 for -glucosidase B.     

New ganglioside analogs that inhibit influenze virus sialidase

Synthetic thioglycoside-analogs of gangliosides such as Neu5Ac)2-S-6)Glc-(1-1)Ceramide (1) and the GM3 analog Neu5Ac(2-S-6)Gal-(1–4)Glc(1-1)Ceramide (2), competitively inhibited GM3 hydrolysis by the sialidase of different subtypes of human and animal influenza viruses with an apparent K_i value of 2.8×10^–6 and 1.5×10^–5 M, respectively. The inhibitory activity of the ganglioside GM4 analog [Neu5Ac-(2-S-6)Gal-(1-1)Ceramide (3)], in which the glucose of 1 was substituted by galactose, was lower than that of 1 (K_i =1.0×10^–4 M). The thioglycoside-analogs (1, 2, 3) of the gangliosides were nonhydrolyzable substrates for influenza virus sialidase. The inhibitory activity of 1 to bacterial sialidases fromClostridium perfringens andArthrobacter ureafaciens was considerably lower than that to influenza virus sialidase, indicating that the structure of the active site in bacterial and influenza virus sialidase may be different and the analogs may be useful to determine the orientation of the substrate to the active site of sialidases, especially of influenza viruses.Abbreviations Cer ceramide - GM3 Neu5Ac(2–3)Gal(1–4)Glc(1-1)Cer - GM4 Neu5Ac(2–3)Gal(1-1)Cer Gangliosides were abbreviated according to Svennerholm [1] and the recommendation of the IUPAC-IUB Commission on Biochemical Nomenclature [2].     

α-and β-Glycosidases in maize roots

Four glycosidases were analyzed in 10 mm apical segments prepared from growing roots (15 mm) of Zea mays L. The pH optima were found to be 5.8 for -glucosidase, 4.4 for -galactosidase, 6.4 for -glucosidase and 6.0 for -galactosidase. The -glucosidase showed 4-fold higher activity than the -galactosidase. The distribution of the -glucosidase activity was signifcantly different from that of the -galactosidase, -glucosidase and -galactosidase.Abbreviations -Glu -glucosidase - -Gal -galactosidase - -Glu -glucosidase - -Gal -galactosidase     

Structure of a new nonasaccharide isolated from human milk: VI2Fuc,V4Fuc,III3Fuc-p-lacto-N-hexaose

The structure of a new nonasaccharide isolated from human milk has been investigated. By using methylation analysis, FAB-MS and¹H-and¹³C-NMR spectroscopy as basic methods of structural investigation, this oligosaccharide was identified as VI²--Fuc,V⁴-Fuc,III³--Fuc-p-lacto-n-hexaose: Fuc1-2Gal1-3[Fuc1-4]GlcNAc1-3Gal1-4[Fuc1-3]GlcNAc1-3Gal1-4Glc.Abbreviations COSY correlation spectroscope - DP degree of polymerisation - FAB-MS fast atom bombardment-mass spectrometry - HPLC high performance liquid chromatography - NMR nuclear magnetic resonance - GLC gas-liquid chromatography     

Single mid-chain GlcNAcβ1-6Galβ1-4R sequences of linear oligosaccharides are resistant to endo-β-galactosidase ofBacteroides fragilis

Endo--galactosidase (EC 3.2.1.103) ofBacteroides fragilis, at 250 mU ml^–1, did not cleave the internal galactosidic linkage of the linear radiolabelled trisaccharide GlcNAc1-6Gal1-4GlcNAc, or those of the tetrasaccharides Gal1-4GlcNAc1-6Gal1-4GlcNAc and Gal1-4GlcNAc1-6Gal1-4Glc. The isomeric glycans which contained the GlcNAc1-3Gal1-4GlcNAc/Glc sequence were readily cleaved.Abbreviations GlcNAc 2-acetamido-2-deoxy-d-glucose - Lact lactose - MT maltotriose - MTet maltotetraose - R _MTet chromatographic migration rate in relation to that of maltotetraose     

Structures of the carbohydrate units of polysialoglycoproteins isolated from the eggs of four species of salmonid fishes

Fish egg polysialoglycoprotein (PSGP) is a novel type of 200 kDa-glycoprotein containing more than 50% sialic acid by weight and about 90O-glycosidically-linked sialoglycan units per molecule. Of about 100 different molecular species assumed to be present in a sialoglycan mixture obtained by alkaline borohydride treatment ofSalvelinus leucomaenis pluvius PSGP, 23 mono- to tetrasialylglycans were isolated by anion-exchange chromatography and preparative column chromatography on porous silica, and their structures were determined. Core asialo-oligosaccharides were obtained from PSGP of four species of salmonid fishes by exhaustive enzymatic desialylation of sialoglycan mixtures and the structures of purified compounds were determined. Two complete types of asialopentasaccharide core structures, Fuc1-3GalNAc1-3Gal1-4 Gal1-3GalNAcOL, and GalNAc1-4GalNAc1-3Gal1-4Gal1-3GalNAcOL, and all of the possible biosynthetic precursors of these pentasaccharide cores were found in every PSGP examined. All types of oligosaccharide chains, both complete and incomplete, were found to occur in highly sialylated forms in PSGP.Abbreviations NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - GalNAcOL N-acetylgalactosaminitol - PSGP polysialoglycoprotein - SRO sialidase-resistant oligosaccharide     

1H and13C-NMR analysis of four pentasaccharides from urine of blood-group A and B,Leb adults. Confirmation of the structure of two new oligosaccharides: Fuc(α1–2)[GalNAc(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc and Fuc(α1–2)[Gal(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc

The major pentasaccharides Fuc(1-2)[GalNAc(1-3)]Gal(1-4)[Fuc(1-3)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-4)[Fuc(1-3)]Glc, which are normally present in the urine of bloodgroup A Le^b and B Le^b healthy subjects, were each found to be contaminated by a minor component when analysed by¹H-NMR. The determination of these structures, Fuc(1-2) [GalNAc(1-3)]Gal(1-3)[Fuc(1-4)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-3)[Fuc(1-4)]Glc, was based on the results of methylation analysis and¹H/¹³C-NMR spectroscopy.Abbreviations HPLC high performance liquid chromatography - GLC gas liquid chromatography - NMR nuclear magnetic resonance - COSY correlation spectroscopy - Gal d-galactopyranose - GalNAc 2-acetamido-2-deoxy-d-galactopyranose - Glc d-glucopyranose - Fuc l-fucopyranose - LNDFH I lacto-N-difucohexaose I (Le^b determinant     

The structure of the incompatibility factors of Schizophyllum commune: Constitution of the three classes of B factors

Summary The B factors of Schizophyllum commune are of 3 classes: The high recombining class I has 7 alleles and 7 alleles; the low recombining classes are class II with 7 allels and probably 2 alleles and class III with probably 2 (or also 2) alleles and 7 allels. A fourth hypothetical class (-) was not found and either does not exist or is indistinguishable from class III by the tests employed. The and alleles differ from and by either (a) mutations affecting both mating specificity and recombination frequency, or (b) deletions involving most of the B region.The research was supported by a grant from the Atomic Energy Commission of the U.S. No. (30-1)-3875 and was performed at the Biological Laboratories, Harvard University, Cambridge, Mass., U.S.A.     

Cytological characterization,powdery mildew resistance and storage protein composition of tetraploid and hexaploid 1BL/1RS wheat-rye translocation lines

Summary Progenies of a tetraploid 1BL/1RS wheat-rye translocation line, CV 256, selected from the cross Cando x Veery, were analyzed by means of Giemsa C-banding. CV 256 is cytologically stable for the presence of the 1BL/1RS translocation but still segregating for A- and B-genome chromosomes of Cando and Veery. In CV 256, nucleolar activity of the 1RS NOR locus is suppressed, as judged by the absence of a secondary constriction in that rye segment and the capability of organizing nucleoli. PAGE analysis of prolamins confirmed the presence of two 1RS secalins in all single seeds analyzed. SDS-PAGE analysis of reduced glutenins of single seeds indicated that some seeds contained the Cando Glu-B1 locus (subunits 6+8), some contained the Veery Glu-B1 locus (subunits 7+9) while others contained all four subunits, indicating that the material was heterozygous. Pm8 resistance is expressed in the tetraploid 1BL/1RS translocation line based on the reactions of six well-defined powdery mildew isolates. However, Pm8 resistance is not expressed in the hexaploid wheat cultivars Olymp, Heinrich and Florida, which also contain the 1BL/1RS translocation. Obviously, the existence of the 1BL/1RS translocation is not a proof for the expression of the associated genes. PAGE results did not show a clear linkage between powdery mildew resistance and the presence of 1RS secalins.     

Theβ-subunit of human chorionic gonadotropin containsN-glycosidic trisialo tri- and tri′-antennary carbohydrate chains

TheN-linked carbohydrate chains of the-subunit of highly purified urinary human chorionic gonadotropin have been re-investigated. The oligosaccharides were released enzymatically by peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F, and fractionated by a combination of FPLC and HPLC. As a result of the application of improved fractionation methods, apart from the earlier reported carbohydrate chains, also small amounts of trisialo tri- and tri-antennary oligosaccharides were found. The primary structures of the latter carbohydrate chains have been determined by 500-MHz¹H-NMR spectroscopy to beAbbreviations hCG human chorionic gonadotropin - hCG- -subunit - hCG- -subunit - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F (E.C. 3.5.1.52) - endo-F endo--N-acetylglucosaminidase-F (E.C. 3.2.1.96) - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - CBB coomassie brilliant blue R 250 - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - Man mannose - Gal galactose - Fuc fucose     

Purification and properties ofβ-fructofuranosidase from Aspergillus japonicus

-Fructofuranosidase fromAspergillus japonicus, which produces 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose, was purified to homogeneity by fractionation with calcium acetate and ammonium sulphate and chromatography with DEAE-Cellulofine and Sephadex G-200. Its molecular size was estimated to be about 304,000 Da by gel filtration. The enzyme was a glycoprotein which contained about 20% (w/w) carbohydrate. Optimum pH for the enzymatic reaction was 5.5 to 6. The enzyme was stable over a wide pH range, from pH 4 to 9. Optimum reaction temperature for the enzyme was 60 to 65°C and it was stable below 60°C. The K_m value for sucrose was 0.21m. The enzyme was inhibited by metal ions, such as those of silver, lead and iron, and also byp-chloromercuribenzoate.     

Polynucleotides and Their Components in the Processes of Aromatic Nucleophilic Substitution: II.1 Nucleophilic Modification of 3′,5′-Bis-O- (α,β,α′,β′-tetrafluoropyrid-γ-yl)thymidine

The interaction of 3,5-bis-O-(,,,-tetrafluoropyrid--yl)thymidine with various nucleophilic reagents was studied to evaluate the possibility of molecular design of new types of nucleic acid analogues using S _NAr reactions. The reactions with morpholine and sodium azide led to the introduction of one and two nucleophilic residues into each of the polyfluorinated pyridine rings. The nucleophilic polycondensation with bifunctional reagents ethylenediamine and hexamethylenediamine depended on the nature of nucleophile and reaction conditions and resulted in the formation of supramolecules containing about five or more than 20 pyrimidine bases.     

Production of β-fructofuranosidase byAspergillus japonicus

A newly isolated strain, MU-2, which produces very high -fructofuranosidase activity, was identified asAspergillus japonicus. For enzyme production by the strain, sucrose at 20% (w/v) was the best carbon source and yeast extract at 1.5 to 3% (w/v) the best nitrogen source. Total enzymatic activity and cell growth were at maximum after 48 h, at 1.57×10⁴ U/flask and 0.81 g dry cells/flask, respectively. The optimum pH value of the enzymatic reaction was between 5.0 and 5.5 and the optimum temperature 60 to 65°C. The enzyme produced 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose by fructosyl-transferring activity. The strain was found to be very useful for industrial production of -fructofuranosidase.     

Glutamine synthetase isoenzymes of Phaseolus vulgaris L.: subunit composition in developing root nodules and plumules

In the legume Phaseolus vulgaris L., glutamine synthetase (GS) (EC.6.3.1.2.) occurs as three cytosolic polypeptides, , and , and a plastidic polypeptide, . This paper describes the subunit composition of active octameric GS isoenzymes from root nodules and plumules using ionexchange high-performance liquid chromatography followed by two-dimensional denaturing gel electrophoresis and Western immunodetection. Root nodules contained four separable GS activities, three of which were composed mainly of cytosolic , / and GS polypeptides, whereas the fourth activity, consisted of plastidic GS polypeptides. The increase in GS activity during nodulation was due largely to the appearance of -containing isoenzymes, and to a lesser extent on the isoenzyme, whereas the -isoenzyme activity remained approximately constant throughout. Plumule GS from imbibed seeds was found to be composed of separate and isoenzymes, but 2 d after germination, plumule GS consisted of a mixture of , / and isoenzymes. The results from both nodules and plumules indicate that different cytosolic GS polypeptides in P. vulgaris are able to assemble into both homo-octameric and heterooctameric isoenzymes. Moreover, the changes in the patterns of isoenzymes observed during nodule development and plumule growth are interpreted to be caused both by temporal changes in the denovo synthesis of the polypeptides and also by their spatial separation in different cell types.Abbreviations 1D, 2D one-, two-dimensional - GS glutamine synthetase - GS_s GS semibiosynthetic activity - GS_t GS transferase activity - IEX-HPLC ion-exchange high-performance liquid chromatography - kDa kilodaltons - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis