Structures of asparagine linked oligosaccharides of immunoglobulins (IgY) isolated from egg-yolk of Japanese quail

Structures of the Asn linked oligosaccharides of quail egg-yolk immunoglobulin (IgY) were determined in this study. Asn linked oligosaccharides were cleaved from IgY by hydrazinolysis and labelled withp-aminobenzoic acid ethyl ester (ABEE) afterN-acetylation. The ABEE labelled oligosaccharides were then fractionated by a combination of Concanavalin A-agarose column chromatography and anion exchange, normal phase and reversed phase HPLC before their structures were determined by sequential exoglycosidase digestion, methylation analysis, HPLC, and 500 MHz¹H-NMR spectroscopy. Quail IgY contained only neutral oligosaccharides of the following categories: the glucosylated oligomannose type (0.6%, Glc1-3Glc1-3Man₉GlcNAc₂; 35.6%, Glc1-3Man_7–9GlcNAc₂). oligomannose type (15.0%, with the structure Man_5–9GlcNAc₂) and biantennary complex type with core structures of-Man1-3(-Man1-6)Man1-4GlcNAc1-4GlcNAc (9.9%),-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4GlcNAc (25.1%) and-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4(Fuc1-6)GlcNAc (11.4%). Although never found in mammalian proteins, glucosylated oligosaccharides (Glc₁Man_7–9GlcNAc₂) have been located previously in hen IgY.Abbreviations IgG, IgM, IgA, IgY immunoglobulin G, M, A and Y, respectively - ABEE p-aminobenzoic acid ethyl ester     

Isolation and characterization of a Forssman antigen-binding lectin from velvet bean (Mucuna derringiana) seeds

A Forssman antigen (GalNAc1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer)-binding lectin has been purified from velvet bean (Mucuna derringiana) seeds by a combination of affinity chromatography and reversed phase HPLC. This lectin agglutinates both native and trypsin-treated sheep erythrocytes as well as trypsinized rabbit erythrocytes, but neither native rabbit nor human erythrocytes, irrespective of blood group type. SDS-PAGE and gel filtration chromatography reveal the lectin to be a homodimer consisting of two 54 kDa subunits linked by non-covalent bonds. The results obtained by quantitative precipitation, haemagglutination inhibition and TLC overlay assays indicate that theMucuna lectin specifically recognizes Forssman antigen and Forssman disaccharide (GalNAc1-3GalNAc)-related structures. Abbreviations: The abbreviations and the trivial names used are: AH, 6-aminohexyl; BSA, bovine serum albumin; Cer, ceramide; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; PBS, 10mm phosphate-buffered saline, pH 7,2, containing 0.15m NaCl; PMSF, phenyl methyl sulfonyl fluoride; SDS, sodium dodecyl sulphate; TFA, trifluoroacetic acid; TBS, 20mm tris-buffered saline, pH 7.2; TLC, thin-layer chromatography; A disaccharide, GalNAc1-3Gal; A trisaccharide, GalNAc1-3[Fuc1-2]Gal; Forssman disaccharide, GalNAc1-3GalNAc; CDH (ceramide dihexoside or lactosyl ceramide) Gal1-4Glc1-1Cer (LacCer); CTH (ceramide trihexoside or globotriosyl ceramide), Gal1-4Gal1-4Glc1-1Cer (GbOse₃Cer or Gb₃); globoside (globotetraosyl ceramide), GalNAc1-3Gal1-4Gal1-4Glc1-1Cer (GbOse₄Cer or Gb₄); Forssman antigen (globopentaosyl ceramide), GalNAc1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer (GbOse₅Cer).     

Testis-specific β2 tubulins are identical in Drosophila melanogaster and D. hydei but differ from the ubiquitous β1 tubulin

In Drosophila as in many organisms tubulins are encoded by a gene family. We have determined the complete nucleotide sequences coding for the 1 and 2 tubulins of Drosophila melanogaster and the 2 tubulin of D. hydei, and found these insect tubulins to be highly conserved and like tubulins of other organisms. This is discussed with reference to the possible functional domains of these proteins. — The 1 tubulin gene of Drosophila is constitutively expressed, whereas the 2 tubulin is expressed specifically in the testes. In D. melanogaster the amino acid sequences of these proteins are 95% homologous, differing at only 25 positions. In the testes the 2 tubulin participates in different microtubules as shown by genetic analysis (Kemphues et al. 1982). Interestingly, all of the amino acids characteristic of the testis-specific 2 tubulin are also present in the corresponding gene of D. hydei. Of special interest is the high degree of conservation of the carboxy-terminal domain in these functionally equivalent tubulins.     

Inoculation of sugar mill by-products compost with N2-fixing bacteria

Inoculation of sugar mill by-products compost with N₂-fixing bacteria may improve its quality by increasing total N and available P. Compost was inoculated with Azotobacter vinelandii(ATCC 478), Beijerinckia derxii (ATCC 49361), and Azospirillumsp. TS8, each alone and all three together. Numbers of all N₂-fixing bacteria in compost declined from an initial population of 5×10⁵cellsg^–1 during incubation. The population of Azotobacter declined to approximately 2×10²cellsg^–1 and the population of Beijerinckia and Azospirillum declined to approximately 9×10³ and 3.5×10⁴cellsg^–1 respectively, at day 50. Inoculation with N₂-fixing bacteria increased acetylene reduction, total N by 6–16 and available P by 25–30% in comparison to the uninoculated control. Increasing the N content and P availability of compost increases its value and there may be additional benefit from providing N₂ fixing bacteria.     

Effect of gibberellic acid on pod set in soybean

Field-grown soybean plants (Glycine max (L.) Merr. cv. Evans) were treated with gibberellic acid (GA₃; 10gl^–1) and/or (2-chloroethyl)-trimethylammonium chloride (CCC; 0.8gl^–1) in 1983 and 1984, and subsequent anthesis, pod set, seed size, seed number, and seed yield were determined at one node. The treatments were applied to five leaves in the center of each plant (typically leaves 7–11) and reproductive development at the node in the center of those leaves was monitored. Gibberellin A₃ applied Early (about 3d before anthesis of the first flower at the monitored node) had no effect on the number of flowers produced, but decreased the fraction of flowers that set pods in both experimental years (by 32% in 1983 and 76% in 1984). Seed size was slightly decreased by the GA₃ treatment in 1983 but not in 1984. The Middle GA₃ treatment (applied about 3 days after the Early treatment) slightly decreased the number of pods set; and Late treatments (9 days after) had no effect. None of the monitored parameters were affected by CCC.The Early experiments were repeated with two additional genotypes, Lincoln and T210. Genotype T210 is a single-gene, dwarf mutant of Lincoln whose stem elongation and leaf expansion are insensitive to GA₃. Gibberellin A₃ affected the reproductive parameters in Lincoln very similarly to Evans but those in T210 were unaffected. This indicates that GA₃ exerts its effect by increasing the mass of vegetative tissue and thus diverting assimilates away from the pods. However, since the mutation in T210 might affect a receptor that is in flowers as well as shoots, it is possible that GA₃ exerted its effect on the normal genotypes directly on the developing pods, rather than indirectly by diverting photoassimilates.     

Microanalysis of C and O isotopes of azooxanthellate and zooxanthellate corals by ion microprobe

We have determined the ¹⁸O and ¹³C values of azooxanthellate (Lophelia pertusa) and zooxanthellate (Porites lutea) corals at a micrometer scale using an ion microprobe (SIMS—secondary ion mass spectrometry). In P. lutea, centers of calcification are small (10 to 15 m) and difficult to locate during measurements. In L. pertusa, they are large (50 m) and arranged in lines of centers of calcification. Our results show that centers of calcification in L. pertusa have a restricted range of variation in ¹⁸O [-2.8±0.3 (PDB)], and a larger range in ¹³C [14.3 to 10.9 (PDB)]. Surrounding skeletal fibers exhibit large isotopic variation both for C and O (up to 12), and ¹³C and ¹⁸O are positively correlated. The C and O isotopic compositions of the center of calcification deviate from this linear trend at the lightest ¹⁸O values of the surrounding fibers. Ion microprobe results on P. lutea demonstrate also a large range of variation for the ¹⁸O values (up to 10). No correlation is found with C isotopes that exhibit, in comparison with L. pertusa, a small range of variation (2). This variation of ¹⁸O at a micrometer scale is probably the result of two processes: (1) an isotopic equilibrium calcification with 1 pH unit variation in the calcification fluid as indicated by direct measurements of coelenteron pH in the coral Galaxea fascicularis (Al-Horani et al. 2003) and (2) a kinetic fractionation. The ¹³C apparent disequilibrium in P. lutea may be the result of mixing between metabolic CO₂ (respiration) and dissolved inorganic carbon (DIC) coming directly from seawater.     

Purification and properties of β-ketothiolase from Zoogloea ramigera

-Ketothiolase from Zoogloea ramigera I-16-M was purified 140-fold to electrophoretic homogeneity. The bacterium appeared to contain a single isoenzyme of -ketothiolase with a molecular weight of 190000, as determined by Sephadex G-200 gel filtration. The monomer molecular weight was 44000, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The native enzyme thus appeared to be a tetramer with identical subunits.The enzyme showed a pH optimum of 7.5 in the condensation reaction, and 8.5 in the thiolysis reaction. The enzyme employed a Bi Bi ping pong mechanism for the forward thiolysis reaction. The apparent K _m value for acetoacetyl coenzyme A in the thiolysis reaction was 10 M, and that for coenzyme A was 8.5 M. The apparent K _m value for acetyl coenzyme A in the condensation reaction was 0.33 mM. The condensation reaction was inhibited by coenzyme A concentrations lower than 0.1 mM.The enzyme was stable in the presence of dithiothreitol and other SH-compounds, but was strongly inhibited by 0.4 mM p-chloromercuribenzoate.Non-Standard Abbreviation PHB poly--hydroxybutyrate     

The energy transmission in ATP synthase: From the γ-c rotor to the α3β3 oligomer fixed by OSCP-b stator via the βDELSEED sequence

ATP synthase (F₀F₁) is driven by an electrochemical potential of H⁺ (H⁺). F₀F₁ is composed of an ion-conducting portion (F₀) and a catalytic portion (F₁). The subunit composition of F₁ is ₃₃. The active ₃₃ oligomer, characterized by X-ray crystallography, has been obtained only from thermnophilic F₁ (TF₁). We proposed in 1984 that ATP is released from the catalytic site (C site) by a conformational change induced by the DELSEED sequence via -F₀. In fact, cross-linking of DELSEED to stopped the ATP-driven rotation of in the center of ₃₃. The torque of the rotation is estimated to be 420 pN·å from the H⁺ and H⁺-current through F₀F₁. The angular velocity () of is the rate-limiting step, because H⁺ increased theV _max of H⁺ current through F₀, but not theK _m (ATP). The rotational unit of F₀ (=ab₂c₁₀) is /5, while that in ₃₃ is 2/3. This difference is overcome by an analog-digital conversion via elasticity around DELSEED with a threshold to release ATP. The distance at the C site is about 9.6 å (2,8-diN₃-ATP), and tight Mg-ATP binding in ₃₃ was shown by ESR. The rotational relaxation of TF₁ is too rapid (=100 nsec), but the rate of AT(D)P-induced conformational change of ₃₃ measured with a synchrotron is close to . The ATP bound between the P-loop and E188 is released by the shift of DELSEED from RGL. Considering the viscosity resistance and inertia of the free rotor (-c), there may be a stator containing OSCP (= of TF₁) and F₀-d to hold free rotation of ₃₃.     

Laser kinetic spectroscopy studies of the bimolecular oxygenation of alpha- and beta-subunits within the R-state of human hemoglobin

Bimolecular oxygenation of tri-liganded R-state human hemoglobin (HbA) is described by bi-exponential kinetics with association rate constants k = 27.2 ± 1.3 (M·sec)^-1 and k = 62.9 ± 1.6 (M·sec)^-1. Both the observed processes have been assigned to the bimolecular oxygenation of - and -subunits of the native tetrameric protein by molecular oxygen. The quantum yields of photodissociation within the completely oxygenated R-state HbA are = 0.0120 ± 0.0017 and = 0.044 ± 0.005 for - and -subunits, respectively. The oxygenation reactions of isolated ^PCMB- and ^PCMB-hemoglobin chains are described by mono-exponential kinetics with the association rate constants k = 44 ± 2 (M·sec)^-1 and k = 51 ± 1 (M·sec)^-1, respectively. The quantum yields of photodissociation of isolated ^PCMB- and ^PCMB-chains (0.056 ± 0.006 and 0.065 ± 0.006, respectively) are greater than that observed for appropriate subunits within the R-state of oxygenated HbA.     

Expression,purification, characterization,and deletion mutations of phosphorylase kinase γ subunit: identification of an inhibitory domain in the γ subunit

A catalytic fragment, _1-298, derived from limited chymotryptic digestion of phosphorylaseb kinase (Harris, W.R.et al., J. Biol. Chem., 265: 11740–11745, 1990), is reported to have about six-fold greater specific activity than does the subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the subunit, full-length wild-type and seven truncated forms of were expressed inE. coli. Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column. Four out of seven truncated mutants show similar ( _1-353 and _1-341) or less ( _1-331 and _1-276) specific activity than does the full-length wild-type , _1-386. Three truncated forms, _1-316, _1-300, and _1-290 have molar specific activities approximately twice as great as those of the full-length wild-type and the nonactivated holoenzyme. All recombinant s exhibit similarK _m values for both substrates, i.e., about 18M for phosphorylaseb and about 75 M for MgATP. Three truncated s, _1-316, _1-300, and _1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (V _max/K _m) than that of the full-length wild-type and a 3.5- to 4.5-fold greater efficiency than that of the truncated _1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of , which is located at _301-331· _1-290, but not _1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the subunit to exhibit phosphotransferase activity. Both _1-290 and _1-300 have several properties similar to full-length wild-type , including metal ion responses (activation by free Mg²⁺ and inhibition by free Mn²⁺) pH dependency, and substrate specificities.     

Stimulation of the differential rate of beta-galactosidase synthesis in E. coli by trimethoprim

Summary It was found that partial inhibition of peptide chain initiation by trimethoprim causes a significant increase in the differential rate of -galactosidase in cultures of E. coli growing in the presence of suboptimal concentrations of a galactoside inducer. This stimulation is not produced by inhibitors of peptide chain growth, such as chloramphenicol and puromycin. Trimethoprim, furthermore, does not cause a significant increase in the differential rate of -galactosidase synthesis in E. coli cultures (a) of i ⁺ genotype which are fully induced, (b) of fully constitutive i ^- genotype, (c) of partially constitutive o ^c genotype, or (d) of noninducible i ^s genotype. These findings are compatible with the idea that the differential rate of -galactosidase synthesis is regulated by means of an inducer-dependent growth period of a regulatory polypeptide.     

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     

Paramagnetic spin catalysis of a radical recombination reaction

Recombination of the triplet state radical pair consisting of two hydrogen atoms catalysed by molecular oxygen is considered as a simulating example of a paramagnetic-exchange catalytic process. Intermolecular exchange interaction in the collision complex between the H₂ and O₂ molecules is calculatedab initio in STO-6G and 6–31 G^* basis sets with complete active space configuration interaction. Calculations are done at a fixed O–H distance (3 Å), scanning the H–H bond length from 0.6 till 12 Å at the linear geometry of collision. The mixture of the triplet (T)³ _u ⁺ and singlet (S)¹ _g ⁺ states of the hydrogen moiety is possible because both states have the same triplet symmetry in the collision complex with O₂ (³ _g ^– ). A strong mixture of the S (¹ _g ⁺ , H₂ +³ _g ^– , O₂) and T) and T (³ _u ⁺ , H₂ +³ _g ^– , O₂) states is actually obtained even at large H–H distances. The quintet and singlet states^5,1(³ _u ⁺ , H₂ +³ _g ^– , O₂) are also considered for comparison of the exchange potentials. Atr(H–H)4.4 Å the S-T splitting is approximately constant (12 cm^-1 in the STO-6G basis set; 55.5 cm^-1 in the 6–31 G^* basis set) and is determined by the exchange interaction between O₂ and the nearest hydrogen atom in the O–O...H fragment. The paramagnetic catalyst can accelerate radical recombination through the triplet-singlet nonadiabatic transition to the lowest S reactive state when the radical encounter takes place in the vicinity of the catalyst. Though we do not consider the radical dynamics in a real solvent, which modulates the exchange potentials and the T-S transitions, the nature of this mechanism of spin catalysis is obvious. The electric polarization and charge transfer are important in the analysis of the exchange interaction and radical recombination potentials for all multiplets. In accordance with the concept of spin catalysis, the electronic spin-uncoupling mechanism, induced by O₂ perturbation, has the same nature as other known catalytic processes of paramagnetic-exchange type.     

Shifts in carbon isotope ratios of two C3 halophytes under natural and artificial conditions

Summary The total carbon ¹³C values of two C₃ halophytes,Salicornia europaea L. ssp.rubra (Nels.) Breitung andPuccinellia muttalliana (Schultes) Hitch., native to inland saline areas of Alberta, Canada, were determined for plants grown under controlled conditions of supplied NaCl in the nutrient solution, and for plants found growing in the field. Field specimens were collected along line transects which ran from areas of high salinity to areas of low salinity across the pattern of species zonation. The ¹³C value of the two species seemed to reflect the water potential of the soil ( _w ^soil ) as measured arbitrarily at a depth of 10 cm, becoming less negative as the _w ^soil decreased. Over a linear distance of 5.55 m,S. europaea spp.rubra showed a shift of +5.3 as the _w ^soil went from-25x10² kPa to a minimum of-73x10² kPa. ForP. nuttalliana, the ¹³C values differed by 3.4 over a distance of 7.45 m where the maximum difference in _w ^soil was 12.7x10² kPa. However, ¹³C values ofP. nuttalliana only roughly reflected the spatial trends in _w ^soil at the time of collection. In the growth chamber, the ¹³C value ofS. europaea ssp.rubra changed by a maximum of +8.0 when the solute potential of the nutrient solution ( _w ^soil ) was dropped from-0.25x10² kPa to-64.25x10² kPa; while the ¹³C value ofP. nuttalliana changed by a maximum of +10.8 when the _w ^soil was dropped from-0.25x10² kPa to-40.25x10² kPa. Linear regression analyses indicated that the ¹³C values of both species were strongly correlated (P<0.2%) with _w ^soil . The observed shifts in ¹²C may represent changes in the mode of photosynthetic CO₂ fixation. However, a number of other explanations, some of which are discussed in the text, are also possible. A proper ecophysiological interpretation of such shifts in ¹³C values of C₃ plants awaits a better understanding of the isotope fractionation mechanisms involved.     

Identification of a GDP-Fuc:Galβ1–3GalNAc-R (Fuc to Gal)α1–2 fucosyltransferase and a GDP-Fuc:Galβ1–4GlcNAc (Fuc to GlcNAc)α1–3 fucosyltransferase in connective tissue of the snailLymnaea stagnalis

Connective tissue of the freshwater pulmonateLymnaea stagnalis was shown to contain fucosyltransferase activity capable of transferring fucose from GDP-Fuc in 1–2 linkage to terminal Gal of type 3 (Gal1–3GalNAc) acceptors, and in 1–3 linkage to GlcNAc of type 2 (Gal1–4GlcNAc) acceptors. The 1–2 fucosyltransferase was active with Gal1–3GalNAc1-OCH₂CH=CH₂ (K _m=12 mM,V _max=1.3 mU ml^–1) and Gal1–3GalNAc (K _m=20 mM,V _max=2.1 mU ml^–1), whereas the 1–3 fucosyltransferase was active with Gal1–4GlcNAc (K _m=23 mM,V _max=1.1 mU ml^–1). The products formed from Gal1–3GalNAc1-OCH₂CH=CH₂ and Gal1–4GlcNAc were purified by high performance liquid chromatography, and identified by 500 MHz¹H-NMR spectroscopy and methylation analysis to be Fuc1–2Gal1–3GalNAc1-OCH₂CH=CH₂ and Gal1–4(Fuc1–3)GlcNAc, respectively. Competition experiments suggest that the two fucosyltransferase activities are due to two distinct enzymes.Abbreviations 2Fuc-T 1–2 fucosyltransferase - 3Fuc-T 1–3 fucosyltransferase - MeO-3Man 3-O-methyl-D-mannose - MeO-3Gal 3-O-methyl-D-galactose     

Effects of L-Glutamate Transport Inhibition by a Conformationally Restricted Glutamate Analogue (2S,1'S,2'R)-2-(Carboxycyclopropyl)Glycine (L-CCG III) on Metabolism in Brain Tissue In Vitro Analysed by NMR Spectroscopy

(2S,1'S,2'R)-2-(Carboxycyclopropyl)glycine (L-CCG III) was a substrate of Na⁺-dependent glutamate transporters (GluT) in Xenopus laevis oocytes (IC₅₀ 13 and 2 M for, respectively, EAAT 1 and EAAT 2) and caused an apparent inhibition of [³H]L-glutamate uptake in mini-slices of guinea pig cerebral cortex (IC₅₀ 12 M). In slices (350 M) of guinea pig cerebral cortex, 5 M L-CCG III increased both the flux of label through pyruvate carboxylase and the fractional enrichment of glutamate, GABA, glutamine and lactate, but had no effect on total metabolite pool sizes. At 50 M L-CCG III decreased incorporation of ¹³C from [3-¹³C]-pyruvate into glutamate C4, glutamine C4, lactate C3 and alanine C3. The total metabolite pool sizes were also decreased with no change in the fractional enrichment. Furthermore, L-CCG III was accumulated in the tissue, probably via GluT. At lower concentration, L-CCG III would compete with L-glutamate for GluT and the changes probably reflect a compensation for the missing L-glutamate. At 50 M, intracellular L-CCG III could reach > 10 mM and metabolism might be affected directly.     

Subunit structures of multiple hemoglobins in carp

Three hemoglobin components in carp designated CI, CII, and CIII, were isolated by DEAE-Toyopearl ion-exchange chromatography. Constituent globin chains, ¹, ², ¹ and ², were analyzed by urea-Triton acid polyacrylamide gel electrophoresis and isolated by high performance liquid chromatography with a reversed-phase column. Tryptic peptide mapping indicated that the -globin chains of the three hemoglobin components have slightly different structures. In addition, N-terminal amino acid sequence analysis indicated that the ¹-globin chain has a primary structure different from that of the ²-chain. A series of hybridization experiments between isolated hemoglobins, together with such structural properties of globin chains, suggested that the three hemoglobins have the following compositions: CI (^{1 2} ₂ ¹ ), CII (^{1 2 1 2}), and CIII (^{1 2} ₂ ² ). Hemoglobin CII was a hybrid between the two types each of - and -chain and could be constructed in vitro from two hemoglobin components CI and CIII.Abbreviations a-a amino acid - Hb hemoglobin - HPLC high performance liquid chromatography - P ₅₀ oxygen pressure at half saturation - PAGE polyacrylamide gel electrophoresis - TFA trifluoroacetic acid     

Purification and characterization of β‐methylaspartase from Fusobacterium varium

Methylaspartase (EC 4.3.1.2) was purified 20fold in 35% yield from Fusobacterium varium, an obligate anaerobe. The purification steps included heat treatment, fractional precipitation with ammonium sulfate and ethanol, gel filtration, and ion exchange chromatography on DEAESepharose. The enzyme is dimeric, consisting of two identical 46 kDa subunits, and requires Mg²⁺ (K_m = 0.27 ± 0.01 mM) and K⁺ (K_m = 3.3 ± 0.8 mM) for maximum activity. Methylaspartasecatalyzed addition of ammonia to mesaconate yielded two diastereomeric amino acids, identified by HPLC as (2S,3S)3methylaspartate (major product) and (2S,3R)3methylaspartate (minor product). Optimal activity for the deamination of (2S,3S)3methylaspartate (K_m = 0.51 ± 0.04 mM) was observed at pH 9.7. The Nterminal protein sequence (30 residues) of the F. varium enzyme is 83% identical to the corresponding sequence of the clostridial enzyme.     

Conformational energy of the 5-membered ring. Implication of geometric and energetic properties in the conformational characteristics

In a previous article (8) a geometrical study of the five-membered ring showed that: a) for the case of the 20 symmetrical C₂ and C_s conformations, the pseudorotation formulae for the torsion angles are a geometrical property of the ring; b) geometrical considerations alone are unable to define the puckering amplitude, the bond angle values, and the pathway between two symmetrical conformations. Here we examine how the energy equations enable us to define the deformation amplitude _m, establish the bond angles expressions and check the energy invariability along the pseudorotation circuit. The problem is next developed fully in the case where the bond and torsional energy only are considered: the literal expression¹ of _m is then given as a function of the bond angle which cancels out the bond angle energy. A numerical application is carried out on cyclopentane and the values of the parameters K^t, K¹ and used in the Conformational energy calculations are considered.Notations used 1 _i bond lengths 1 in the case of the regular ring - _i torsional angles - _i bond angles - 3/5 = 108 - 4/5 = 144 - , _{i i} – = complement to the 108 bond angle _i - _T - E Conformational energy of the 5-membered ring - E Conformational energy difference between planar and deformed ring - A _n Coefficients of the energy development in terms of - E _i ^l Bond energy relative to atom i (associated with angle _i) - K _i ^l Bond constant relative to atom i (associated with angle _i) - E _i ^l Torsional energy relative to the i ^th bond (associated with angle _i) - k _i ^l Torsional constant relative to the i ^th bond (associated with angle _i) - _i Angle _i value corresponding to zero bond energy E _i ^l (when the 5 atoms of the ring are identical, _i ) - r _ij Distance between atoms i and j - q _i Charge carried by atom i - e Constant of proportionality including the effective dielectric constant - A _ij, B_ij, d_ij Coefficients dependent on the nature of the atoms i and j and accounted for in the Van der Waals energy and hydrogen bond expressions - S (r _ij) Electrostatic contribution to the hydrogen bond energy - P Pseudorotation phase angle - _m Maximum torsional angle value characterising the deformation amplitudeM     

The linear tetrasaccharide,Galβ1-4GlcNAcβ1-6Galβ1-4GlcNAc,isolated from radiolabeled teratocarcinoma poly-N-acetyllactosaminoglycan resists the action ofE. freundii endo-β-galactosidase

A novel linear tetrasaccharide, Gal1-4GlcNAc1-6Gal1-4GlcNAc, was isolated from partial acid hydrolysates of metabolically labeled poly-N-acetyllactosaminoglycans of murine teratocarcinoma cells. It was characterized by exo-glycosidase sequencing and by mild acid hydrolysis followed by identification of all partial cleavage products. The tetrasaccharide, and likewise labelled GlcNAc1-6Gal1-4GlcNAc, resisted the action of endo--galactosidase (EC 3.2.1.103) fromE. freundii at a concentration of 125 mU/ml, while the isomeric, radioactive teratocarcinoma saccharides Gal1-4GlcNAc1-3Gal1-4GlcNAc and GlcNAc1-3Gal1-4GlcNAc were cleaved in the expected manner.Abbreviations WGA wheat germ agglutinin - BSA bovine serum albumin - [³H]GlcNAc1-4-GlcNAc1-4GlcNAcOL N,N,NN'-triacetylchitotriose reduced with NaB³H₄