Biodegradation Kinetics of Four Substituted Chlorobenzoic Acids by Enterobacter aerogenes

Enterobacter aerogenes is generally found in soil, sewage plants, and human gastrointestinal tract. Thus, this study was conducted to evaluate the ability of Enterobacter aerogenes to degrade four chlorobenzoic acid compounds (2-chlorobenzoic acid (2-CBA), 3-chlorobenzoic acid (3-CBA), 4-chlorobenzoic acid (4-CBA), and 3,4-dichlorobenzoic acid (3,4-dCBA)) in minimal salt medium. Enterobacter aerogenes was partially able to degrade and dechlorinate these CBAs at concentration of 3.5 mM within 72 h of incubation. According to Haldane single-substrate model, the values of maximum predicted growth rate (μ_max), half saturation constant (K _s), and inhibition constant (K _i) fell in the range of 0.2–0.8 h^?1, 8–41 mM, and 5–53 mM, respectively. Based on the estimated values of both α, a growth-associated constant, and β, a non–growth-associated constant, the production of chloride was predominantly growth associated, since negligible values of the β were determined. Haldane model gave a good prediction of the CBA substrate utilization and degradation, and was in a very good agreement with the experimental data. Because of the capability of Enterobacter aerogenes to utilize these aromatic compounds as carbon and energy sources, this microorganism can be a valuable and promising candidate for use in the biotreatment of wastewater and soil samples contaminated with mixtures of chlorobenzoates.     

ON THE STRUCTURE OF A NEW, FUCOSE CONTAINING GANGLIOSIDE FROM PIG CEREBELLUM

A new ganglioside, provisionally named GLIVa, was isolated in pure form from pig cerebellum. Ganglioside GLIVa is a disialoganglioside containing fucose. Its basic neutral glycosphingolipid core is the gangliotetraose ceramide: Gal, β 1 → 3 GalNAc, β 1 → 4 Gal, β 1 → 4 Glc, β 1 → Cer. Fucose is α-glycosidically linked to the 2-position of external galactose and one N-acetylneuraminic acid is linked to the other one by an α, 2 → 8 linkage. Thus the total structure of ganglioside GLIVa is the following: Fuc, α 1 → 2 Gal, β 1 → 3 GalNAc, β 1 → 4 (NeuAc, α 2 48 NeuAc, α 2 → 3) Gal, β 1 → 4 Glc, β 1 → Ceramide. According to the IUPAC-IUB Commission on Biochemical Nomenclature is indicated as II³α(NeuAc)₂ IV²αFuc-GgOse₄Cer.     

Synthèse et étude R.M.N. de disaccharides et trisaccharides dans la série du l-rhamnose

Various di- and tri-saccharides containing l-rhamnose were synthesized by condensation of 2,3,4-tri-O-acetyl- or 2,3,4-tri-O-benzoyl-α-l-rhamnopyranosyl bromide with an unblocked glycopyranoside. The determination of the anomeric configuration of l-rhamnose saccharides by n.m.r. is difficult because structure has a greater effect on the spectra than does configuration. The α and β configurations and the position of the substitution may be assigned from the chemical shifts of H-5 and CH₃. In all the compounds having a β configuration, a shielding of the methyl group and a deshielding of the H-5 proton have been observed as compared to the compounds having an α configuration. The H-5 proton and the methyl group of peracetylated, (1→3)-linked α-l derivatives always resonate at higher fields than the corresponding protons of (1→6)-linked α-l derivatives.     

Proteolytic analysis of polymerized maize tubulin: regulation of microtubule stability to low temperature and Ca2+ by the carboxyl terminus of β-tubulin

To obtain information on plant microtubule stability to low temperature and Ca²⁺, the regulatory domain of polymerized tubulin from maize (Zea mays ev. Black Mexican Sweet) was dissected by limited proteolysis with subtilisin. Tubulin in taxol-stabilized microtubules was cleaved in a subtilisin concentration- and time-dependent manner. Immunoblotting of microtubules with antibodies having mapped epitopes on α- and β-tubulins revealed that cleavage initially removed ≤15 residues from the β-tubulin carboxyl terminus to produce αβ_s-microtubules. Subsequent cleavage occurred at an extreme site and an internal site within the α-tubulin carboxyl terminus. Electron microscopy revealed that αβ_s-microtubules were ultra structurally indistinguishable from uncleaved control αβ-micro-tubules. Quantitative polymer sedimentation showed that low temperature treatment (0°C) caused significant depolymerization of αβ-microtubules, but little depolymerization of αβ_s-microtubules. Ca²⁺ enhanced the cold-induced depolymerization of both αβ- and αβ_s-microtubules. However, αβ_s-microtubules were significantly more stable to depolymerization by cold and Ca²⁺ than were αβ-micro-tubules. The results showed that maize microtubules containing shortened β-tubulin carboxyl termini are relatively resistant to the combined depolymerizing effects of cold and Ca²⁺. Thus, the extreme carboxyl terminus of β-tubulin is a crucial element of the plant tubulin regulatory domain and may be involved in the modulation of microtubule stability during the chilling response in plants.     

Studies on the Decarboxylation of Amino Acids with Glyoxal

Decarboxylation of about twenty kinds of α, β and γ-amino acids in the reaction with glyoxal or ninhydrin was investigated. The decarboxylation rate of amino acids proved that steric and polar effects had important roles in the reaction.

From the data of pK₂ values and decarboxylation rates of amino acids, it can be concluded that under a similar steric environment, the decarboxylation rate depends on the anion concentration of amino acids.

Besides carbon dioxide, acetaldehyde, 2-propanone and propionaldehyde were respectively detected from the reaction of β-alanine, β and γ-amino-n-butyric acids with glyoxal or ninhydrin. The decarboxylation mechanism of these amino acids seemed to take place through the corresponding β- or γ-keto acid.

Oxygen absorption was also observed from the reaction of amino acids with dicarbonyl compounds.     

Identification of a region in alcohol dehydrogenase that binds to α-crystallin during chaperone action

α-Crystallin, the major eye lens protein and a member of the small heat-shock protein family, has been shown to protect the aggregation of several proteins and enzymes under denaturing conditions. The region(s) in the denaturing proteins that interact with α-crystallin during chaperone action has not been identified. Determination of these sites would explain the wide chaperoning action (promiscuity) of α-crystallin. In the present study, using two different methods, we have identified a sequence in yeast alcohol dehydrogenase (ADH) that binds to α-crystallin during chaperone-like action. The first method involved the incubation of α-crystallin with ADH peptides at 48 °C for 1 h followed by separation and analysis of bound peptides. In the second method, α-crystallin was first derivatized with a photoactive trifunctional cross-linker, sulfosuccinimidyl-2[6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]ethyl-1,3di-thiopropionate (sulfo-SBED), and then complexed with ADH at 48 °C for 1 h in the dark. The complex was photolyzed and digested with protease, and the biotinylated peptide fragments were isolated using an avidin column and then analyzed. The amino acid sequencing and mass spectral analysis revealed the sequence YSGVCHTDLHAWHGDWPLPVK (yeast ADH_40–60) as the α-crystallin binding site in ADH. The interaction was further confirmed by demonstrating complex formation between α-crystallin and a synthetic peptide representing the binding site of ADH.     

Similarity Between Rat Brain Nicotinic α-Bungarotoxin Receptors and Stably Expressed α-Bungarotoxin Binding Sites

Abstract: The present results demonstrate stable expression of α-bungarotoxin (α-BGT) binding sites by cells of the GH₄C₁ rat pituitary clonal line. Wild-type GH₄C₁ cells do not express α-BGT binding sites, nor do they contain detectable mRNA for nicotinic receptor α2, α3, α4, α5, α7, β2, or β3 subunits. However, GH₄C₁ cells stably transfected with rat nicotinic receptor α7 cDNA (α7/GH₄C₁ cells) express the transgene abundantly as mRNA, and northern analysis showed that the message is of the predicted size. The α7/GH₄C₁ cells also express saturable, high-affinity binding sites for ¹²⁵I-labeled α-BGT, with a K_D of 0.4 nM and B_max of 3.2 fmol/10⁶ intact cells. ¹²⁵I-α-BGT binding affinities and pharmacological profiles are not significantly different for sites in membranes prepared either from rat brain or α7/GH₄C₁ cells. Furthermore, K_D and K_i values for ¹²⁵I-α-BGT binding sites on intact α7/GH₄C₁ cells are essentially similar to those for hippocampal neurons in culture. Sucrose density gradient analysis showed that the size of the α-BGT binding sites expressed in α7/GH₄C₁ cells was similar to that of the native brain α-BGT receptor. Chronic exposure of α7/GH₄C₁ cells in culture to nicotine or an elevated extracellular potassium concentration induces changes in the number of α-BGT binding sites comparable to those observed in cultured neurons. Collectively, the present results show that the properties of α-BGT binding sites in transfected α7/GH₄C₁ cells resemble those for brain nicotinic α-BGT receptors. If the heterologously expressed α-BGT binding sites in the present study are composed solely of α7 subunits, the results could suggest that the rat brain α-BGT receptor has a similar homooligomeric structure. Alternatively, if α-BGT binding sites exist as heterooligomers of α7 plus some other previously identified or novel subunit(s), the data would indicate that the α7 subunits play a major role in determining properties of the α-BGT receptor.     

Study of the reactivity of quinohemoprotein alcohol dehydrogenase with heterocycle-pentacyanoferrate(III) complexes and the electron transfer path calculations

The reactivity of alcohol dehydrogenase IIG (ADH IIG) from Pseudomonas putida HK5 with new heterocycle-pentacyanoferrate(III) complexes and hexacyanoferrate(III) was determined at pH 7.2. The pentacyanoferrate(III) complexes contained imidazole, pyrazole, pyridine, their derivatives and 2-aminobenzothiazole as the sixth ligand. The largest reactivity of the complexes with ADH IIG was estimated for the complex containing pyridine. An apparent bimolecular constant (k _ox ) for this complex was 8.7 × 10⁵ M⁻¹s⁻¹. The lowest value of k _ox was estimated for the complex with benzotriazole (k _ox = 3.1 × 10⁴ M⁻¹s⁻¹). The investigation of the hexacyanoferrate(III) enzymatic reduction rate at different ionic strength gave a single negative charge of reduced ADH IIG. Docking calculations revealed two binding sites of the complexes in ADH-IIG structure. The first one is located at the entrance to the PQQ pocket, and the second is at the site of cytochrome domain. The calculations of electron transfer (ET) path indicated that the most effective ET takes place from heme to the complex docked at the entrance to the PQQ pocket. This shortest path is constructed of amino acids Ser607 and Cys606.     

Purification and Characterization of an Enzyme That Catalyzes Ring Cleavage of Aspergillic Acid,from Tvichoderma koningii ATCC 76666

The aspergillic acid degrading enzyme (ADE) that catalyzes the cleavage of the pyrazine ring in aspergillic acid (AA, l-hydroxy-3-isobutyl-6-sec-butyl-2-pyrazinone) was purified to electrophoretic homogeneity from extracts of Trichoderma koningii ATCC 76666. ADE was a homodimeric protein with a molecular mass of 112kDa, contained lmol of FAD per mol of subunit, and required NAD(P)H and molecular oxygen for its activity. ADE had an isoelectric point of around 5.3, and an optimum pH of 7.0–8.0. p-Chloromercuribenzoate and HgCl₂ completely inhibited ADE activity, while metal chelating reagents, α, α′-dipyridyl and o-phenanthroline, were not inhibitors. The substrate specificity among AA-related compounds was that hydroxyaspergillic acid was a poor substrate (16% of the activity for AA) and deoxyaspergillic acid did not serve as a substrate.     

Kinetic study of aroxyl radical-scavenging action of vitamin E in membranes of egg yolk phosphatidylcholine vesicles

Vitamin E is localized in membranes and functions as an efficient inhibitor of lipid peroxidation in biological systems. In this study, we measured the reaction rates of vitamin E (α-, β-, γ-, δ-tocopherols, TocH) and tocol with aroxyl radical (ArO) as model lipid peroxyl radicals in membranes by stopped-flow spectrophotometry. Egg yolk phosphatidylcholine (EYPC) vesicles were used as a membrane model. EYPC vesicles were prepared in the aqueous methanol solution (MeOH:H₂O = 7:3, v/v) that gave the lowest turbidity in samples. The second-order rate constants (k_s) for α-TocH in MeOH/H₂O solution with EYPC vesicles were apparently 3.45 × 10⁵ M⁻¹ s⁻¹, which was about 8 times higher than that (4.50 × 10⁴ M⁻¹ s⁻¹) in MeOH/H₂O solution without EYPC vesicles. The corrected k_s of α-TocH in vesicles, which was calculated assuming that the concentration of α-TocH was 133 times higher in membranes of 10 mM EYPC vesicles than in the bulk MeOH/H₂O solution, was 2.60 × 10³ M⁻¹ s⁻¹, which was one-seventeenth that in MeOH/H₂O solution because of the lower mobility of α-TocH in membranes. Similar analyses were performed for other vitamin E analogues. The k_s of vitamin E in membranes increased in the order of tocol < δ-TocH < γ-TocH ∼ β-TocH < α-TocH. There was not much difference in the ratios of reaction rates in vesicles and MeOH/H₂O solution among vitamin E analogues [k_s(vesicle)/k_s (MeOH/H₂O) = 7.7, 10.0, 9.5, 7.4, and 5.1 for α-, β-, γ-, δ-TocH, and tocol, respectively], but their reported ratios in solutions of micelles and ethanol were quite different [k_s(micelle)/k_s(EtOH) = 100, 47, 41, 15, and 6.3 for α-, β-, γ-, δ-TocH, and tocol, respectively]. These results indicate that the reaction sites of vitamin E analogues were similar in vesicle membranes but depended on hydrophobicity in micelle membranes, which increased in the order of tocol < δ-TocH < γ-TocH ∼ β-TocH < α-TocH.     

Inhibition of Tyrosine Aminotransferase by β-N-Oxalyl-l-α,β-Diaminopropionic Acid, the Lathyrus sativus Neurotoxin

Abstract: Species differences in susceptibility are a unique feature associated with the neurotoxicity of β-N-oxalyl-l -α,β-diaminopropionic acid (l -ODAP), the Lathyrus sativus neurotoxin, and the excitotoxic mechanism proposed for its mechanism of toxicity does not account for this feature. The present study examines whether neurotoxicity of l -ODAP is the result of an interference in the metabolism of any amino acid and if it could form the basis to explain the species differences in susceptibility. Thus, Wistar rats and BALB/c (white) mice, which are normally resistant to l -ODAP, became susceptible to it following pretreatment with tyrosine (or phenylalanine), exhibiting typical neurotoxic symptoms. C57BL/6J (black) mice were, however, normally susceptible to l -ODAP without any pretreatment with tyrosine. Among the various enzymes associated with tyrosine metabolism examined, the activity of only tyrosine aminotransferase (TAT) was inhibited specifically by l -ODAP. The inhibition was noncompetitive with respect to tyrosine (K_i = 2.0 ± 0.1 mM) and uncompetitive with respect to α-ketoglutarate (K_i = 8.4 ± 1.5 mM). The inhibition of TAT was also reflected in a marked decrease in the rate of oxidation of tyrosine by liver slices, an increase in tyrosine levels of liver, and also a twofold increase in the dopa and dopamine contents of brain in l -ODAP-injected black mice. The dopa and dopamine contents in the brain of only l -ODAP-injected white mice did not show any change, whereas levels of these compounds were much higher in tyrosine-pretreated animals. Also, the radioactivity associated with tyrosine, dopa, and dopamine arising from [¹⁴C]tyrosine was twofold higher in both liver and brain of l -ODAP-treated black mice. Thus, a transient increase in tyrosine levels following the inhibition of hepatic TAT by l -ODAP and its increased availability for the enhanced synthesis of dopa and dopamine and other likely metabolites (toxic?) resulting therefrom could be the mechanism of neurotoxicity and may even underlie the species differences in susceptibility to this neurotoxin.     

ADH, α-GPDH and SOD enzyme activities of second and third chromosomal genotypes from two geographically different populations of Drosophila melanogaster

ADH, α-GPDH and SOD enzyme activities have been measured in lines of Drosophila melanogaster homozygous and/or heterozygous for chromosomes extracted from two different populatioi Globally the results demonstrate that factors other than structural genes are determining the observed pattern of enzyme activities. ADH and α-GPDH activities are, however, more affected than SOD by these factors. Geographic origin, sex, chromosome, genetic background of the lines, containing regulatory genes in a broad sense, can be mentioned as the more relevant factors that influencing enzyme activities. A high and significant correlation is detected between ADH and α-GPDH enzyme activities and it can be interpreted as due to linkage disequilibrium among these two loci. SOD activity shows a lesser correlation with ADH and α-GPDH because it is less variable within population, i.e. it is a more canalized character. Finally, a principal component analysis, using the three enzyme systems shows that both populations are clearly separated, with a first principal component explaining 71.1 percent of the observed variance.     

Experimental conformational study of two peptides containing α-aminoisobutyric acid. Crystal structure of N-acetyl-α-aminoisobutyric acid methylamide

Some theoretical studies have predicted that the conformational freedom of the α-aminoisobutyric acid (H-Aib-OH) residue is restricted to the α-helical region of the Ramachandran map. In order to obtain conformational experimental data, two model peptide derivatives, MeCO-Aib-NHMe 1 and Bu^tCO-LPro-Aib-NHMe 2 , have been investigated. The Aib dipeptide 1 crystallizes in the monoclinic system (a = 12.71 Å, b = 10.19 Å, c = 7.29 Å, β = 110.02°, Cc space group) and its crystal structure was elucidated by x-ray diffraction analysis. The azimuthal angles depicting the molecular conformation (? = ?55.5°, ψ = ?39.3°) fall in the α-helical region of the Ramachandran map and molecules are hydrogen-bonded in a three-dimensional network. In CCl₄ solution, ir spectroscopy provides evidence for the occurrence of the so-called ₅ and C₇ conformers stabilized by the intramolecular i → i and i + 2 → i hydrogen bonds, respectively. The tripeptide 2 was studied in various solvents [CCl₄, CD₂Cl₂, CDCl₃, (CD₃)₂SO, and D₂O] by ir and pmr spectroscopies. It was shown to accommodate predominantly the βII folded state stabilized by the i + 3 → i hydrogen bond. All these experimental findings indicate that the Aib residue displays the same conformational behavior as the other natural chiral amino acid residues.     

Cloning of the Fatty Acid Synthetase β Subunit from Fission Yeast, Coexpression with the α Subunit, and Purification of the Intact Multifunctional Enzyme Complex

We have cloned and sequenced the fission yeast (Schizosaccharomyces pombe)fas1⁺gene, which encodes the fatty acid synthetase (FAS) β subunit, by applying a PCR technique to conserved regions in the β subunit of the α₆β₆types of FAS among different organisms. The deduced amino acid sequence of the Fas1 polypeptide, consisting of 2073 amino acids (M_r= 230,616), exhibits the 48.1% identity with the β subunit from the budding yeast (Saccharomyces cerevisiae). This subunit, with five different catalytic activities, bears four distinct domains, while the α subunit, the sequence of which was previously reported by Saitohet al.(S. Saitohet al.,1996,J. Cell Biol.134, 949–961), carries three domains. We have developed a co-expression system of the FAS α and β subunits by cotransformation of two expression vectors, containing thelsd1⁺/fas2⁺gene and thefas1⁺gene, into fission yeast cells. The isolated FAS complex showed quite high specific activity, of more than 4000 mU/mg, suggesting complete purification. Its molecular weight was determined by dynamic light scattering and ultracentrifugation analysis to be 2.1–2.4 × 10⁶, and one molecule of the FAS complex was found to contain approximately six FMN molecules. These results indicate that the FAS complex fromS. pombeforms a heterododecameric α₆β₆structure. Electron micrographs of the negatively stained molecule suggest that the complex adopts a unique barrel-shaped cage architecture.     

The solubilization of cytoskeletons of human erythrocyte membranes by p-mercuribenzene sulphonate

The disruption of erythyrocyte membrane cytoskeletons brought about by treatment with p-mercuribenzene sulphonate (PMBS) has been followed by measurements of turbidity and the binding of ²⁰³Hg-labelled PMBS. After pretreatment with N-ethylmaleimide to block readily reactive sulphydryl groups, incubation with [²⁰³Hg]PMBS showed incorporation of approximately 4 moles radiolabel per mole of spectrin and one per mole of actin. The incorporation of radiolabel paralleled the decrease in turbidity, and the labelling of spectrin paralleled that of actin. The kinetics were pseudo first order, and the pH dependence of the observed rate constant indicated a normal pK_a value for the sulphydryl group involved. The calculated second-order rate constant for the reaction of the sulphydryl anion with PMBS, however, was several orders of magnitude less than expected from model compound studies. The results suggest that association between spectrin and actin may result in the steric hindrance of reactivity of a limited number of sulphydryl groups in each protein. Disruption of the spectrin-actin association may then be linked to the modification of the sulphydryl groups.     

Variation in alcohol dehydrogenase activity in vitro in flies from natural populations ofDrosophila melanogaster

Alcohol dehydrogenase (ADH) activity variation in male flies taken directly from seven natural populations ofDrosophila melanogaster is largely accounted for by segregation of alleles at theAdh structural gene locus. There was little overlap in the ADH activities ofAdh ^F andAdh ^s homozygotes. Body weights varied only slightly betweenAdh genotypes and contributed little to ADH variation. Between and within population variation in ADH activity and ADH protein in flies in the wild is mainly due to the relative frequencies ofAdh ^F andAdh ^s.     

Purification of an alcohol dehydrogenase involved in the conversion of methional to methionol in <Emphasis Type="Italic">Oenococcus oeni</Emphasis> IOEB 8406

Oenococcus oeni, the major lactic acid bacteria involved in malolactic fermentation (MLF) in wine, is able to produce volatile sulfur compounds from methionine. Methional reduction is the last enzymatic step of methionol synthesis in methionine catabolism. Alcohol dehydrogenase (ADH) activity was found to be present in the soluble fraction of O. oeni IOEB 8406. An NAD(P)H-dependent ADH involved in the reduction of methional was then purified to homogeneity. Sequencing of the purified enzyme and amino acid sequence comparison with the database revealed the presence of a conserved sequence motif specific to the medium-chain zinc-containing NAD(P)H-dependent ADHs. Despite the great importance of ADH activities in wine flavor modification, this is the first report of the purification of an ADH isolated from O. oeni. The purified ADH does not seem to be involved in the modification of buttery and lactic notes or to be involved in the specific formation of volatile alcohols during MLF. The enzyme was not strictly specific of methional reduction and the highest reducing activity was obtained with acetaldehyde as substrate. The function of the purified ADH remains unclear, although the role of the sulfur atom in methional molecules in the interaction between enzyme and substrate was evidenced.     

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.     

Cholera Toxin Induces Cyclic AMP-Independent Down-Regulation of Gsα and Sensitization of α2-Autoreceptors in Chick Sympathetic Neurons

Abstract: The role of the stimulatory GTP-binding protein (G_S) in the α₂-autoinhibitory modulation of noradrenaline release was investigated in cultured chick sympathetic neurons. The α₂-adrenoceptor agonist UK 14,304 caused a concentration-dependent reduction of electrically evoked [³H]noradrenaline release with half-maximal effects at 14.0 ± 5.5 nM. In neurons treated with 100 ng/ml cholera toxin for 24 h, the half-maximal concentration was lowered to 3.2 ± 1.4 nM without changes in the maximal effect of UK 14,304. The pretreatment with cholera toxin also increased the inhibitory action of 10 nM UK 14,304 when compared with the inhibition of noradrenaline release in untreated cultures derived from the same cell population. In cultures treated with either 10 µM forskolin or 100 µM 8-bromo-cyclic AMP, neither the half-maximal concentration nor the maximal effect of UK 14,304 was altered. Cholera toxin, forskolin, and 8-bromo-cyclic AMP all induced an increase in spontaneous outflow and a reduction in electrically evoked overflow, effects not observed after a pretreatment with dideoxyforskolin. Exposure of neurons to cholera toxin, but not to forskolin or 8-bromo-cyclic AMP, induced a translocation of α-subunits of G_s (G_sα) from particulate to soluble fractions and led ultimately to a complete loss of G_sα from the neurons. In contrast, no effect was seen on the distribution of either α-subunits of G_i- or G_o-type G proteins or of β-subunits. These results indicate that cholera toxin causes a selective, cyclic AMP-independent down-regulation of G_sα. This down-regulation of G_sα is associated with the sensitization of α₂-autoreceptors.