Effects of propylene and oxygen on the ethylene-producing system of apples

Hypobaric conditions and treatments with ethylene and the ethylene analogue propylene were used to investigate effects of oxygen and elhylene on 1-aminocyclopropane-1-carboxylic acid (ACC) content, ACC synthase activity and ethylene production of apples ( Malus sylveslris Mill. cv. Golden Delicious). Prcclimacteric apples were stored in air at 6.6 kPa (reduced pressure); 6.6 kPa ventilated with pure O₂; 6.6 kPa ventilated with 2600 μl 1⁻¹ C₂H₄; and in air at 101.3 kPa (atmospheric pressure) for 4 months at 4°C. No ACC synthase activity was detectable in apples stored at 6.6 kPa, whereas ACC synthase activity was induced in apples stored at 6.6 kPa and ventilated with either O₂ or C₂H₄. In a further experiment, preclimacteric apples were stored for 14 days either in air at 20 kPa or at 20 kPa ventilated with pure O₂. Both treatments were supplied with 58 500 μl 1⁻¹ propylene from day 0 to day 9 or from day 9 to day 12. Ethylene production of apples treated with propylene from day 0 to day 9 increased earlier than ethylene production of untreated apples. Propylene treatment from day 9 to day 12 did not stimulate ethylene production. Ethylene and propylene induced and stimulated extractable ACC synthase activity and ACC formation of apples. Oxygen enhanced this effect. The results also suggest inhibition of in vivo ACC synthase activity by propylene.     

Chromium-induced inhibition of ethylene evolution in bean (Phaseolus vulgaris) leaves

The influence of chromium concentration on ethylene production in bean plants ( Phaseolus vulgaris L. cv. Contender) was investigated. A Cr ion-induced inhibition of ethylene synthesis from endogenous 1-aminocyclopropane-1-carboxylic acid (ACC) was observed within both leaf discs floated on 2 m M CrO²⁻₄ or Cr³⁺ and leaf discs from plants cultured in nutrient solutions containing 10, 20 or 40 μ M CrO²⁻₄. However, Cr ions supplied either to plants with the nutrient solution or to discs with the incubation medium rather increased the conversion of exogenous ACC to ethylene. Primary leaves of plants exposed to CrO²⁻₄-containing nutrient solutions showed a statistically insignificant decrease of ACC-synthase activity. In the trifoliolate leaves of plants exposed to 10 μ M CrO²⁻₄, in which a significant decrease of ethylene production from endogenous ACC was observed, a substantial increase of ACC synthase was found. These results indicate that Cr ion-induced inhibition of ethylene production is not due to a breakdown of membrane integrity, which is necessary for ethylene forming enzyme activity, but caused by metabolic alterations leading to decreased ACC availability. Chromium ions may act by inhibiting ACC synthase activity or by diverting a metabolic step prior to the ACC synthase catalyzed reaction.     

Binding Characteristics and Interactions of Depressant Drugs with [35S]t-Butylbicyclophosphorothionate, a Ligand that Binds to the Picrotoxinin Site

Abstract: [³⁵S]r-Butylbicyclophosphorothionate (TBPT), a cage convulsant with picrotoxinin-like activity, binds to rat brain membranes to a single site with an apparent K_D of 25.1 ± 5.6 n M and a B_max of 1.40 ± 0.22 pmol/mg protein. TBPT binding to rat brain membranes was inhibited by a variety of convulsant, depressant, anxiolytic, and anticonvulsant drugs that had previously been shown to inhibit [³H]a-dihydropicrotoxinin binding. Depressant drugs such as pentobarbital and the nonbarbiturate (+)etomidate inhibited TBPT binding in an uncompetitive manner. Thus, pentobarbital and (+)etomidate decreased both the affinity and the number of binding sites of TBPT to whole brain membranes. The IC₅₀ values of (+)etomidate (9 μ M ) and pentobarbital (90 μ M ) are similar to the EC₅₀ values at which they enhance both [³H]-γ-aminobutyric acid and [³H]diazepam binding in cerebral cortex membranes. RO5–4864, which has recently been shown to be a convulsant, also inhibited TBPT binding (IC₅₀= 10 μ M ). These results suggest that TBPT binds to the picrotoxinin site and further supports the notion that the picrotoxinin site is an important modulatory site at the benzodiazepine-GABA receptor-ionophore complex.     

Purification and characterization of a feruloyl esterase from the fungus Penicilliumexpansum

An extracellular phenolic acid esterase produced by the fungus Penicillium expansum in solid state culture released ferulic and ρ-coumaric acid from methyl esters of theacids, and from the phenolic-carbohydrate esters O-[5-O-(trans-feruloyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose (FAXX) and O-[5-O-((E)-ρ-coumaroyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose(PAXX). The esterase was purified 360-fold in successive stepsinvolving ultrafiltration and column chromatography by gel filtration, anion exchange andhydrophobic interaction. These chromatographic methods separated the phenolic acid esterasefrom α- l -arabinofuranosidase, pectate and pectin lyase, polygalacturonase,xylanase and β- d -xylosidase activities. The phenolic acid esterase had an apparentmass of 65 kDa under non-denaturing conditions and a mass of 57·5 kDa underdenaturing conditions. Optimal pH and temperature were 5·6 and 37 °C,respectively and the metal ions Cu2+ and Fe³+ atconcentrations of 5 mmol l⁻¹ inhibited feruloyl esterase activity by 95% and44%, respectively, at the optimum pH and temperature. The apparent K_m and V_max of the purified feruloyl esterase for methyl ferulate at pH 5·6 and 37 °Cwere 2·6 mmol l⁻¹ and 27·1 μmol min⁻¹ mg⁻¹. The corresponding constants of ρ-coumaroylesterase for methyl coumarate were 2·9 mmol l⁻¹ and 18·6μmol min−1 mg⁻¹.     

Citrate synthases from germinating castor bean seeds – II. Time course of synthesis and immunochemical comparison

The time course of total citrate synthase activity in castor bean ( Ricinus communis L., type Sanzibariensis) endosperm showed a 7-fold increase during the initial 5 days of germination and a decrease thereafter. All citrate synthase activity in the ungerminated seeds was due to the mitochondrial isoenzyme. After two days of germination the glyoxysomal isoenzyme began to appear. After 5 days the glyoxysomal citrate synthase represented 50 to 55% of the total activity and the mitochondrial enzyme the remainder. This was estimated from (a) inactivation of the glyoxysomal citrate synthase by 5,5'-dithiobis(2-nitrobenzoic acid); (b) solid phase adsorption of the glyoxysomal synthase by a specific antiserum; (c) separation of isoenzymes by (NH₄)₂SO₄ gradient solubilization.
The increase of both citrate synthases during the initial 4 days of germination could be prevented by 10 μg cycloheximide ml⁻¹, but not by 40 or 400 μg chloramphenicol ml⁻¹, indicating a synthesis on 80 S ribosomes. Actinomycin D completely inhibited the appearance of the glyoxysomal enzyme while the mitochondrial enzyme was not affected. Antisera against the two isoenzymes revealed major structural differences between two citrate synthases, however, also some common determinants. No cross-reaction was observed with the citrate synthase from pig heart or E. coli.     

l-Myo-inositol-1-phosphate synthase from lower plant groups: Partial purification and properties of the enzyme from Euglena gracilis

A survey for the enzyme L-myo-inositol-1-phosphate synthase (EC 5.5.1.4) has been conducted among various members of the lower plant groups, mainly algac, bryophytes and fungi; some properties of the partially purified enzyme from Euglena gracilis Z . are presented. The enzyme was detected in Chloropycean algae, Marchantiales and the Basidiomycetous fungi. The enzyme from Euglena had a pH optimum at 7.5. The Km for glucose-6-P was 2.1 m M and for NAD⁺ 80 μ M . When assayed in the absence of added NAD⁺, the enzyme showed a basal activity suggesting the presence of bund NAD⁺ in the system. NH₄Cl increased the enzyme activity two-fold, altough the enzyme was inactivated by (NH₄)₂SO₄.     

Modulation of Human Glutamate Transporter Activity by Phorbol Ester

Abstract: Termination of synaptic glutamate transmission depends on rapid removal of glutamate by neuronal and glial high-affinity transporters. Molecular biological and pharmacological studies have demonstrated that at least five subtypes of Na⁺-dependent mammalian glutamate transporters exist. Our study demonstrates that Y-79 human retinoblastoma cells express a single Na⁺-dependent glutamate uptake system with a K _m of 1.7 ± 0.42 µ M that is inhibited by dihydrokainate and dl - threo -β-hydroxyaspartate (IC₅₀ = 0.29 ± 0.17 µ M and 2.0 ± 0.43 µ M , respectively). The protein kinase C activator phorbol 12-myristate 13-acetate caused a concentration-dependent inhibition of glutamate uptake (IC₅₀ = 0.56 ± 0.05 n M ), but did not affect Na⁺-dependent glycine uptake significantly. This inhibition of glutamate uptake resulted from a fivefold decrease in the transporter's affinity for glutamate, without significantly altering the V _max. 4α-Phorbol 12,13-didecanoate, a phorbol ester that does not activate protein kinase C, did not alter glutamate uptake significantly. The phorbol 12-myristate 13-acetate-induced inhibition of glutamate uptake was reversed by preincubation with staurosporine. The biophysical and pharmacological profile of the human glutamate transporter expressed by the Y-79 cell line indicates that it belongs to the dihydrokainate-sensitive EAAT2/GLT-1 subtype. This conclusion was confirmed by western blot analysis. Protein kinase C modulation of glutamate transporter activity may represent a mechanism to modulate extracellular glutamate and shape postsynaptic responses.     

The role of NADH- and NADPH-linked acetoacetyl-CoA reductases in the poly-3-hydroxybutyrate synthesizing organism Alcaligenes eutrophus

Abstract Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Alcaligenes eutrophus . Incorporation of [1-¹⁴C]-acetyl-CoA into poly-3-hydroxybutyrate (PHB) by systems reconstituted from purified preparations of either 3-ketothiolase, AcAc-CoA reductase and PHB synthase, occurred only when NADPH-AcAc-CoA reductase was present. The NADH reductase was active with all of the d (−)- and l (+)-3-hydroxyacyl-CoA substrates tested (C₄-C₁₀), whereas the NADPH reductase was only active with d (−)-3-hydroxyacyl-CoAs (C₄-C₆). The products of AcAc-CoA reduction by the NADH- and NADPH-linked enzymes were l (+)-3-hydroxybutyryl-CoA and d (−)-3-hydroxybutyryl-CoA, respectively. The NADH-linked enzyme had an M _r of 150,000 (containing identical M _r 30,000 sub-units) and the NADPH-linked enzyme appeared to be a tetramer ( M _r 84,000) with identical sub-units ( M _r 23,000). K _m^app values of 22 μM and 5 μM for AcAc-CoA and 13 μM (NADH) and 19 μM (NADPH) for the coenzymes were determined for the NADH- and NADPH-linked enzymes, respectively.     

Occurrence and activity of hydrogenase in symbiotic Frankia from field-collected Alnus incana

Occurrence and activity of the hydrogen uptake enzyme were studied in root nodule homogenates made from plants of Alnus incana (L.) Moench collected from field sites in the northern part of Sweden. Nitrogenase (EC 1.7.99.2) activity (estimated by acetylene reduction) and hydrogen evolution were studied in excised nodules. All Frankia sources showed acetylene reduction activity, and possessed a hydrogen uptake system. Hydrogen uptake in nodule homogenates from the Frankia sources measured at 23.8 μM H₂ ranged from 0.04 to 5.0 μmol H₂ (g fresh weight nodule)⁻¹ h⁻¹. The H₂ uptake capacity of nodule homogenates from one of the Frankia sources was almost 8 times higher than the hydrogen evolution from nitrogenase, both expressed on a nodule fresh weight basis. Frankia sources from field sites 6 and 11 showed K_m for H₂ of 13.0 and 23.6 μM H₂, respectively. This indicates similarities in the hydrogen uptake enzymes in the two Frankia sources. It is concluded that hydrogen uptake is a common characteristic in Frankia.     

De novo shoot morphogenesis and plant growth of mustard (Brassica juncea) in vitro in relation to ethylene

Role of ethylene in de novo shoot morphogenesis from explants and plant growth of mustard ( Brassica juncea cv. India Mustard) in vitro was investigated, by culturing explants or plants in the presence of the ethylene inhibitors aminoethoxyvinylglycine (AVG) and AgNO₃. The presence of 20 μ M AgNO₃ or 5 μ M AVG in culture medium containing 5 μ M naphthaleneacetic acid and 10 μ M benzyladenine were equally effective in promoting shoot regeneration from leaf disc and petiole explants. However, AgNO₃ greatly enhanced ethylene production which reached a maximum after 14 days, whereas ethylene levels in the presence of AVG remained low during 3 weeks of culture. The promotive effect of AVG on shoot regeneration was overcome by exogenous application of 25 μ M 2-chloroethylphosphonic acid (CEPA), but AgNO_3-induced regeneration was less affected by CEPA. For whole plant culture, AVG did not affect plant growth, although it decreased ethylene production by 80% and both endogenous levels of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC by 70–80%. In contrast, AgNO₃ stimulated all 3 parameters of ethylene synthesis. Both AgNO₃ and CEPA were inhibitory to plant growth, with more severe inhibition occuring in AgNO₃. Leaf discs derived from plants grown with AVG or AgNO₃ were highly regenerative on shoot regeneration medium without ethylene inhibitor, but the presence of AgNO₃ in the medium was inhibitory to regeneration of those derived from plants grown with AgNO₃.     

An extracellular β-galactosidase secreted from cell suspension cultures of carrot. Its purification and involvement in cell wall-polysaccharide hydrolysis

β-Galactosidase (β-Galase, EC 3.2.1.23) activity has been detected in a culture medium of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The extracellular β-Galase (β-Galase-II) was purified to electrophoretic homogeneity from the concentrated medium using ammonium sulfate precipitation, chromatography on CM-Sephadex C-50. DEAE-Sepharose CL-6B and Sephacryl S-200HR, and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 65 kDa by Sephacryl S-200HR gel-permeation, and 60 kDa by SDS-PAGE after treatment with SDS and 2-mercaptoethanol. The pI was 6.5. The K_m and V_max values for p -nitrophenyl (PNP)-β-D-galactopyranoside were 0.17 m M and 31.9 μmol (mg protein)^-1, h^-1, respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.0–4.4. The enzyme activity was inhibited by Co²⁴, Cu²⁺, Hg^2-, p -chloromercuribenzoate (PCMB) and D-galactono-1,4-lactone. The enzyme acted on citrus galactan and larchwood arabinogalactan in an exo-fashion, and was slightly involved in the hydrolysis of an acidic pectic polymer containing arabinosyl and galactosyl residues and in the breakdown of cell walls isolated from carrot cell cultures.     

Opioid Modulation of Extracellular Signal-Regulated Protein Kinase Activity Is Ras-Dependent and Involves Gβγ Subunits

Abstract: Although it is well-established that G protein-coupled receptor signaling systems can network with those of tyrosine kinase receptors by several mechanisms, the point(s) of convergence of the two pathways remains largely undelineated, particularly for opioids. Here we demonstrate that opioid agonists modulate the activity of the extracellular signal-regulated protein kinase (ERK) in African green monkey kidney COS-7 cells transiently cotransfected with μ-, δ-, or κ-opioid receptors and ERK1- or ERK2-containing plasmids. Recombinant proteins in transfected cells were characterized by binding assay or immunoblotting. On treatment with corresponding μ- ([ d -Ala²,Me-Phe⁴,Gly-ol⁵]enkephalin)-, δ- ([ d -Pen², d -Pen⁵]enkephalin)-, or κ- (U69593)-selective opioid agonists, a dose-dependent, rapid stimulation of ERK1 and ERK2 activity was observed. This activation was inhibited by specific antagonists, suggesting the involvement of opioid receptors. Pretreatment of cells with pertussis toxin abolished ERK1 and ERK2 activation by agonists. Cotransfection of cells with dominant negative mutant N17-Ras or with a βγ scavenger, CD8-β-adrenergic receptor kinase-C, suppressed opioid stimulation of ERK1 and ERK2. When epidermal growth factor was used to activate ERK1, chronic (>2-h) opioid agonist treatment resulted in attenuation of the stimulation by the growth factor. This inhibition was blocked by the corresponding antagonists and CD8-β-adrenergic receptor kinase-C cotransfection. These results suggest a mechanism involving Ras and βγ subunits of G_i/o proteins in opioid agonist activation of ERK1 and ERK2, as well as opioid modulation of epidermal growth factor-induced ERK activity.     

Polygalacturonase biosynthesis by Lactobacillus plantarum: effect of cultural conditions on enzyme production

Lactobacillus plantarum was found to produce extracellular polygalacturonase (EC 3.2.1.15.). Maximum enzyme production was obtained in a medium containing 0.5% glucose and 1.5% low methyl-pectin as inducer at 27°C at an initial pH of 6.8. Enzyme production was strongly inhibited by 5 μmol/l NiCl₂, 5 μmol/l CoCl₂, 5 μmol/l CuSO₄, and 10 μmol/l ZnCl₂. MnSO₄ and MgSO₄ at 200 μmol/l and 50 μmol/l respectively seemed to enhance enzyme biosynthesis. The optimal pH and temperature for enzyme activity were 4.5 and 30°C respectively. Enzyme production in batch culture accompanied growth.     

Interaction of α-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor

α-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. α-Conotoxins blocking muscle-type or α7 nAChRs compete with α-bungarotoxin. However, α-conotoxin ImII, a close homolog of the α7 nAChR-targeting α-conotoxin ImI, blocked α7 and muscle nAChRs without displacing α-bungarotoxin ( Ellison et al. 2003, 2004 ), suggesting binding at a different site. We synthesized α-conotoxin ImII, its ribbon isomer (ImII iso ), 'mutant' ImII(W10Y) and found similar potencies in blocking human α7 and muscle nAChRs in Xenopus oocytes. Both isomers displaced [¹²⁵I]-α-bungarotoxin from human α7 nAChRs in the cell line GH₄C₁ (IC₅₀ 17 and 23 μM, respectively) and from Lymnaea stagnalis and Aplysia californica AChBPs (IC₅₀ 2.0–9.0 μM). According to SPR measurements, both isomers bound to immobilized AChBPs and competed with AChBP for immobilized α-bungarotoxin ( K _d and IC₅₀ 2.5–8.2 μM). On Torpedo nAChR, α-conotoxin [¹²⁵I]-ImII(W10Y) revealed specific binding ( K _d 1.5–6.1 μM) and could be displaced by α-conotoxin ImII, ImII iso and ImII(W10Y) with IC₅₀ 2.7, 2.2 and 3.1 μM, respectively. As α-cobratoxin and α-conotoxin ImI displaced [¹²⁵I]-ImII(W10Y) only at higher concentrations (IC₅₀≥ 90 μM), our results indicate that α-conotoxin ImII and its congeners have an additional binding site on Torpedo nAChR distinct from the site for agonists/competitive antagonists.     

Toxicity of organotins towards cyanobacterial photosynthesis and nitrogen fixation

Abstract Inhibition of photosynthesis by a range of organotin compounds in Plectonema boryanum was concentration-dependent and decreased in the order tributyltin (Bu₃SnCl) > tripropyltin (Pr₃SnCl) ≥ dibutyltin (Bu₂SnCl₂) ≥ triphenyltin (Ph₃SnCl) > triethyltin (Et₃SnCl) > trimethyltin (Me₃SnCl) > monobutyltin (BuSnCl₃). IC₅₀ values were determined for the most toxic organotin species and varied from approximately 1.2 μM for Bu₃SnCl to approximately 13 μM for Ph₃SnCl. A similar order of inhibition of photosynthesis was observed in Anabaena cylindrica , although here IC₅₀ values were slightly lower (e.g. approximately 1 μM for Bu₃SnCl and 5 μM for Ph₃SnCl).Nitrogenase activity was generally more sensitive to inhibition by organotin compounds than photosynthesis in A. cylindrica and this was particularlyy evident for Bu₂SnCl₂; approximate IC₅₀ values for Bu₂SnCl₂ were 3 and 9 μM, as estimated by nitrogenase activity and photosynthesis, respectively. These results indicate that organotin compounds have the potential to inhibit cyanobacterial metabolism in aquatic systems.     

Uptake kinetics of iron-phytosiderophores in two maize genotypes differing in iron efficiency

Iron inefficiency in the maize ( Zea mays L.) mutant ysl is caused by a defect in the uptake system for Fe-phytosiderophores. To characterize this defect further, the uptake kinetics of Fe-phytosiderophores in ysl was compared to the Fe-efficient maize cultivar Alice. Short-term uptake of ⁵⁹Fe-labeled Fe-deoxymugineic acid (Fe-DMA) was measured over a concentration range of 0.03 to 300 μM. Iron uptake in Fe-deficient plants followed Michaelis-Menten kinetics up to about 30 μM and was linear at higher concentrations, indicating two kinetically distinct components in the uptake of Fe-phytosiderophores. The saturable component had similar K_m (∼ 10 μM) in both genotypes. In contrast. V_max was 5.5 μmol Fe-DMA g⁻¹ dry weight [30 min]⁻¹ in Alice, but only 0.6 μmol Fe-DMA g⁻¹ dry weight [30 min]⁻¹ in _ysl. Uptake experiments with double-labeled ⁵⁹Fe-[¹⁴C]DMA suggest that in both cultivars Fe-DMA was taken up by the roots as the intact chelate. The results indicate the existence of a high-affinity and a low-affinity uptake system mediating Fe-phytosiderophore transport across the root plasma membrane in maize. Apparently, the mutation responsible for Fe inefficiency in ysl affected high-affected uptake and led to a decrease in activity and/or number of Fe-phytosiderophore transporters.     

Investigations on Myelination In Vitro: Regulation of 2',3'-Cyclic Nucleotide 3'-Phosphohydrolase by Thyroid Hormone in Cultures of Dissociated Brain Cells from Embryonic Mice

Abstract: The direct influence of l -3,3',5-triiodothyronine (T₃) on the development of 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37, CNPase) is demonstrated by using an in vitro culture system of dissociated embryonic mouse brain cells. Serum from a thyroidectomized calf, which contained low levels of T₃ (31 ng/100 ml), and thyroxine, T₄ (<1 μg/ml), was used in the culture medium in place of normal calf serum (T₃, 103 ng/100 ml; T₄, 5.7 μg/ml) to render the culture responsive to exogenously added T₃. The lower levels of enzyme activity observed in the presence of such a deficient medium could be restored to normal values by T₃ supplementation. Half-maximal effect was obtained with 2.5 ± 10⁻⁹ m -T₃. Three days of hormone treatment resulted in the maximal stimulation of CNPase. T₄ was less effective in inducing CNPase activity and the inactive analog of the hormone, reverse T₃ (3,3',5'-T₃) was ineffective. The morphological appearance of the cells was characterized by deformed (smaller size and less in number) reaggregates in the cultures, lacking hormone.     

Functional Reconstitution of a Highly Purified μ-Opioid Receptor Protein with Purified G Proteins in Liposomes

Abstract: A μ-opioid receptor protein (μ-ORP) purified to homogeneity from bovine striatal membranes has been functionally reconstituted in liposomes with highly purified heterotrimeric guanine nucleotide regulatory proteins (G proteins). A mixture of bovine brain G proteins, predominantly G_oA, was used for most of the experiments, but some experiments were performed with individual pure G proteins, G_oA, G_oB, G_i1, and G_i2. Low K _m GTPase was stimulated up to 150% by μ-opioid receptor agonists when both μ-ORP and a G protein (either the brain G protein mixture or a single heterotrimeric G protein) were present in the liposomes. Stimulation by a selective μ-agonist was concentration dependent and was reversed by the antagonist (−)-naloxone, but not by its inactive enantiomer, (+)-naloxone. The μ selectivity of μ-ORP was demonstrated by the inability of δ and κ agonists to stimulate GTPase in this system. High-affinity μ-agonist binding was also restored by reconstitution with the brain G protein mixture and with each of the four pure G_i and G_o proteins studied. The binding of μ agonists is sensitive to inhibition by GTPγS and by sodium.     

THE SYNTHESIS OF ACETYLCHOLINE FROM ACETYL-CoA, ACETYL-DEPHOSPHO-CoA AND ACETYLPANTETHEINE PHOSPHATE BY CHOLINE ACETYLTRANSFERASE

Abstract— The synthesis of ACh by choline acetyltransferase (ChAc) has been examined using acetyl-CoA, acetyl-dephospho-CoA and acetylpantetheine phosphate. At pH 7.5 K_m values of 25.7 μ m for acetyl-CoA, 54.8 μ m for acetyl-dephospho-CoA and 382 μ m for acetylpantetheine phosphate were obtained and are similar to those at pH 6.0. This indicates that the 3-phosphate may not be required for binding the substrate to the enzyme unlike carnitine acetyltransferase.
Inhibitor constants ( K_i ) for CoA, dephospho-CoA and pantetheine phosphate were also measured and when considered with the K_m values obtained for the acetyl derivatives it is concluded that acetyl-dephospho-CoA could be a successful acetyl donor in the synthesis of ACh.
Acetyl-dephospho-CoA was found to be less satisfactory as a substrate for citrate synthase.     

MJ0400 from Methanocaldococcus jannaschii exhibits fructose-1,6-bisphosphate aldolase activity

The central carbon metabolism is well investigated in bacteria, but this is not the case for archaea. MJ0400-His₆ from Methanocaldococcus jannaschii catalyzes the cleavage of fructose-1,6-bisphosphate (FBP) to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate with a V _max of 33 mU mg⁻¹ and a K _m of 430 μM at 50 °C. MJ0400-His₆ is inhibited competitively by erythrose-4-phosphate with a K _i of 380 μM and displays heat stability with a half-life of c . 1 h at 100 °C. Hence, MJ0400 is the second gene encoding for an FBP aldolase in M. jannaschii . Previously, MJ0400 was shown to act as an 2-amino-3,7-dideoxy- d - threo -hept-6-ulosonic acid synthase. This indicates that MJ0400 is involved in both the carbon metabolism and the shikimate pathway in M. jannaschii .