Some properties of glutamine synthetase and glutamate synthase from Chlorobium vibrioforme f. thiosulfatophilum

The phototrophic green sulphur bacterium Chlorobium vibrioforme f. thiosulfatophilum assimilated ammonia via glutamine synthetase and glutamate synthase when grown with ammonia up to 30 mM, but above this level glutamate dehydrogenase was the key enzyme. Glutamine synthetase purified 42-fold was found to be adenylylated. The -glutamyltransferase activity of the enzyme was markedly inhibited by alanine, glycine, serine and lysine, and these amino acids in various combinations showed cumulative inhibition. Adenine nucleotides also inhibited enzyme activity, especially ATP. Glutamate synthase purified 222-fold had a maximum absorption at 440 nm which was reduced by sodium dithionite, and the enzyme was inhibited by atebrin indicating the presence of a flavin component. The enzyme had specific requirements for NADH, -ketoglutarate and l-glutamine, the K _m values for these were 13.5, 270 and 769 M respectively. Glutamate synthase was sensitive to feedback inhibition by amino acids, adenine nucleotides and other metabolites and the combined effects of these inhibitors was cumulative.Abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamic dehydrogenase     

Arsenic‐induced NFκβ transactivation through Erks‐ and JNKs‐dependent pathways in mouse epidermal JB6 cells

Carnosine, a alanylLhistidine dipeptide with antioxidant properties is present at high concentrations in skeletal muscle tissue. In this study, we report on the antioxidant activity of carnosine on muscle lipid and protein stability from both in vitro and in vivo experiments. Carnosine inhibited lipid peroxidation and oxidative modification of protein in muscle tissue prepared from rat hind limb homogenates exposed to in vitro Fenton reactant (Fe²⁺, H₂O₂)generated free radicals. The minimum effective concentrations of carnosine for lipid and protein oxidation were 2.5 and 1 mM, respectively. Histidine and alanine, active components of carnosine, showed no individual effect towards inhibiting either lipid or protein oxidation. Skeletal muscle of rats fed a histidine supplemented diet for 13 days exhibited a marked increase in carnosine content with a concomitant reduction in muscle lipid peroxidation and protein carbonyl content in skeletal muscle caused by subjecting rats to a Fenitrilotriacetate administration treatment. This significant in vitro result confirms the in vivo antioxidant activity of carnosine for both lipid and protein constituents of muscle under physiological conditions.     

Role of ubiquitin‐proteasome‐dependent proteolytic process in degradation of muscle protein from diabetic rabbits

The activity of ATP, ubiquitin (Ub)dependent proteases partially purified from skeletal muscle (psoas) from alloxan diabetic rabbits was determined at different periods of insulin deficiency. Two days after alloxan injection, no change was observed in the activity of ATP, Ubdependent proteases, but this activity increased 3 and 5 days after diabetes induction, attaining 181% of control values on the 5th day. However, after this early rise, the activity of muscle ATP, Ubdependent proteases decreased, returning to values that did not differ significantly from controls 7 and 10 days after alloxan injection. After 15 days, the activity of these proteases was 57% lower than in muscle from control rabbits. Both the initial increase and the subsequent fall in the activity of the enzymes were prevented by insulin treatment of alloxan diabetic rabbits. The data suggest that Ubproteasomedependent proteolysis have an important role in the control of muscle protein degradation and may be regulated by insulin.     

Purification and properties of cAMP independent glycogen synthase kinase and phosvitin kinase from human leukocytes

Summary cAMP independent glycogen synthase kinase and phosvitin kinase activity was purified from the 180 000 × g supernatant of human polymorphonuclear leukocytes by ammonium sulphate precipitation and phosphocellulose chromatography. The cAMP independent glycogen synthase kinase eluted from the phosphocellulose at 0.54 m NaCl (peak A) separate from the major phosvitin kinase eluting at 0.68 m NaCl (peak B). The kinase activity of both peaks tended to form aggregates, but in the presence of 0.6 m NaCl, the peak B enzyme had M_r 250 000, 7.2S and the peak A enzyme M_r 38 000, 3.8S. The ratio between synthase kinase and phosvitin kinase activity in peak A was 1:3.2 and in peak B 1:31.4. In addition the kinase activities differed with respect to sensitivity to temperature, ionic strength and CaCl₂. It is suggested that the peak A enzyme represents the cAMP independent glycogen synthase kinase of leukocytes, whereas the peak B enzyme is a phosvitin kinase, which is insignificantly contaminated with some synthase kinase (peak A) and contains a separate, second synthase kinase.Synthase kinase had K _m ^app 4.2 m for muscle glycogen synthease I and K _m ^app 45 m for ATP. GTP was a poor substrate. The activity was not influenced by cyclic nucleotides, Ca²⁺, or glucose-6-P. Synthase I from muscle and leukocytes was phosphorylated to a ratio of independence of less than 0.05.Abbreviations cAMP adenosine cyclic 3:5-monophosphate - DTT dithiothreitol - EGTA ethylene glycol-bis-(-amino-ethylether)-N,N-tetraacetic acid - PMSF phenylmethylsulfonylfluoride - PKI protein kinase inhibitor - RI ratio of independence for glycogen synthase - SDS sodium dodecyl sulphate     

Ribulose bisphosphate carboxylase from Thiobacillus A2. Its purification and properties

Ribulose bisphosphate carboxylase (EC 4.1.1.39) from Thiobacillus A2 has been purified to homogeneity on the basis of polyacrylamide gel electrophoresis and U.V. analysis during sedimentation velocity studies. The enzyme had an optimum pH of about 8.2 with Tris-HCl buffers. The molecular weight was about 521000 with an S _rel. of 16.9. K _m for RuBP was 122 M, for total CO₂ it was 4.17 mM, and for Mg²⁺ 20.0 M. The absolute requirement for a divalent cation was satisfied by Mg²⁺ which was replaceable to a certain extent by Mn²⁺. Activity was not significantly affected by SO ₄ ^2- , SO ₃ ^2- , or S₂O ₃ ^2- at 1.0 mM. At this concentration S^2- caused a 27% stimulation. All mercurials tested were inhibitory. pHMB was the most potent causing about 60% inhibition at 0.01 mM. This inhibition was reversible by low concentrations of cysteine. Cyanide was also inhibitory. Its mode of inhibition with respect to RuBP was un-competitive and with a K _i of 20 M. Lost activity could be restored partially by GSH or Cu²⁺. Although azide at the concentration tested had no significant effect on enzyme activity, 2,4-dinitrophenol at 1.0 mM caused 91% inhibition. Finally, activity was also affected by energy charge.Abbreviations ATP adenosine-5-triphosphate - GAPDH glyceraldehyde phosphate dehydrogenase - GSH (reduced) glutathione - G6P glucose-6-phosphate - NAD⁺ nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - pHMB parahydroxymercuribenzoate - 6PG 6-phosphogluconate - 3-PGA 3-phosphoglycerate - PGK phosphoglyceratekinase - RuBP ribulose-1,5-bisphosphate     

The influence of temperature on glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and the regulation of these enzymes in a mesophilic and a thermophilic cyanobacterium

The activities and kinetics of the enzymes G6PDH (glucose-6-phosphate dehydrogenase) and 6PGDH (6-phosphogluconate dehydrogenase) from the mesophilic cyanobacterium Synechococcus 6307 and the thermophilic cyanobacterium Synechococcus 6716 are studied in relation to temperature. In Synechococcus 6307 the apparent K _m's are for G6PDH: 80M (substrate) and 20M (NADP⁺); for 6PGDH: 90M (substrate) and 25M (NADP⁺). In Synechococcus 6716 the apparent K _m's are for G6PDH: 550M (substrate) and 30M (NADP⁺); for 6PGDH: 40M (substrate) and 10M (NADP⁺). None of the K _m's is influenced by the growth temperature and only the K _m's of G6PDH for G6P are influenced by the assay temperature in both organisms. The idea that, in general, thermophilic enzymes possess a lower affinity for their substrates and co-enzymes than mesophilic enzymes is challenged.Although ATP, ribulose-1,5-bisphosphate, NADPH and pH can all influence the activities of G6PDH and 6PGDH to a certain extent (without any difference between the mesophilic and the thermophilic strain), they cannot be responsible for the total deactivation of the enzyme activities observed in the light, thus blocking the pentose phosphate pathway.Abbreviations G6PDH glucose-6-phosphate, dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - G6P glucose-6-phosphate - 6PG 6-phosphogluconate - RUDP ribulose-1,5-bisphosphate - Tricine N-Tris (hydroxymethyl)-methylglycine     

Redox modulation of reductase and phosphatase activities in human erythrocytes

Summary We have previously reported that ferricyanide reductase activity in human erythrocytes depended on glycolysis and could be modulated by several compounds including oxidants and hormones like insulin. Insulin could activate glycolysis, probably as a consequence of tyrosine phosphorylation of protein band 3, implicating phosphorylation reactions as an important signal for activation of the reductase by insulin. Reversible phosphorylation of cellular proteins is also believed to play a key role in the action of insulin. Cytosolic acid phosphatase activity has been found in human erythrocytes. To further extend initial reports, we studied the effect of modulators on the cytosolic erythrocyte acid phosphatase. Mild oxidants like ferricyanide (1 mM), vanadate (1 mM), Mn²⁺ (0.5 and 1 mM), and phenylarsine oxide (10 and 100 M) inhibited the phosphatase activity. Similarly, insulin at concentrations that stimulate ferricyanide reduction (500, 1000 IU/ml) inhibited the activity of the phosphatase enzyme. The overall results indicated that oxidants are able to inhibit the acid phosphatase and stimulate the redox enzyme. In addition, a significant negative correlation (r = –0.400; P = 0.006) was observed between phosphatase and reductase activities. The observations discussed here, together with previous ones, emphasize that a close association between reductase and phosphatase enzymes may exist and also suggest a role for redox reactions in tyrosine phosphorylation/dephosphorylation-mediated signal transduction pathways.     

The role of glutamine synthetase in the regulation of nitrogenase activity (“switch off” effect) in Rhodospirillum rubrum

We have studied the changes in the activities of both nitrogenase (switch off) and glutamine synthetase in Rhodospirillum rubrum upon addition of ammonium ions or glutamine to nitrogen fixing cultures. Both activities decrease drastically and return in a parallel manner when added ammonia is metabolized. The decrease in glutamine synthetase activity does not seem to be primarily due to adenylylation of the enzyme. Addition of glutamine to cells starved for nitrogen results in inactivation of glutamine synthetase but nitrogenase is only partially switched off.Abbreviations CeMe₃NBr Cetyltrimethylammonium bromide - Hepes N-2-hydroxyethyl-piperazine-N-2 sulfonic acid - MSO methionine-D,L-sulfoximine - Tea-Dmg triethanol amine-3,3-dimethylglutaric acid     

Three glucose 6-phosphate dehydrogenase variants found in Japan

Summary Three Japanese glucose 6-phosphate dehydrogenase (G6PD) variants were investigated. G6PD Mediterranean-like had markedly decreased activity, normal electrophoretic mobility, low Km G6P, low Km NADP, increased utilization of all three substrate analogues (2-deoxy-G6P, Gal-6P, and deamino-NADP) and slightly decreased heat stability and slightly biphasic pH curve. G6PD Ogori had markedly decreased activity, but otherwise normal characteristics. G6PD Hofu had moderately decreased activity, normal electrophoretic mobility, slightly reduced Km G6P, normal Km NADP, normal utilization of 2-deoxy-G6P and Gal-6P, but increased utilization of deamino-NADP and normal heat stability as well as normal pH curve.     

UDP-GlcNAc: Galβ3GalNAc-Mucin: (GlcNAc → GalNAc) β6-N-Acetylglucosaminyltransferase and UDP-GlcNAc: Galβ3(GlcNAcβ6) GalNAc-Mucin (GlcNAc → Gal)β3-N-Acetylglucosaminyltransferase from Swine Trachea Epithelium

Summary Two specific -N-acetylglucosaminyltransferases involved in the branching and elongation of mucin oligosaccharide chains, namely, a 1,6 N-acetylglucosaminylsaminyltransferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3GalNAc-Mucin to yield Gal3(GlcNAc6)GalNAc-Mucin and a 3-N-acetylglucosaminyl transferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3(GlcNAC6)GalNAc-mucin to yield GlcNAc3Gal3 (GlcNAc6)GalNAc-Mucin were purified from the microsomal fraction of swine trachea epithelium. The 1,6-N-acetylglucosaminyltransferase was purified about 21,800-fold by procedures which included affinity chromatography on DEAE columns containing bound asialo Cowper's gland mucin glycoprotein with Gal1,3GalNAc side chains. The apparent molecular weight estimated by gel filtration was found to be about 60 Kd. The purified enzyme showed a high specificity for Gal1,3GalNAc chains and the most active substrates were mucin glycoproteins containing these chains. The apparent Km of the 6-glucosaminyltrans-ferase for Cowper's gland mucin glycoprotein containing Gal1,3GalNAc chains was 0.53 µM; for UDP-N-acetylglucosamine, 12 µM; and for Gal 1,3GalNAc NO₂ø, 4 mM. The activity of the 6-glucosaminyltransferase was dependent on the extent of glycosylation of the Gal3GalNAc chains in Cowper's gland mucin glycoprotein.The best substrate for the partially purified 3-Glucosaminyltransferase was Cowper's gland mucin glycoprotein containing Gal1,3(GlcNAc6)GalNAc side chains. This enzyme showed little or no activity with intact sialylated Cowper's gland mucin glycoprotein or derivatives of this glycoprotein containing GalNAc or Gal1,3GalNAc side chains.The radioactive oligosaccharides formed by these enzymes in large scale reaction mixtures were released from the mucin glycoproteins by treatment with alkaline borohydride, isolated by gel filtration on Bio-Gel P-6 and characterized by methylation analysis and sequential digestion with exoglycosidases. The oligosaccharide products formed by the 6- and 3-glucosaminyltransferases were shown to be Gal3(GlcNAC6) GalNAc and GlcNAc3 Gal3(GlcNAC6)GalNAc respectively.Taken collectively, these results demonstrate that swine trachea epithelium contains two specific N-acetylglucosaminyltransferases which catalyze the initial branching and elongation reactions involved in the synthesis of O-linked oligosaccharide chains in respiratory mucin glycoproteins. The first enzyme a 6-glucosaminyltransferase converts Gal3GalNAc chains in mucin glycoproteins to Gal3(GlcNAc6)GalNAc chains. This product is the substrate for a second 3-glucosaminyltransferase which converts the Gal3(GlcNAc6)GalNAc chains to GlcNAc3Gal(GlcNAc6)GalNAc chains in the glycoprotein. The 3-glucosaminyltransferase did not utilize Gal3GalNAc chains as a substrate and this results in an ordered sequence of addition of N-acetylglucosamine residues to growing oligosaccharide chains in tracheal mucin glycoproteins.Abbreviations NeuNAc N-acetylneuraminic acid - GalNAcol N-acetylgalactosaminitol - CGMG Cowper's gland mucin glycoprotein - GalNAc-CGMG Cowper's gland mucin glycoprotein containing GalNAc side chains O-glycosidically linked to serine or threonine - Gal3GalNAc-CGMC Cowper's gland mucin glycoprotein containing Gal3GalNAc side chains - MES 2-(N-morpholino) Ethane Sulfonic acid - PBS Phosphate Buffered Saline     

Calcium ions and polyamines activate the plasma membrane-located 1,3-β-glucan synthase

Sucrose-density-gradient centrifugation and partitioning in a polyethylene glycol/dextran two-phase system were used to isolate plasmamembrane vesicles from microsomal preparations of soybean cell suspension cultures. Both methods resulted in the enrichment of the activity of a 1,3--glucan synthase which forms a polymer consisting of more than 99% of 1,3-linked glucose (callose). Digitonin increases the 1,3--glucan synthase activity in the various membrane fractions to a different degree, supporting the suggestion that this enzyme is vectorially arranged in the plasma membrane. The enzyme is greatly activated either by poly-l-ornithine or synergistically by Ca²⁺ and spermine, indicating that the same enzyme is affected and exhibits the regulatory properties necessary for callose synthesis.Abbreviations GSI glucan synthase I - 1,3--GS 1,3--glucan synthase (EC 2.4.1.34) - IDPase inosine 5-diphosphatase - poly-l-Orn poly-l-ornithine - PEG polyethyleneglycol - SGT sterol-glucosyl-transferase - UDPG uridine 5-diphosphoglucose Dedicated to W. Nultsch on the occasion of his 60th birthday     

A dynamic model of a two-synapse feedback loop in the vertebrate retina

The theoretical properties of synapses such as those in the retina which operate on graded potentials are developed using work on tetrodotoxin-treated synapses as a basis. A linearized model of a two-synapse negative feedback loop analogous to the bipolaramacrine feedback loop in the retina possesses a frequency response which developes an increasingly prominent resonance peak at higher input levels and under some circumstances shows instability. Psychophysical studies have shown that the visual system also exhibits this behaviour suggestive of progressive underdamping in a harmonic oscillator. Evidence in favor of the hypothesis that resonance originates in the loop is presented, the conclusions being that the loop functions to tune the retina to a range of temporal frequencies.Symbol Table V millivolts depolarisation relative to resting membrane potential - V _n , V _out pre-synaptic, post-synaptic depolarisation respectively - V _e , V _i reversal potential or e.m.f. of post-synaptic battery of excitatory, inhibitory synapses respectively - V _out (max) maximal post-synaptic depolarisation defined by Eq.(10c) - V ₀ input depolarisation for feedback loop - depolarisation potential normalised with respect to V _out(max) - I milliamperes of depolarising current - I _s post-synaptic membrane current - I _c cable current - I ₀ input depolarising current for feedback loop - I _max maximal physiological value for I ₀ =V _e ·G ₀ - i depolarising current normalised with respect to I_max - e reversal potential normalised with respect to V _e - r _i specific resistivity of internal medium - R _m membrane resistance - C _m membrane capacitance - cable space constant = R _m /2R _i - g ₀ characteristic cable conductance = 2/R _m ·R _i - G conductance of post-synaptic membrane - G _s maximal post-synaptic membrane conductance - g fraction of receptors occupied by transmitter = G/G _s - r the ratio G _s/G ₀ - membrane time constant = R _m·C_m - ₁ time constant of transmitter release in response to presynaptic depolarisation [Eq. (6)] - ₂ time constant of decay of g [Eq. (7a)] - _{2 2}·[1+k·exp(b·v _in)]^–1 - k equilibrium constant for transmitter-receptor interaction [Eq. (7a)] - b constant determining increase in rate of transmitter release with pre-synaptic depolarisation [Eq. (6)] - c concentration of transmitter in synaptic cleft normalised with respect to resting concentration - H _jk (s) linearised transfer function for synaptic transmission from neurone j to neurone k - G(s) H₁₂(s) - H(s) -H₂₁(s) - F(s) linearised closed-loop transfer function - _x 2 times spatial frequency of counterphase grating pattern - the ratio (1+s)/(_x)₂ - a the product (1+r)·k - d density of bipolars per unit area     

Structure of tanacetan,a pectic polysaccharide from tansy Tanacetum vulgare L

Tanacetan TVF was found to have a branched structure with a backbone of linear -1,4-D-galacturonan. The ramified regions consist of linear -1,2-L-rhamno--1,4-D-galacturonan as the core. The side chains appear to attach to the 4-position of the L-rhamnopyranose residues. They are present as single -galactopyranose residues or a branching -1,4-galactopyranan bearing 4,6-substituted -D-galactopyranose residues as branched points. In addition, the ramified regions contain side chains of a branched -1,5-arabinofuranan possessing 2,5- and 3,5-substituted -L-arabinofuranose residues as branching points. Some side chains of rhamnogalacturonan appear to be arabinogalactan which contains branched sugar chains of -1,5-arabinofuranan attached to the linear chains of -1,4-galactopyranan by 1,3- and 1,6-linkages. The residues of -L-arabinofuranose seem to occupy the terminal positions of the arabinogalactan side chains.     

Lipoxygenase is an abundant protein in cucumber exudates

The presence of lipoxygenase (LOX) has been reported in many plant organs. High LOX activity (1–2 katal/mg protein) was detected in exudates from cut cucumber (Cucumis sativus L.) stems and petioles. Exudate LOX had a pH optimum of 5.0, an estimated molecular weight of 95 kDa and cross-reacted on sodium-dodecyl-sulfate gels with anti-LOX antibodies raised against soybean leaf LOX isoenzymes. Lipoxygenase activity was detected on native gels stained with o-dianisidine using linoleic acid as a substrate. Enzyme activity was similar with linoleic and linolenic acid and 2 times greater with arachidonic acid as substrate. At pH 6.8, LOX metabolized linoleic acid into 13- and 9-hydroperoxides at a ratio of 12. Linolenic acid was preferentially oxidized at carbon 13. Lipoxygenase activity was inhibited by n-propyl gallate (IC₅₀ 300 nM) and nordihydroguaiaretic acid (IC₅₀ 25 nM), but not by nonsteroidal anti-inflammatory drugs. LOX activity was enhanced 4.5-fold by 300 mM Ca²⁺. Spermine at 1 mM, and putrescine and spermidine at 2 mM completely inhibited LOX activity, but at low concentrations spermine (100 mM) and spermidine (100–500 mM) significantly stimulated LOX activity: 8- and 4.5-fold, respectively. Tissue printing of stem, petiole and hypocotyl sections with subsequent incubation with the antiserum raised against soybean leaf LOX revealed the presence of LOX in the internal and external phloem and in the sieve tubes.Abbreviations DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid - 9(S)-HpOD 9-(S)-hydroperoxy-(E,Z)10,12-octadecadienoic acid - 13(S)-HpOD 13-(S)-hydroperoxy(Z,E)-9,11-octadecadienoic acid - IC inhibition constant - IEF isoelectrofocusing - LOX lipoxygenase - NDGA nordihydroguaiaretic acid - PBS phosphate buffered saline - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate We would like to thank Ulla Jarlfors for exellent technical assistance with the histological analysis. The research reported in this paper was supported in part by grants to J.K. from the R.J. Reynolds Tobacco Company and Cooperative Agreement 43YK-5-0030 of the USDA-ARS. Journal paper 93-11-12 of the Kentucky Agricultural Experiment Station, Lexington.     

Characterization of the in vitro kinase activity of a partially purified soluble GST/JAK2 fusion protein

In vivo effects of insulin and vanadium treatment on glycogen synthase (GS), glycogen synthase kinase-3 (GSK-3) and protein phosphatase-1 (PP1) activity were determined in Wistar rats with streptozotocin (STZ)-induced diabetes. The skeletal muscle was freeze-clamped before or following an insulin injection (5 U/kg i.v.). Diabetes, vanadium, and insulin in vivo treatment did not affect muscle GSK-3 activity as compared to controls. Following insulin stimulation in 4-week STZ-diabetic rats muscle GS fractional activity (GSFA) was increased 3 fold (p < 0.05), while in 7-week diabetic rats it remained unchanged, suggesting development of insulin resistance in longer term diabetes. Muscle PP1 activity was increased in diabetic rats and returned to normal after vanadium treatment, while muscle GSFA remained unchanged. Therefore, it is possible that PP1 is involved in the regulation of some other cellular events of vanadium (other than regulation of glycogen synthesis). The lack of effect of vanadium treatment in stimulating glycogen synthesis in skeletal muscle suggests the involvement of other metabolic pathways in the observed glucoregulatory effect of vanadium.     

Purification of p53/55 kinase from nuclear ribonucleoproteins of Namalwa cells

Summary A nuclear p53/55 protein kinase has been isolated from nuclear ribonucleoprotein particles from human tumor cells. The enzyme was purified approximately 2200-fold from cell nuclei by sequential ribonuclease digestion of the RNP particles, DEAE cellulose and phosphocellulose chromatography. The kinase which was CAMP independent, catalyzed the phosphorylation of rabbit muscle glycogen synthase in the amino terminal domain, and conversion of the I to D form. The D synthase had a phosphorylation stoichiometry of 8 moles ³²P, per mole of synthase subunit with maximal specificity for ATP as phosphate donor; its K_m was 30 M. An antinucleolar antibody inhibited enzyme activity by 80%. Substrates for most other kinases were inactive. The kinase was essentially unaffected by the Walsh inhibitor, EGTA, regulatory subunits of protein kinase, calmodulin, trifluoperazine or heparin. Its activity was lost at 1 mM polyamine, but was enhanced 3-fold by MnC12 and 4- to 9-fold by deoxymononucleotides.The nuclei of HeLa cells contained 64% of the total kinase of which 11% were in nucleoli; the specific activity of the nucleolar kinase was twice that of the nuclear supernatant and four times that of the cytoplasmic kinase. These results indicate that nucleolar ribonucleoprotein particles of human tumor cells contain a CAMP-independent protein kinase which is similar to glycogen synthase kinase.Abbreviations RNP ribonucleoprotein - PMSF phenyl methyl sulfonyl fluoride - EGTA ethylene glycol-bis-(-aminoethyl ether) N,N-tetra acetic acid - TCA trichloroacetic acid - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - ELISA enzyme-linked immunosorbent assay     

Human HE2 (μB) and μA motifs show the same function as whole IgH intronic enhancer in transgenic mice

In the murine IgH gene intronic enhancer (ENH_iH), two major functional domains were reported. One is the E4/octomer region and another includes the A and B motifs. In the human ENH_iH, it was reported that the HE2, which corresponds to the murine B, and E6 motifs play an important role in an enhancer activity and a tissue-specificity at cellular level. Here we examined thein vivo function of the E6, A and HE2 motifs within the human ENH_iH by using the transgenic mice technique. The A and HE2 motifs together revealed almost the same enhancer function as the whole human ENH_iH, but the E6 motif had lesser enhancer acitivty and tissue-specificity.     

Separation of membranes by flotation centrifugation for in vitro synthesis of plant cell wall polysaccharides

Summary Membranes from etiolated maize seedlings were isolated using sucrose gradients for in vitro studies of polysaccharide synthesis. Following downward centrifugation, flotation centrifugation improved the purity of membrane fractions, in particular the Golgi apparatus. Based on naphthylphthalamic acid binding to plasma membrane and inosine-5-diphosphatase activity in Golgi apparatus, flotation centrifugation removed about 70% of the plasma membrane which cosedimented with the Golgi apparatus in downward centrifugation. The addition of chelators during flotation centrifugation allowed separation of the Golgi apparatus from endoplasmic reticulum, as indicated by NADH cytochromec reductase activity. Glucan and xylan synthase activities were measured as the radioactivity incorporated from either UDP-¹⁴C-glucose or UDP-¹⁴C-xylose into 80% ethanol insoluble materials. Glucan synthase activity at a substrate concentration of 1 mM UDP-glucose without CaCl₂ was greatest in fractions enriched in Golgi apparatus, but in the presence of 3 mM CaCl₂ the activity was greatest in fractions enriched in plasma membrane. Glucan synthase activity at a substrate concentration of 10M UDP-glucose in the presence of 3 mM MnCl₂ was greatest in fractions enriched in plasma membrane, but was also high in fractions enriched in Golgi apparatus. Xylan synthase activity, at a substrate concentration of 1 M UDP-xylose in the presence of 3 mM MnCl₂, was greatest in fractions enriched in Golgi apparatus. To further characterize these synthase reactions, the glycosyl linkages of the products formed were analyzed with a gas chromatograph coupled to a radiogas proportional counter. With the substrate, UDP-¹⁴C-glucose, and fractions enriched in Golgi apparatus, both (13)- and (14)-radioactive glucosyl linkages were formed, whereas the main linkage formed by fractions enriched in plasma membrane was (13)-glucosyl. With the substrate, UDP-¹⁴C-xylose, mostly (14)-xylosyl and some terminal-xylosyl linkages were formed by fractions enriched in Golgi apparatus. Only xylan synthase activity copurified with Golgi apparatus and, because plasma membrane lacked this activity, xylan synthase may be used as a reasonable indicator of Golgi apparatus.Abbreviations ATP adenosine-5-triphosphate - CR crude fraction from downward centrifugation - FL purified fraction from flotation centrifugation - GC gas chromatography - GC-RPC gas chromatography-radiogas proportional counting - IDP inosine-5-disphosphate - NPA naphthylphthalamic acid - UDP uridine-5-diphosphate - TEM transmission electron microscopy     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Ethanol fermentation using a fructose-negative mutant of Zymomonas mobilis

Summary The fermentation of an equimolar mixture of glucose and fructose into ethanol and sorbitol by a fructose negative mutant of Zymomonas mobilis is analysed using a recently described methodology (Ait-Abdelkader and Baratti, Biotechnol. Tech. 1993,329–334) based on polynomial fitting and calculation of instantaneous and overall parameters. These parameters are utilized to describe this mixed-substrate mixed-product fermentation.Nomenclature X biomass concentration, g/l - S total sugar concentration, g/l - Glu glucose concentration, g/l - Fru fructose concentration, g/l - Sor sorbitol concentration, g/l - P ethanol concentration, g/l - t fermentation time, h - specific growth rate, h^-1 - q_s specific sugar uptake rate, g/g.h - q_g specific glucose uptake rate, g/g.h - q_F specific fructose uptake rate, g/g.h - q_P specific ethanol productivity, g/g.h - q_Sor specific sorbitol productivity, g/g.h - Y_X/S biomass yield on total sugar, g/g - Y_P/S ethanol yield on total sugar, g/g - Y_Sor/S sorbitol yield on total sugar, g/g - Y_Sor/F sorbitol yield on fructose, (g/g) - Y_P/G ethanol yield on glucose, (g/g)