bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and Properties of a S‐Adenosylmethionine: Isoflavone 4′‐O‐Methyltransferase from Cell Suspension Cultures of Cicer arietinum L.
Authors: WENGENMAYER Herta , EBEL Jürgen , GRISEBACH Hans 
Published: March 2005

An O‐methyltransferase catalyzing the transfer of the S‐methyl group of S‐adenosyl‐l‐methionine to the 4′‐hydroxyl group of isoflavones has been detected in seedlings of Cicer arietinum L. and in cell suspension cultures of this plant.
After transfer of the cell cultures to fresh medium the activity of the methyltransferase rose after a lag period of about 2 h and reached a maximum after about 20 h and then declined. Illumination of the cell cultures had no effect on the enzyme activity.
The enzyme has been purified 360‐fold by (NH4)2SO4 fractionation, chromatography on DEAE‐cellulose and hydroxyapatite and Sephadex G‐200 gel filtration.
The S‐adenosylmethionine:isoflavone 4′‐O‐methyltransferase was isolated as a soluble enzyme with a pH optimum of 9 in glycine‐NaOH buffer. It requires–SH‐protecting agents for activity. The molecular weight of the enzyme was estimated by the elution volume from a Sephadex G‐200 column to be about 110000.
The enzyme is specific for the methylation of 4′‐hydroxyisoflavones. Intermediates in the bio‐synthetic pathway to isoflavones, e.g. p‐hydroxycinnamic acid and 5, 7, 4′‐trihydroxyflavone, were not methylated. The Km values for daidzein (7, 4′‐dihydroxyisoflavone) and S‐adenosylmethionine were determined to be 80 μM and 0.16 mM, respectively. Because of the high specificity of the methyltransferase for isoflavones it can be concluded that methylation is the last step in the biosynthesis of 4′‐methoxyisoflavones.
The enzyme was inhibited by S‐adenosylhomocysteine. The Ki values for S‐adenosylhomocysteine versus daidzein and S‐adenosylmethionine were determined to be 0.3 mM and 30 μM, respectively. From the inhibition kinetics it can be concluded that the reaction follows an Ordered BiBi mechanism with S‐adenosylmethionine and S‐adenosylhomocysteine as leading reaction partners.
Role of Uridine Diphosphate Glucose in the Biosynthesis of Starch
Authors: LAVINTMAN Nelly , TANDECARZ Juana , CARCELLER Marta , MENDIARA Sara , CARDINI Carlos E. 
Published: March 2005

A potato particulate preparation, which is constituted mainly by proplastids, was capable of glucose transfer from micromolar concentrations of uridine diphosphate [14C]glucose into an endogenous protein molecule to form a 14C‐labelled glucoprotein. This glucoprotein had short radioactive glucan chains and could act as acceptor for the synthesis of larger radioactive glucan chains from labelled uridine diphosphate glucose, either at millimolar concentrations or in the presence of glucose 1‐phosphate or adenosine diphosphate glucose at micromolar concentrations. This last step required Mn2+ or Mg2+ ions.The enzyme responsible for the formation of the glucoproteic acceptor was named uridine diphosphate glucose‐protein transglucosylase I and its properties were studied.The uridine diphosphate glucose‐protein transglucosylase activity, that was detected at micromolar uridine diphosphate glucose concentrations in the presence of glucose 1‐phosphate, could be distinguished from uridine diphosphate glucose–protein transglucosylase I by the following criteria: The glucose 1‐phosphate dependent activity required Mn2+ or Mg2+ ions, was sensitive to temperature and formed radioactive glucan chains totally hydrolyzed by β‐amylase. Conversely, uridine diphosphate glucose–protein transglucosylase I was active in the absence of Mn2+ or Mg2+ ions, was resistent to temperature and formed radioactive glucan chains scarcely attacked by β‐amylase.Sequential analysis of the glucose incorporation from uridine diphosphate [3H]glucose and uridine diphosphate [14C]glucose into the glucoprotein and sodium dodecylsulphate–acrylamide gel electrophoresis of the labelled glucoproteins formed in the presence and in the absence of glucose 1‐phosphate were carried out.On the basis of the data obtained a simple scheme of reactions is proposed to explain the involvement of the uridine diphosphate glucose–protein transglucosylase I and glucose 1‐phosphate dependent activities in the synthesis of α‐1,4 glucan chains of the starch type.
DNA Polymerase I from Acinetobacter calcoaceticus: Enzymatic Properties
Authors: NES Ingolf F. , LILLEHAUG Johan R. , KLEPPE Kjell 
Published: March 2005

Certain enzymatic properties of homogenous DNA polymerase I from Acinetobacter calcoaceticus have been investigated. The enzyme requires an initiator DNA for normal DNA repair synthesis. It is, however, also capable of de novo synthesis in the presence of dATP and dTTP or dGTP and dCTP. The enzyme was only partially inhibited by the–SH‐group‐blocking reagent p‐hydroxymercuribenzoate.DNA polymerase I from Acinetobacter calcoaceticus contained both a 3′→ 5′ and a 5′→ 3′ exonuclease activity. The products of the former was 5′‐deoxymononucleotide phosphates. The 5′→ 3′ nuclease gave mononucleotides with T7 DNA and with d(A‐T)n the products were mono, di, and trinucleotides, the ratio depending on the concentration of Mg2+· The 3′→ 5′ exonuclease activity appeared to be slightly more active on single‐stranded than double‐stranded DNA. The opposite was the case for the 5′→ 3′ nuclease which had an absolute requirement for double‐stranded DNA. The present work offers further evidence for the view that DNA polymerase I enzymes in microorganisms have similar structural features and enzymatic properties.
On the Mechanism and Control of the Malonyl‐CoA‐Dependent Chain Elongation of Fatty Acids. The Malonyl‐Transfer Reaction
Authors: PODACK Eckhard R. , SAATHOFF Gisela , SEUBERT Werner 
Published: March 2005

The enoyl‐CoA reductase activity of the purified microsomal chain‐elongation system of rat liver is inhibited non‐competitively by long‐chain acyl‐CoA and competitively by malonyl‐CoA.
The multienzyme complex catalyzes the transfer of the malonyl residue from malonyl‐CoA to pantetheine and CoASH with high affinities for the physiological acceptor and donator CoASH (Km= 20 μM) and malonyl‐CoA (Km= 22 μM), respectively.
The malonyl transfer is competitively inhibited by octanoyl‐CoA, 2,3‐trans‐octenoyl‐CoA and 3‐Oxo‐octanoyl‐CoA.
From the above data a common transferase catalyzing the exchange of the acyl moieties of malonyl enzyme and of the various enzyme‐bound intermediates of chain elongation with free coenzyme A is deduced.
Former observations by other laboratories, suggesting a microsomal chain elongation at the level of the CoA derivatives are explained by a rapid exchange of enzyme‐bound intermediates of the chain elongation process with free CoASH.
Pyruvate Formate‐Lyase of Escherichia coli: the Acetyl‐Enzyme Intermediate
Authors: KNAPPE Joachim , BLASCHKOWSKI Hans P. , GRÖBNER Peter , SCHMTTT Thomas 
Published: March 2005

The reaction pyruvate + CoA ⇌ acetyl‐CoA + formate, catalyzed by pyruvate formate‐lyase of Escherichia coli, occurs by the succeeding half‐reactions (a) E + pyruvate ⇌ E‐acetyl + formate; (b) E‐acetyl + CoA ⇌ E + acetyl‐CoA.Making use of coupled optical assays, a ‘ping‐pong mechanism’ was derived from the complete kinetic investigation of the forward and reverse reactions. The thermodynamic equilibrium constant of the overall reaction was calculated from the kinetic constants to be K= 750 (30°C, pH 8.1), which agrees with chemically determined values.The intermediate acetyl‐enzyme, which had been previously proposed from the [14C]formate‐pyruvate exchange, was detected by product‐pulse experiments with [2‐14C]pyruvate and trapped by acid precipitation. The acetyl group is linked to a sulfhydryl group of the protein.The value of the equilibrium constant of the first half‐reaction is about 50, as directly measured and calculated from the kinetic data. It was concluded that the standard free energy of hydrolysis of acetyl‐enzyme is about 1.7 kcal (7.1 kJ) more negative than that of acetyl‐CoA.The intermediate was found to react with dithiothreitol with a second‐order rate constant at 30°C and pH 7.6 of 1160 M−1× min−1. It resulted in a half‐life of 4 s (or 20 s at 0°C) in the particular buffer which was required for enzyme stabilization.The enzyme (about 60 U/mg) was prepared by carrying its purified inactive form through the enzyme‐II‐dependent activation reaction, employing photoreduced flavodoxin along with the effector compounds S‐adenosylmethionine and oxamate (as a pyruvate analogue).
Studies on tRNA Nucleotidyltransferase from Baker's Yeast
Authors: RETHER Benoît , BONNET Jacques , EBEL Jean‐Pierre 
Published: March 2005

A rapid purification method for tRNA nucleotidyltransferase from baker's yeast has been worked out. The enzyme, purified 900‐fold with a yield of 20%, is electrophoretically pure.ATP and CTP protect the enzyme against heat inactivation and are competitive inhibitors for each other in the reaction of CCA attachment. Differences in the protection properties, as well as in the kinetic constants, suggest that the ATP and CTP sites on the enzyme are different.The enzyme is also protected by high concentrations of tRNA without the CCA terminus (tRNA‐X), but not by the intact tRNA. Differences between tRNA's with or without the CCA triplet were also found when their inhibitory effect on the attachment of the CCA terminus was studied. It was found that tRNA‐X is a competitive inhibitor in the AMP attachment to tRNA‐XCC, whereas tRNA‐XCCA acts as a non‐competitive inhibitor both for CMP attachment to tRNA‐X and AMP attachment to tRNA‐XCC. Two interpretations for these differences in the behaviour of tRNA with or without the CCA terminus are discussed: (a) either a conformational change of the tRNA when the terminal A is reattached, or (b) the occurrence of a sequential mechanism involving the binding of ATP or CTP to the enzyme prior to the binding of tRNA.
Studies on tRNA Nucleotidyltransferase from Baker's Yeast
Authors: RETHER Benoît , GANGLOFF Jean , EBEL Jean‐Pierre 
Published: March 2005

Under special conditions, tRNA nucleotidyltransferase is able to add unusual terminal sequences to yeast tRNAPhe whose CCA end has been sequentially removed. By incubating tRNA‐XCCPhe, tRNA‐XCPhe or tRNA‐XPhe with ATP alone, the following tRNAPhe species are obtained: tRNA‐XCCAPhe (normal sequence), tRNA‐XCAPhe and tRNA‐XAPheWhen the degraded tRNAs are incubated with CTP alone, in all cases the sequence tRNA‐XCCCPhe is synthesized, which is able to accept one AMP, thus leading to tRNA‐XCCCAPhe. This compound can be aminoacylated by phenylalanyl‐tRNA synthetase, in contrast to tRNA‐XCAPhe and to tRNA‐XAPhe.
Source of Amino Acids Used for Protein Synthesis in HeLa Cells
Authors: VAN VENROOIJ Walther J. , MOONEN Hans , VAN LOON‐KLAASSEN Lucy 
Published: March 2005

HeLa cells, prelabeled with [3H]leucine for several generations, were pulse‐labeled with [14C]‐leucine. The 14C/3H ratio of the leucine in the medium, in the total amino acid pool and bound to transfer RNA was determined.It was found that the 14C/3H ratio of the leucyl‐tRNA was identical with that of the extracellular medium while that of the amino acid pool was much lower. These results confirm previous reports that selection of amino acids for protein synthesis takes place before the amino acids are released intracellularly.Further, it was concluded that amino acids resulting from intracellular proteolysis are not reutilized directly under the conditions of our experiments.A possible model of intracellular amino acid pathways is discussed.
The Influence of Respiration and ATP Hydrolysis on the Proton‐Electrochemical Gradient across the Inner Membrane of Rat‐Liver Mitochondria as Determined by Ion Distribution
Authors: NICHOLLS David G. 
Published: March 2005

A technique is described, based on the distribution of rubidium, acetate and methylammonium ions, for the simultaneous estimation of membrane potential and pH gradient across the inner membrane of mitochondria. The technique requires less than 0.5 mg mitochondrial protein and is independent of many factors which interfere with electrode determinations of protonmotive force (Δp).
With a limiting matrix volume of 0.4 μl/mg mitochondrial protein, the indicated value of Δp for rat liver mitochondria is 228 mV in state 4, 170 mV in state 3, and –0.6 mV in the presence of rotenone and uncoupler. The relative contributions of the pH gradient and membrane potential are dependent on the availability of electrophoretically and electroneutrally translocatable species in the incubation medium.
In a sucrose‐based medium containing 0.5 mM KCl, rotenone and uncoupler, the technique indicated a membrane potential of + 85 mV and a pH gradient of + 1.46 (acidic in the matrix compartment). In state 4, under no conditions examined did the pH gradient contribute more than 50% of the total protonmotive force.
The hydrolysis of ATP generates an optimal Δp of 220 mV.
The proton conductance of the inner membrane is potential dependent, increasing when Δp is greater than 200 mV.
The extra‐mitochondrial phosphate potential sustainable by respiration was found to change in parallel to Δp, but to exceed the latter parameter when based upon a stoichiometry of two protons translocated per ATP synthesised.
On the Mechanism of Action of Alkylguanidines on Oxidative Phosphorylation in Mitochondria
Authors: PAPA Sergio , TUENA DE GÓMEZ‐PUYOU Marietta , GÓMEZ‐PUYOU Armando 
Published: March 2005

The effect of octylguanidine and oligomycin on the oxygen uptake of rat liver mitochondria and on the ATPase activity of “sonic” submitochondrial particles has been studied.

Octylguanidine inhibits state 3 respiration with glutamate‐malate and succinate as substrates, but much lower concentrations are required to inhibit oxygen uptake with the former substrates. State 4 respiration is unaffected by octylguanidine.
The titration‐curve for the octylguanidine inhibition of glutamate‐malate oxidation is hyperbolic and apparently biphasic, half‐maximal inhibition is obtained at 30 μM octylguanidine. The octylguanidine‐curve for inhibition of succinate ocidation is sigmoid with half‐maximal inhibition at about 250 μM.
Octylguanidine and oligomycin show additive inhibitory action on state 3 respiration with both glutamate plus malate and succinate as respiratory substrates.
Concentrations of oligomycin or octylguanidine, which added separately are ineffective on state 3 respiration, become inhibitory when the two inhibitors are added together.
Octylguanidine inhibits the ATPase activity of sonic submitochondrial particles with a hyperbolic titration‐curve analogous to that obtained for oligomycin inhibition. The inhibitory actions of octylguanidine and oligomycin on the ATPase activity are additive.
It is concluded that octylguanidine acts directly on the ATPase complex and that its binding at the action site is mutually exclusive with the binding of oligomycin. A kinetic explanation is given for the reported higher sensitivity of site I phosphorylation to octylguanidine.
Rat‐Liver Cholesterol 7α‐Hydroxylase
Authors: VAN CANTFORT Jacques , GIELEN Jacques , RENSON Jean 
Published: March 2005

A new assay is described to measure the activity of cholesterol 7α‐hydroxylase and compared to the conventional 14C method used by other investigators. This method is based on the mechanism of the enzymic hydroxylation, i.e. a direct and stereospecific substitution of the 7α‐hydrogen by a hydroxyl group. [7α3‐H]Cholesterol is incubated at 37 °C and in the presence of molecular O2, in a medium buffered by potassium phosphate at pH 7.4 and containing liver microsomes (or 9000 ×g supernatant), NADPH, MgCI2 and cysteamine. Tween‐80 (1.5 mg/ml) is used to introduce enough substrate (300 μ) in the incubation mixture to saturate the enzyme (Km= 100 μ).Under these conditions the tritiated water released into the incubation medium reflects accurately the enzymic activity. The results obtained with this method are similar to the one obtained with a [4‐14C]cholesterol technique (r= 0.96; P < 0.001).The main advantage of the [7α‐3H]cholesterol method is a complete independence from further metabolism of the first enzymic product, the 7α‐hydroxycholesterol, the tritiated water representing the entire cholesterol 7α‐hydroxylase activity.
Regulation of Protein Secretion and Metabolism in Rat Salivary Glands
Authors: HERMAN Geneviève , ROSSIGNOL Bernard 
Published: March 2005

Carbachol and norepinephrine induce glycogen breakdown in rat submaxillary glands incubated in vitro. These effects are suppressed by specific inhibitors of cholinergic receptors (atropine, a muscarinic blocker) and of adrenergic receptors (propranolol, a β‐blocker and phentolamine, an α‐blocker). The β‐adrenergic effect seems to be mediated through the adenosine 3′:5′‐monophosphate system, whereas the α‐adrenergic and cholinergic effects are dependent of Ca2+ influx into cell. In the two latter systems, it is suggested that a direct activation of phosphorylase b kinase by calcium takes place.These data clearly indicate that glycogenolysis in rat submaxillary glands in vitro can be independently affected by sympathetic and parasympathetic agents via specific receptors.
Early Nucleolar Preribosomal RNA · Protein in Mammalian Cells
Authors: BACHELLERIE Jean‐Pierre , NICOLOSO Monique , ZALTA Jean‐Pierre 
Published: March 2005

Early steps of ribosomal maturation have been studied by analysis of nucleolar extracts using different extraction procedures. Early 45‐S nucleolar RNA is found to be associated with slowly sedimenting elements, distinct from previously described 80‐S and 55‐S nucleolar preribosomes.This early 45‐S RNA has been shown to be of preribosomal type according to the following criteria. (a) When hybridized with nucleolar DNA, competition with rRNA can be observed; (b) its biosynthesis is sensitive to low doses of actinomycin D; (c) it is methylated at an early stage; (d) it contains no linked poly(A) segments.This early 45‐S RNA seems to be specifically linked with proteins. The protein content of these early RNA‐protein complexes is significantly lower than that for 80‐S preribosomes. 45‐S RNA sensitivity to nucleolytic activities that can take place in the course of preribosome extraction has been found to be higher in these RNA‐protein complexes than in 80‐S preribosomes.Identical results were obtained when different mammalian cell species were studied (rat, hamster, or HeLa cells).
Specific Deuterium Labelling and Computerized Gas Chromatography‐Mass Spectrometry in Studies on the Metabolism in vivo of a Steroid Sulphate in the Rat
Authors: BAILLIE Thomas A. , ERIKSSON Håkan , SJÖVALL Jan , HERZ Josef E. 
Published: March 2005

The metabolism of 3β‐hydroxy‐5α‐pregnan‐20‐one sulphate was studied in bile fistula rats and in isolated perfused livers. Computerized gas chromatography‐mass spectrometry, in combination with specific deuterium‐labelling, was employed to follow the metabolic transformations.Male animals excreted metabolites into bile more rapidly than females, a finding which could be correlated with the preferential formation of glucuronide conjugates in the male liver.The major metabolic pathway in male rats involved the steps: hydrolysis, 2α‐hydroxylation, oxidoreduction at C‐3 and glucuronide conjugation, yielding 2α,3α‐dihydroxy‐5α‐pregnan‐20‐one glucuronide as the major metabolite. Only traces of the injected steroid sulphate were detected in bile from male animals. In contrast, the administered compound was the major steroid excreted in bile of female rats, where the main metabolite was identified as 3β,15β‐dihydroxy‐5α‐pregnan‐20‐one sulphate. A minor metabolite, 3β,16α‐dihydroxy‐5α‐pregnan‐20‐one, was found as a mono‐sulphate in female rats and as both a disulphate and a glucuronide conjugate in male rats. The deuterium content of the sulphated 15β‐ and 16α‐hydroxylated metabolites was consistent with metabolic pathways involving direct hydroxylation of the injected steroid sulphate.The results obtained from the liver perfusions were essentially the same as those from the experiments with bile fistula animals. This indicates that all the observed metabolic reactions took place in the liver.
Sequence Determination of 5′‐Terminal and 3′‐Terminal T1 Oligonucleotides of 18‐S Ribosomal RNA of a Mouse Cell Line (L 5178 Y)
Authors: ELADARI Marthe‐Elisabeth , GALIBERT Francis 
Published: March 2005

The 5′ and 3′‐terminal oligonucleotides of 18‐S ribosomal RNA of L 5178 Y (a mouse cell line) obtained after total T1 ribonuclease hydrolysis were isolated by a diagonal procedure. They were localized on the fingerprint of T1‐ribonuclease‐hydrolysed 18‐S RNA.These two oligonucleotides were partially hydrolysed by snake venom and spleen phosphodiesterases and resulting products were fractionated bidimensionally. Their base compositions were determined by total hydrolysis with piperidine or snake venom phosphodiesterase. From these results the following sequences were deduced: pU‐A‐C‐C‐U‐G for the 5′‐terminal oligonucleotide and G‐A‐U‐C‐A‐U‐U‐Aoh for the 3′‐terminal oligonucleotide.Quantitative studies indicated that these sequences represent at least 70% for the 5′ oligonucleotide and 85% for the 3′ oligonucleotide of the terminal sequences of the 18‐S RNA.
Crystalline l‐Histidine Ammonia‐Lyase of Achromobacter liquidum
Authors: SHIBATANI Takeji , KAKIMOTO Toshio , CHIBATA Ichiro 
Published: March 2005

Crystalline l‐histidine ammonia‐lyase of Achromobacter liquidum was prepared with a 24% recovery of the activity. The specific activity of the pure enzyme (63 μmol of urocanic acid min−1 mg−1) is similar to those so far reported for the enzyme from other sources.The purified enzyme appeared to be homogeneous by analytical disc electrophoresis and isoelectric focusing (pI= 4.95). The molecular weight determined by Sephadex G‐200 gel filtration is 200 000. The optimum pH is 8.2, and the optimum temperature is 50 °C. The enzyme showed strict specificity to l‐histidine (Km= 3.6 mM). Several histidine derivatives are not susceptible to the enzyme but do inhibit the enzyme activity competitively; the most effective inhibitors are l‐histidine methyl ester (Ki= 3.66 mM) and β‐imidazole lactic acid (Ki= 3.84 mM). l‐Histidine hydrazide (Ki= 36 mM) and imidazole (Ki= 6 mM) noncompetitively inhibited the enzyme. EDTA markedly inhibited enzyme activity and this inhibition were reversed by divalent metal ions such as Mn2+, Co2+, Zn2+, Ni2+, Mg2+, and Ca2+. These results suggest that the presence of divalent metal ions is necessary for the catalytic activity of histidine ammonia‐lyase. Sodium borohydride and hydrogen peroxide inhibited the enzyme activity.
Selenium Binding to Beef‐Kidney Rhodanese
Authors: CANNELLA Carlo , PECCI Laura , AGRÒ Alessandro FINAZZI , FEDERICI Giorgio , PENSA Bernardo , CAVALLINI Doriano 
Published: March 2005

The reaction of beef kidney rhodanese with selenosulfate was studied. The selenium‐treated enzyme shows an absorption spectrum with a maximum at 375 nm attributable to a sulfoselenide group. This absorption is bleached by addition of cyanide. After cyanide treatment stoichiometric amount of selenocyanate can be found.The intrinsic fluorescence of rhodanese is quenched by addition of stoichiometric selenosulfate. This effect can be reversed by cyanide or sulfite but not by selenite or glutathione.By comparison with model complexes the selenium‐rhodanese intermediate was identified as a cysteinyl‐selenium derivative.
Hydroxylation of p‐Coumaric Acid by Illuminated Chloroplasts
Authors: HALLIWELL Barry 
Published: March 2005

Chloroplasts isolated from leaves of spinach‐beet (Beta vulgaris L. ssp. vulgaris) do not catalyse the hydroxylation of p‐coumaric acid in the dark unless a reductant (such as ascorbate, NADH or NADPH) is added. Superoxide dismutase has no effect on this reaction.
Illuminated chloroplasts catalyse the hydroxylation in the absence of added reductant. This reaction is completely inhibited by superoxide dismutase, but catalase has little effect.
Both hydroxylation in the light and hydroxylation in the dark in the presence of reductants are inhibited by diethyldithiocarbamate, EDTA, cyanide and 2‐mercaptoethanol.
It is proposed that O2.− generated by illuminated chloroplasts is involved in the provision of a reductant to the enzyme phenolase.