bluuebunny/crossref_metadata_2025 huggingface dataset:
Primary Structure of ϰ Light Chain from a Human Myeloma Protein
Authors: MILSTEIN Celia P. , DEVERSON Edward V.
Published: March 2005

The primary structure of the light chain of myeloma protein Car was studied. The light chain was fully reduced and alkylated with iodo[14C]acetic acid and digested with trypsin. The tryptic peptides were purified and sequenced. Peptides that overlapped the tryptic peptides were isolated from a peptic digest. The object was primarily to sequence the variable part of the chain, whereas the peptides of the constant part were only partially sequenced. The amino acid sequence of the variable part coincides with the basic sequence of the ϰIb subgroup, except for few substitutions. Some of the above‐mentioned substitutions are unique and some have been observed already in other ϰI chains. Two unusual features of this light chain are the presence of carbohydrate covalently bound to the asparagine residue occupying position 28 and the deletion of one amino acid at position 95.
Nitrate Reductase as a Product‐Inducible Enzyme
Authors: KAPLAN Drora , ROTH‐BEJERANO Nurit , LIPS Herman
Published: March 2005

Nitrite is a very effective inducer of nitrate reductase. Nitrate induces the enzyme in darkness only upon addition of suitable sources of reducing equivalent which permit the formation of nitrite; nitrite itself brings about induction of the enzyme in darkness without supplementation of exogenous reducing cofactors. Inhibition of constitutive nitrate reductase by tungstate prevents induction of the enzyme by nitrate but not by nitrite. Since nitrate induces the enzyme only under conditions allowing production of nitrite while the induction capacity of nitrite is free of this constraint, nitrate reductase be considered a product‐inducible rather than a substrate‐inducible enzyme.
The Amino‐Acid Sequence of a Polypeptide from the Venom of Dendroaspis viridis
Authors: SHIPOLINI Rudolph A. , BANKS Barbara E. C.
Published: March 2005

The primary structure of a major polypeptide component from the venom of Dendroaspis viridis has been determined unambiguously. The molecule (60 amino acids, four disulphide bridges) closely resembles a relatively non‐toxic component of the venom of Dendroaspis angusticeps. The pharmacological effects of neither of these are understood. The component from Dendroaspis viridis does not block neuromuscular transmission in frogs, is without effect on the excitatory or inhibitor actions of acetylcholine on snail neurons and does not appear to be a lytic factor.
The Interaction of Liver Phosphorylase a with Glucose and AMP
Authors: STALMANS Willy , LALOUX Monique , HERS Henri‐Géry
Published: March 2005

The effects of glucose and of AMP on several properties of purified dog liver phosphorylase a have been investigated.

Glucose stimulated several‐fold the conversion of phosphorylase a into b, catalyzed by purified phosphorylase phosphatase. The reaction was strongly inhibited by AMP in the absence of glucose, and much less in the presence of glucose. Conversely, the concentration of glucose that gave half‐maximal stimulation was increased several‐fold by the nucleotide. Similar effects were observed when the phosphorylase phosphatase was studied in crude liver preparations. 1,5‐Anhydroglucitol, α‐methylglucoside and 2‐deoxyglucose, quoted in order of decreasing efficiency, also stimulated the phosphorylase phosphatase reaction. Similar results were obtained when the conversion of phosphorylase a into b′ by trypsin was studied.

The effect of glucose on the kinetics of purified liver phosphorylase a was mostly to decrease affinity for glucose 1‐phosphate and to increase cooperativity between glucose 1‐phosphate binding sites. AMP had the opposite effect and each of the two ligands antagonized the binding of the other. Glucose derivatives, in the order of efficiency given above, mimicked the effects of glucose. Glucose and the same glucose analogues, as well as AMP, protected phosphorylase a against denaturation at 55°C.

These results indicate that the stimulation of the phosphorylase phosphatase reaction by glucose is secondary to the binding of the hexose to phosphorylase a, and that this binding is modulated by the concentration of AMP. The physiological implication of these findings is discussed.
Studies of Glutamate Dehydrogenase
Authors: GAUPER Friedrich Peter , MARKAU Klaus , SUND Horst
Published: March 2005

The association‐dissociation equilibrium of beef‐liver glutamate dehydrogenase was investigated in phosphate buffer of different ionic strengths and in the presence of the organic solvents ethylene glycol and dioxane. For some of the solvents the temperature dependence of the equilibrium constant was analyzed. The dependence of the apparent weight‐average molecular weight on enzyme concentration shows always the same general shape as that found earlier in 0.067 M phosphate buffer at pH 7.6. Each curve can be fitted assuming an open association‐dissociation equilibrium with one single equilibrium constant for all steps and with one single value for the second virial coefficient. Within the limit of the experimental error, the second virial coefficient is found to be the same for all systems (8–9 nmol × 1 × g‐2), whereas the equilibrium constants vary by orders of magnitude.

At low ionic strength a very small temperature dependence was found with a reduced association at higher temperatures. At high ionic strength a temperature maximum of the equilibrium constant is observed. Preferential phosphate binding is indicated by the high values of the refractive index increment.

The organic solvents investigated here (ethylene glycol, dioxane) cause a dissociation into the unimers (oligomers). The equilibrium constant decreases from 9 × 105 M‐1 in aqueous phosphate buffer to 5.2 × 103 M−1 in 6 M ethylene glycol and to 2.4 × 105 M−1 in 2 M ethylene glycol. The effects obtained with 5% dioxane (0.57 M) are similar to that with 2 M ethylene glycol. In both cases a sharp decrease of the association with increasing temperature is observed.

The reaction enthalpy and the reaction entropy of the association reaction decrease with decreasing temperature in all solvents, the enthalpy being positive in the absence and negative in the presence of organic solvents. The free enthalpy of reaction, however, increases with increasing temperature in buffers of high and low ionic strengths but decreases in the presence of organic solvents. The association reaction at high ionic strength appears mainly to be entropy‐driven whereas in the presence of the organic solvents the reaction seems to be energy‐driven at all temperatures. It is suggested that the organic solvents interfere with the hydrophobic interactions between the unimers. The positive reaction enthalpy in the presence of high salt concentrations indicates that the attractive electrostatic forces are largely shielded, whereas shielding is imperfect for the repulsive forces which even seem to be enhanced. This could be due to a preferential binding of anions which would tend to increase the negative net charge and could explain the difference observed between the effects of phosphate and chloride solutions of identical ionic strength.
Effect of DNA Polymerase on Nuclei from Different Phases of Cell Cycle
Authors: BERNARD Ora , MOMPARLER Richard L. , BRENT Thomas P.
Published: March 2005

DNA replication was investigated in isolated HeLa cell nuclei from different phases of the cell cycle. The addition of S‐phase cytoplasm to S‐phase nuclei restored the ability of these nuclei to synthesize DNA. The S‐phase cytoplasm requirement could be replaced by partially purified calf‐thymus DNA polymerases A and B. The activity of both the S‐phase cytoplasm and purified DNA polymerases in the nuclear system was ATP‐dependent. Using nuclei prelabelled with bromodeoxyuridine and CsCl equilibrium centrifugation, the DNA synthesis in vitro was shown to be of the replicative, not repair type when supported by either S‐phase cytoplasm or DNA polymerase A. DNA polymerase B, but not DNA polymerase A, was capable of using G1 nuclei to synthesize DNA; however, this synthesis was not ATP‐dependent. Gel‐filtration chromatography of HeLa cell‐free extracts have shown that DNA polymerases B and C are present in S‐phase and missing in G1‐phase cells.
Hamster Brown‐Adipose‐Tissue Mitochondria
Authors: NICHOLLS David G.
Published: March 2005

The rate of controlled respiration of hamster brown‐adipose‐tissue mitochondria is dependent on the pH of the incubation medium, and on the presence of albumin and exogenous purine nucleotide. The steady‐state protonmotive force (Δp) across the inner membrane is dependent on the same parameters. The freshly prepared mitochondria fail to maintain a proton electrochemical gradient during respiration. The maximal Δp which may be attained is 220 mV, in the presence of both albumin and purine nucleotide. The concentration of purine nucleotide required to raise ΔP is dependent on the pH of the medium and on the presence of albumin. The rate of controlled respiration is an inverse linear function of Δp over the range 220 mV to 166 mV. Albumin, purine nucleodites and medium pH control Δp by modulating the effective proton conductance of the inner membrane. At pH 7.1, the effective proton conductance of the freshly prepared mitochondria is greater than 35 nequiv. H+× min−1× mg−1× mV−1 at 23°C, a figure which is reduced to 7–10 in the presence of albumin, to 2 in the presence of 1 mM GDP, and to 1 in the presence of both inhibitors of proton conductance. In the presence of albumin, the concentration of GDP required for 50% inhibition of the proton conductance is 10 μM at pH 7.1. The additional proton conductance induced by the addition of carbonyl cyanide p‐trifluoromethoxyphenylhydrazone is a linear function of the uncoupler added. The significance of these findings is discussed in terms of theories of energy transduction, and the possible physiological mechanisms controlling respiration in brown adipose tissue in vivo.
Purification and Specificity of Aspergillus sojae DNase
Authors: SUZUKI Masaru , SAKAGUCHI Kenji
Published: March 2005

An endo‐type deoxyribonuclease isolated from autolysate of Aspergillus sojae was purified 175‐fold. The purified enzyme migrated as a single band during electrophoresis on polyacrylamide gel and hydrolysed only deoxyribonucleic acids. Optimal hydrolysis of DNA by the enzyme was observed at pH 9.4. The enzyme was strongly activated by Mg2+. Mn2+ and Zn2+ also enhanced the enzymatic activity, but to a lesser extent. Heat‐denatured DNA was found to be a better substrate than native DNA. The molecular weight of the enzyme determined by gel filtration on Sephadex G‐100 was 15600.

A limited amount of the enzyme preferentially splits the phosphodiester bonds of purine nucleotides in DNA. The mononucleotides liberated are P‐5′‐dGuo (major) and P‐5′‐dAdo (minor). The oligonucleotides are terminated mostly either with P‐5′‐dGuo or P‐5′‐dAdo and partly with P‐5′‐dCyd at the 5′‐phosphate termini, and with P‐5′‐dGuo and P‐5′‐dAdo at the 3′‐hydroxyl termini, respectively.
Structure and Synthesis of a Lipid‐Containing Bacteriophage Preparation of Virus and Localization of the Structural Proteins
Authors: HINNEN Rosmarie , SCHÄFER Rolf , FRANKLIN Richard M.
Published: March 2005

The four structural proteins of bacteriophage PM2 have been related to the morphological structures in the virion. Protein I can be selectively removed from the virus by degradation with bromelain, a proteolytic enzyme. From electron microscopic studies and from experiments showing that the virus cannot attach to its host cell after bromelain treatment, we conclude that protein I forms the outer spikes at the vertices of the icosahedron. Protein II can be labeled with [35S]sulfanilic acid diazonium salt. As shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, proteins I and II can be removed if the virus is treated with 1 M urea. From the above studies and from packing considerations, we conclude that protein II forms the outer shell of the virus.

The dissociation of the virus in 1 M urea leads to: (a) a nucleocapsid core containing viral lipid, DNA, protein III and IV, (b) a second fraction which consists of the same elements obtained by treatment of the virus with bromelain. Both particles have been identified by electron microscopy. The dissociation of the virus in 1 M urea plus 0.5% Triton X‐100, a nonionic detergent, leads to a third fraction representing an empty shell which contains all of the lipid, proteins II and III, and about 50% of protein IV, but no DNA. We have also been able to isolate a second type of nucleocapsid core by dissociation of the virus in the presence of 4.5 M urea. This core, which has an approximate s20,w, = 133 S and which has been identified in the electron microscope as a core with icosahedral shape, contains proteins III and IV, as well as the DNA, but not phospholipid. In the presence of 4.5 M urea this core is resistant to Triton X‐100 concentrations up to 0.1%. Higher concentrations of the nonionic detergent dissociate the core into DNA and the solubilized proteins 111 and IV. In the absence or in the presence of low concentrations of urea (1 M) the core is resistant to Triton X‐100 concentrations up to 0.5%. The DNA in the core is digestible by pancreatic DNAase I and the core structure is broken down after DNAase treatment. Protein IV interacts with the DNA as shown by transformation of non‐covalent interactions between DNA and protein into covalent bonds induced by a nucleophilic reagent. In conclusion, protein I forms the spikes, protein II the outer icosahedral shell and protein III the inner icosahedral shell around the DNA, which is closely associated with protein IV. The phospholipid bilayer is sandwiched between these two shells. Protein III may also form the protein bridges to protein I which connect the inner and outer shells through the bilayer.
Deoxyribonucleic Acid Polymerases from Yeast
Authors: WINTERSBERGER Erhard
Published: March 2005

Yeast DNA polymerases A and B were purified 5000 – 10000‐fold by chromatography on DEAE‐cellulose, phosphocellulose, DNA‐agarose and DEAE‐Sephadex. In acrylamide gel electrophoresis in the presence of sodium dodecylsulfate, both enzymes give rise to three main bands. The enzymes are equally susceptible to inhibition by the – SH reagents N‐ethylmaleimide and p‐chloromercuri‐benzoate but differ in their sensitivity to cytosine‐arabinoside triphosphate, polymerase A being considerably more sensitive to this nucleotide analog. Whereas DNA polymerase A prefers nicked DNA and poly[d(A‐T)] as template‐primer, polymerase B is most active with poly(dA) · (dT)10. Km values for deoxyribonucleoside triphosphates were determined as 3.7–3.9 μM for enzyme A and 1.8–2.4 μM for enzyme B. During active growth of cells polymerase activity increases about 1.4‐fold over the amount found in resting cells, which is mainly if not exclusively due to an increased level of DNA polymerase A. This together with other properties suggests a role of this enzyme in DNA replication.
New Sialic Acids
Authors: BUSCHER Hans‐Peter , CASALS‐STENZEL Jorge , SCHAUER Roland
Published: March 2005

Sialic acids were released by dilute acids or by neuraminidase from glycoproteins of bovine and equine submandibular glands, from glycoproteins of equine blood plasma and from equine erythrocyte membranes. They were purified by ion‐exchange chromatography and fractionated on cellulose.

The following methods have been adapted to the structural analysis of sialic acids with regard to the nature and position of the N‐acyl and O‐acyl groups.

a) Thin‐layer chromatography in two dimensions with intermediate ammonia treatment to remove the O‐acyl groups leading to the identification of the basic N‐acylneuraminic acids.

b) Thin‐layer chromatography in two dimensions with intermediate hydroxylamine treatment allowing the identification of the O‐acyl groups of sialic acids as hydroxamates.

c) Gas‐liquid chromatography for identification of the different N‐acylated and O‐acylated sialic acids.

The structure of the following new sialic acids have been established:

a) N‐glycoloyl‐8‐O‐acetylneuraminic acid isolated from glycoproteins of bovine submandibular glands;

b) N‐acetyl‐4‐O‐glycoloylneuraminic acid isolated from glycoproteins of equine submandibular glands and also occurring in equine blood plasma and equine erythrocyte membranes;

c) N‐acetyl‐7 or 8‐O‐glycoloylneuraminic acid detected in glycoproteins from bovine submandibular glands.

The O‐acetylated N‐glycoloylneuraminic acid first detected in horse erythrocyte membranes by Hakomori and Saito and in horse serum by Pepper, was determined to be N‐glycoloyl‐4‐O‐acetyl‐neuraminic acid; it was also found to occur in submandibular glands of horse.

The sialic acid patterns from the different biological materials investigated are compared.
Micrococcin: Acceptor‐Site‐Specific Inhibitor of Protein Synthesis
Authors: OTAKA Tadahiko , KAJI Akira
Published: March 2005

Micrococcin inhibited the binding of aminoacyl‐tRNA at 13 mM Mg2+, but not at 6 mM Mg2+. The elongation factor Tu (EF‐Tu)‐dependent binding of aminoacyl‐tRNA was inhibited appreciably by 7 μM micrococcin. The EF‐G‐dependent puromycin reaction of the ribosome‐bound aminoacyl‐tRNA, release of unesterified tRNA concomitant with translocation, and the EF‐G‐dependent evacuation of aminoacyl‐tRNA from the acceptor site of the ribosomes were all sensitive to this antibiotic. It is concluded that micrococcin interacts with the acceptor site specifically and this interaction results in the dual action of this antibiotic, namely inhibition of translocation and the binding of aminoacyl‐tRNA.
Isolation and Characterization of α‐Galactosidase from Lens esculanta
Authors: DEY Prakash M. , WALLENFELS Kurt
Published: March 2005

α‐Galactosidase from Lens esculanta (red lentils) was purified 4090‐fold through a multistep process involving acidification, ammonium sulphate fractionation, Sephadex gel filtration, DEAE‐cellulose and CM‐cellulose chromatography. The purified enzyme is stable at 4°C for nearly six months with only a small loss of activity. It showed a pH optimum of 5.5 with raffinose as the substrate and 6.0 with p‐nitrophenyl‐α‐d‐galactoside. The Km for the former substrate was 40 mM and for the latter, 0.26 mM.

The enzyme failed to liberate galactose from guar gum, tara gum and locust bean gum galactomannans. A study of the hydrolyzability of α‐d‐galactoside, α‐d‐fucoside and β‐l‐arabinoside showed that the presence of primary alcoholic group in the sugar was important for faster hydrolysis and a stronger binding. The substrate specificity studies indicated the absence of any related glycosidase activity in the α‐galactosidase preparation.
Substituent Effects on the Pre‐Steady‐State Kinetics of Oxidation of Benzyl Alcohols by Liver Alcohol Dehydrogenase
Authors: HARDMAN Michael J. , BLACKWELL Leonard F. , BOSWELL Colin R. , BUCKLEY Paul D.
Published: March 2005

The oxidation of benzyl alcohols by liver alcohol dehydrogenase under conditions [S]0≫ [E]0 is a biphasic process with exponential rise to a steady state. The rate constants for the transient phase were determined by a curve‐fitting procedure.

A large isotope effect was obtained for p‐methoxybenzyl alcohol, indicating that hydrogen transfer is rate‐determining for the transient phase. Rate constants for the hydrogen transfer step, k3, were obtained for the various alcohols and correlated with the Hammett σ constant by plotting log k3 against σ. The slope of this line (ϱ=–0.76) is consistent with rate‐limiting hydride transfer; a mechanism is proposed to account for the low magnitude of ϱ.

The pre‐steady‐state rate constants, kb, for all benzyl alcohols (< 20 s−1) are lower than that for ethanol. 2‐Methylpropan‐1‐ol and cyclohexylmethanol have pre‐steady‐state rate constants of about 150 s−1, indicating that the lower kb values for benzyl alcohols are due to the electron‐withdrawing effect of the benzene ring, rather than to steric effects.
Raman Studies on Native, Reduced, and Modified Basic Pancreatic Trypsin Inhibitor
Authors: BRUNNER Horst , HOLZ Michael , JERING Helmut
Published: March 2005

The method of Raman scattering was applied to determine the effect of chemical modification on the conformation of the basic pancreatic trypsin inhibitor in aqueous solution. Cleavage of one of the three disulfide bonds yields no spectral changes except, of course, the manifestation of the reduction, i.e. a lower intensity of the disulfide band and the appearance of a line due to the newly formed sulfhydryl groups. S‐alkylation, however, produces several changes in the spectra as compared to the native inhibitor. In the case of the carboxymethyl derivative the amide III band, shifted to lower frequency, is particularly significant. This observation suggests that the inactivity towards trypsin of the reduced carboxymethyl inhibitor is the result of structural changes near the reactive centre of the molecule.
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‐XAPhe

When 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.
Thyroglobulin Messenger RNA: Translation of a 33‐S mRNA into a Peptide Immunologically Related to Thyroglobulin
Authors: VASSART Gilbert , BROCAS Huguette , LECOCQ Raymond , DUMONT Jacques E.
Published: March 2005

Poly(U)‐Sepharose chromatography of the RNA extracted from a thyroid fraction sedimenting between 800 ×g and 27000×g allows the purification of two RNA fractions amounting each to 1% of the applied material. The first one is loosely bound to the column from which it is eluted at 25°C. It is mainly composed of 16‐S and 12‐S RNA comprising no poly(A) sequences. This could correspond to mitochondrial rRNA. The second one, which is eluted at 50°C, is poly(A)‐rich and represents 33‐S and 17‐18‐S RNA species. The 33‐S RNA resists heating at 80°C, suggesting that it is composed of one polynucleotide chain. When injected into Xenopus oocytes, the 33‐S RNA specifically promotes the synthesis of a peptide with an apparent molecular weight of 185000 and an apparent sedimentation coefficient of 10‐S. This peptide is immunologically related to thyroglobulin and could represent its main precursor. Under the conditions tested it does not polymerize spontaneously into 19‐S thyroglobulin, suggesting that assembly of the molecule could require specific, post‐translational alterations of the precursor and/or the presence of additional lighter subunits.
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).
Morphogenesis of the Membrane‐Bound Electron‐Transport System in Sporulating Bacillus megaterium KM
Authors: WILKINSON Brian J. , ELLAR David J.
Published: March 2005

The properties of electron transport systems present in soluble and particulate fractions of spores of Bacillus megaterium KM have been compared with those of similar fractions prepared from exponential‐phase vegetative cells of this organism. The timing and localization of modifications of the electron transport system occurring during sporulation have been investigated by using a system for separating forespores from mother cells at all stages during development [8]. Spore membranes contained cytochromes a+a3, b and o at lower concentrations than in vegetative membranes, and in addition cytochrome c, which was not found in exponential‐phase vegetative membranes. An NADH oxidase activity of similar specific activity was found in both spore and vegetative membranes but dl‐glycerol 3‐phosphate and l‐malate oxidase activities were found only in vegetative membranes. A soluble NADH oxidase of low specific activity was found in spores and vegetative cells which probably involves a flavoprotein reaction with oxygen because the activity was stimulated by FAD or FMN and difference spectra of concentrated soluble fractions showed spectra typical of a flavoprotein. Particulate NADH oxidase was sensitive to all classical inhibitors of electron transport tested whereas soluble NADH ocidase was insensitive to many of these inhibitors. Cytochrome c was formed between stage I and II of sporulation and this coincided with a five‐fold increase in NADH‐cytochrome c reductase activity. Forespore membranes had lower contents of cytochromes than sporangial cell membranes but similar levels of NADH and l‐malate oxidases; dl‐glycerol 3‐phosphate oxidase activity could not be detected in either membranes by stage III of sporulation. This characterization of spore electron transport systems provides a basis for suggestions concerning initial metabolic events during spore germination and the effect of a number of germination inhibitors.
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.