bluuebunny/crossref_metadata_2025 huggingface dataset:
The Specificity of Yeast Fatty‐Acid Synthetase with Respect to the “Priming” Substrate
Authors: Pirson Wolfgang , Schuhmann Lieselotte , Lynen Feodor
Published: March 2005

Yeast fatty acid synthetase catalyzes the synthesis of palmityl‐CoA and stearyl‐CoA from acetyl‐CoA and malonyl‐CoA. Acetyl‐CoA serves as “primer” of the reaction cycle. The specificity of the fatty acid synthetase with respect to the “priming” substrate was studied on the purified enzyme.

In its function as primer acetyl‐CoA was replaced by homologous saturated acyl‐CoA derivatives (butyryl‐CoA, capronyl‐CoA, etc.). In these experiments nonanoyl‐CoA and decanoyl‐CoA were good primers in low concentration range. This kind of fatty acid synthesis leads to normal end products.

At higher concentrations of priming substrate an inhibitory effect became apparent with all long‐chain acyl‐CoA derivatives. With decanoyl‐CoA the degree of inhibition was dependent on the concentration of malonyl‐CoA. This was interpreted as competition between decanoyl‐CoA and malonyl‐CoA.

The rate of synthesis with dl‐3‐hydroxydecanoyl‐CoA and Δ2‐decenoyl‐CoA as primer was 25–50 times slower than with decanoyl‐CoA. The rate‐limiting step was the transfer of the acyl residue from CoA to the enzyme. The saturated acids C12–C18 were isolated as end products of this synthesis.

Derivatives of decanoyl‐CoA with substitutions at positions more distant from the carboxyl group were not so strongly depressed in their priming activity, e.g.Δ3‐decenoyl‐CoA or 4‐oxo‐decanoyl‐CoA. In these cases the functional groups were retained during chain elongation. As reaction products unsaturated acids or oxo acids respectively were isolated.

The enzymatic reduction of the double bond (Δ3‐decenoyl‐CoA) or the oxo group (4‐oxo‐decanoyl‐CoA) could be demonstrated only as a slow side reaction.
Endonuclease Activity Associated with the DNA · Protein Complex of Minicircular DNA of Escherichia coli 15
Authors: Messing Joachim , Staudenbauer Walter L. , Hofschneider Peter Hans
Published: March 2005

The DNA · protein complex (form I*) of minicircular DNA of Escherichia coli has associated with it an endonucleolytic activity which converts the supercoiled form I into an open circular form II by introducing one single‐stranded break per molecule. This nick can occur with similar frequency in either strand. Prolonged incubation results in the formation of linear duplex molecules (form III). The endonuclease requires a divalent cation (Mg2+ or Mn2+) and is inhibited by high salt concentration and transfer RNA. It might provide a swivel mechanism during the replication of minicircular DNA.
The Anomalous Absorption of Tryptophan
Authors: ElÖDI Pal , Lakatos Susan
Published: March 2005

The influence of polarizable organic molecules, e.g. organic acids and ionic detergents, on the light absorption of indole, tryptophan and its derivatives was studied. The interaction of these compounds with the chromophore, in addition to the red shift due to solvent perturbation effect, results in an “anomalous” blue shift in the 295 to 310‐nm region.

The nature of spectral changes depended on the character of the interacting solvent and solute molecules: a net positive electrostatic field induced red shift, whereas a negative one caused blue shift. The absorption changes increased with the size of nonpolar part of the polarizable molecule. The spectral changes can be assigned to the electrostatic attraction or repulsion of electrons of the indole nitrogen according to the electrostatic character of the polarizable molecule interacting with the chromophore.

The energy of indole chromophore‐polarizable molecule interaction was estimated from the data of thermal perturbation difference spectra. The calculated upper limit of this interaction energy was 0.04 eV, which suggests that in aqueous solution van der Waals interactions are formed between the chromophore and the compounds containing polarizable or charged groups.
The Effect of Temperature on Gel Formation in Pig‐Gastric Mucus
Authors: Snary David , Allen Adrian , Pain Roger H.
Published: March 2005

The viscosity and gel properties of gastric mucus are due to a mucoprotein of protein content of 18% and molecular weight 2 × 106.

At 25°C the reduced specific viscosity of this mucoprotein increases with increasing concentration until the mucoprotein undergoes a reversible sol‐gel transition at concentrations above 4 mg · ml−1.

For a given concentration of mucoprotein the viscosity increases markedly with temperature and at 45°C the threshold concentration for reversible gel formation is reduced to 2 mg · ml−1.

The intrinsic viscosity corrected for zero shear, the shear dependence of the viscosity, and the sedimentation properties of the mucoprotein as a function of temperature have been measured.

These experiments show that the increase of viscosity with temperature is due to increased intermolecular interaction rather than structural changes in the molecule.

The temperature dependence is characteristic of hydrophobic interactions and the results are interpreted to show that the mechanism of gel formation in gastric mucus involves micelle junctions.
The Conformational Transitions in Yeast tRNAPhe as Studied with tRNAPhe Fragments
Authors: Riesner Detlev , Maass Günter , Thiebe Rainer , Philippsen Peter , Zachau Hans G.
Published: March 2005

Conformational transitions of the anticodon branch, the acceptor branch and the tertiary structure of yeast tRNAPhe were investigated using tRNAPhe fragments. Thermodynamic and kinetic methods were employed using the optical absorbance of the fragments and the fluorescence of the Y‐base. Related results of partial nuclease digestions on tRNAPhe fragments are also reported.

Hypochromicity, reaction enthalpy and rate constants of helix‐coil transitions of the anti codon branch agree with the values expected from the cloverleaf model. The dissociation of the branch is accompanied by a quenching of the fluorescence of the Y‐base.

In some isolated fragments the anticodon branch is more stable than in the intact tRNA. The destabilization in the intact tRNA is attributed to electrostatic effects.

The anticodon half Phe 21–57, at low concentrations and temperatures, assumes a structure which is different from a segment of the cloverleaf model. A transition from this structure to the one of a segment of the cloverleaf is observed with increasing temperature. At higher fragment concentrations and in the presence of Mg2+ dimers are formed.

Bimolecular recombinations are observed between fragments Phe 1–18 and Phe 21–76 reconstituting the acceptor and dihydrouridine stems, and between Phe 1–18 and Phe 38–76 where only the base pairs of the acceptor stem are newly formed. The mechanism of the recombination and the relationship of the fragment combination to the intact tRNA are discussed.

The thermodynamic properties of the fragment Phe 1‐69/70 are very similar to those of the intact tRNA. It can be concluded that the tertiary structure is essentially unchanged anti that the terminal nucleotides 70–76 are not directly involved in its formation.

The thermal denaturation process of intact tRNAPhe can now be interpreted as follows: at first the tertiary structure is converted to a cloverleaf‐like structure; this process is followed by the melting of the acceptor and anticodon stems; finally the ribosylthymine and dihydrouridine stems dissociate independently.
Purification and Molecular Properties of an Unspecific Carboxylesterase (E1) from Rat‐Liver Microsomes
Authors: Arndt Rüdiger , Heymann Eberhard , Junge Wolfgang , Krisch Klaus , Hollandt Heinrich
Published: March 2005

The isolation of an unspecific carboxylesterase from rat liver microsomes is reported. The purification is 66‐fold at a yield of about 20%. The enzyme is one of 5 esterase/amidase variants recently separated and corresponds to the fraction designated E1.

The enzyme is homogeneous in disc electrophoresis, dodecylsulfate‐polyacrylamide gel electrophoresis and in analytical ultracentrifugation, but exhibits slight heterogeneity in isoelectric focusing.

Its amino acid composition is presented.

The molecular weight of rat liver esterase E1 is 177000 (equilibrium sedimentation), the subunit weight 61 500 (dodecylsulfate disc electrophoresis), and the equivalent weight 66000 (titration with bis[p‐nitrophenyl]phosphate). After cross‐linking of the enzyme by dimethyl suberimidate and dodecylsulfate‐polyacrylamide gel electrophoresis three principal bands are observed. Therefore, a trimeric structure in which each subunit possesses one active site is proposed.
Catalytic Properties of an Unspecific Carboxylesterase (E1) from Rat‐Liver Microsomes
Authors: Arndt Rüdiger , Krisch Klaus
Published: March 2005

In a study on the influence of the alkyl and acyl chain length of aliphatic esters on kinetic parameters of rat liver carboxylesterase (E1) it was found that V is mainly governed by the length of the acyl residue, whereas on elongation of the alcohol group pKm is increased.

By halogen substitution of ethyl acetate in the α‐position of the acetate, the pKm values are increased in the order F < Cl < Br < I.

Besides aliphatic esters, also aromatic ones, such as l‐tyrosine ester, procaine, p‐nitrophenyl acetate and, as an aromatic amide, butacetoloid (hostacaine®) are hydrolyzed.

The pH‐profile was found to depend on the nature and concentration of the substrate.

In contrast to the results obtained with the purified enzyme, no substrate inhibition and a different pH‐profile were observed on incubation of rat liver microsomes with methyl butyrate.
Hydrolysis of Amino‐Acid Esters by Pig‐Pancreatic Kallikrein
Authors: Fiedler Franz , Leysath Gisela , Werle Eugen
Published: March 2005

Initial velocities of the hydrolysis of α‐N‐acylated esters of 14 different amino acids catalyzed by pig pancreatic kallikrein were determined. Control experiments with kallikrein partially inactivated by controlled treatment with diisopropylphosphorofluoridate demonstrated the enzymic homogeneity of the preparation and the ability of kallikrein to hydrolyse (at decreasing rates) esters of basic, aromatic and neutral aliphatic amino acids, though being predominantly specific for l‐arginine esters. The controversial literature on the substrate specificity of the enzyme has its origin not in the molecular heterogeneity of kallikrein, but in a contamination with other enzymes. Kinetic constants for the kallikrein‐catalyzed hydrolysis of several of the esters have been determined and compared to those of trypsin and clostripain. The preference of kallikrein for an arginine over a lysine ester is still higher than with the latter enzyme. In the α‐N‐benzoylated as well as in the α‐N‐tosylated series, esters of l‐arginine with different alcohol moieties exhibit not only widely different Km, but also Kcat values. The latter parallel the rates of the hydroxyl‐ion‐catalyzed hydrolyses of the α‐N‐benzoyl‐l‐arginine esters which have also been determined.

Syntheses of α‐N‐benzoyl‐l‐lysine methyl ester hydrochloride and of α‐N‐benzoyl‐l‐ornithine methyl ester hydrochloride are described. A value for the rate constant of the hydroxyl‐ion‐catalyzed lactamization of the ornithine ester has been obtained.
Sexual Differences in Hepatic Sulphurylation of Deoxycorticosterone in Rats
Authors: Carlstedt‐Duke Jan , Gustafsson Jan‐Åke
Published: March 2005

The sulphurylation of [1,2‐3H]deoxycorticosterone was studied in the 105000 ×g supernatant fluid obtained from rat liver homogenates. The sulphurylating activity in newborn rats was high; it was considerably lower in rats 7 days of age. Between 7 and 28 days of age the sulphurylating activity did not change and it was the same in male and female rats. From about 28 days of age until the rats were about 56 days of age the sulphurylating activity increased in female rats but decreased in male rats so that finally female animals sulphurylated about five times more efficiently than male animals (P < 0.001). Neonatal or postpubertal ovariectomy did not influence the hepatic sulphurylating activity in the adult female rats. Neonatal testectomy led to complete feminization of the sulphurylating activity in the adult male rats whereas postpubertal testectomy led to an increase in the hepatic sulphurylation but not complete feminization. Administration of testosterone propionate to postpubertally castrated male and female rats suppressed the hepatic sulphurylating activity in both sexes. Administration of oestradiol benzoate did not affect the sulphurylating activity in female rat liver but feminized this activity in male rat liver. The conclusions drawn from all these findings are that the sulphurylating activity in female rats is normally regulated by nongonadal factors and that the sulphurylating activity in male rats is suppressed by testicular androgens in two ways; by irreversible “imprinting” or programming of the liver during the neonatal period, and by reversible suppression postpubertally. The effects of oestradiol benzoate treatment are probably best explained by “de‐imprinting” of the male liver back to the basal “feminine” character with respect to sulphurylating activity.
The Effect of Fluoride on the Spectral and Catalytic Properties of Three Copper‐Containing Oxidases
Authors: BrÄNDÉN Rolf , MalmstrÖM Bo G. , Vänngård Tore
Published: March 2005

The optical spectra of fungal laccase, lacquer tree laccase and ceruloplasmin are changed in a similar way when F– is added to the proteins at pH 5.5.

A change around 320 nm is associated with the binding of F– to type 2 Cu2+ of the proteins as seen from a correlation with electron paramagnetic resonance spectra.

The rate of binding of F– to fungal laccase is first order with respect to uncomplexed enzyme and independent of the F– concentration, indicating that an intramolecular process is rate limiting. The stability constant of the enzyme · F– complex is greater than 10 μM‐1.

Fungal laccase is almost completely inhibited when 1 equivalent of F– is bound to the type 2 Cu2+. Presence of substrate gradually decreases the inhibition, due to the release of F– from a reduced form of the enzyme.
The Mechanism of Action of Methionyl‐tRNA Synthetase
Authors: Blanquet Sylvain , Petrissant Guy , Waller Jean‐Pierre
Published: March 2005

The interactions of three methionine‐specific tRNAs (initiator tRNAfMet from rabbit liver, cross‐linked tRNAfMet and untreated tRNAfMet from Escherichia coli) with methionyl‐tRNA synthetase from E. coli were examined. Binding parameters determined by fluorescence spectroscopy were compared with kinetic parameters derived from the aminoacylation reaction performed under comparable experimental conditions.

Cross‐linked tRNAfMet and untreated tRNAfMet are virtually indistinguishable on the bases of their kinetic parameters in the aminoacylation reaction, but can be differentiated by fluorescence measurements. The lower level of enzyme fluorescence quenching elicited by the cross‐linked species must be related to a structural modification which has no significant repercussion on its performance in the aminoacylation reaction.

The initiator tRNA from rabbit liver differs from that of E. coli in several respects. Its binding parameters, measured by fluorescence, are characterized by a two‐fold higher Ka, a two‐fold lower maximal level of enzyme fluorescence quenching and a less pronounced sensitivity to the addition of KCl. The lower sensitivity to KCl is also reflected in the kinetic parameters measured in the aminoacylation reaction.

Several unspecific tRNAs which are not aminoacylated by the enzyme under standard conditions are able to interact with it to significant extents, as assessed by fluorescence measurements. These interactions are characterized by weak affinities and small effects on protein fluorescence, comparable in magnitude to those observed when specific tRNAs interact with the enzyme in the absence of magnesium ions.
The Synthesis and Stability of Ribosomal RNA in Blue‐Green Algae
Authors: Grierson Donald , Smith Harry
Published: March 2005

The ribosomal RNAs of the blue‐green algae Tolypothrix distorta, Anabaena cyclindrica and Nostoc muscorum have average molecular weights (Mr) of 1.1 × 106 and 0.56 × 106 as determined by acrylamide gel electrophoresis. The larger‐molecular‐weight rRNA is unstable and breaks down to yield a fragment of Mr approximately 0.9 × 106. Pulse‐labelling experiments show that no single high‐molecular‐weight polycistronic precursor to the rRNAs can be detected. The rRNA with Mr 1.1 × 106 is synthesized via a precursor molecule of Mr approximately 1.22 × 106, whilst there are apparently two sequential precursors to the rRNA with Mr 0.56 × 106 having Mr 0.76 × 106 and 0.68 × 106. The DNA of all the blue‐green algae was fractionated into two components on the gels. The significance of this behaviour is not yet understood.
Small‐Angle X‐Ray Scattering Studies on Virus‐Like Protein Particles Derived from Bacteriophage fr
Authors: Zipper Peter , Schubert Dieter , Vogt Jürgen
Published: March 2005

Small‐angle X‐ray scattering and analytical ultracentrifugation were applied to solutions of virus‐like protein particles prepared from bacteriophage fr by alkaline degradation of the phage (artificial top component) as well as by self‐assembly of the monomeric protein subunits in the absence of nucleic acid. The artificial top component sediments with 42 S, while the reaggregated protein particles were found to sediment on the average with 45.4 S. The radial dimensions of the artificial top component are practically identical to those of the protein shell of the virus, the inner radius being 10.6 nm and the outer radius being 13.2 nm. The same outer radius was obtained also for the reaggregated protein particles, except for one out of four samples which showed slight remainders of multishells on the outer surface. For the other samples, the molecular weight was found to be higher than that of the artificial top component by about 8%, whereas the radius of gyration was smaller by about 3.6%. From these results and from the radial net electron density distribution, it is concluded that the reaggregated protein particles contain on the average 14.4 additional protein subunits located in their interior.
Sequential Modifications of DNA‐Dependent RNA Polymerase during Sporogenesis in Bacillus thuringiensis
Authors: Klier André F. , Lecadet Marguerite‐M. , Dedonder Raymond
Published: March 2005

A purification procedure of DNA‐dependent RNA polymerase from vegetative and sporulating cells of Bacillus thuringiensis is described. The subunit composition of the vegetative enzyme has been established as β′βσαa2. It has been shown that two active forms of the enzyme are present in sporulating cells; for one of them the modification of a β subunit has been observed. The relative amounts of the two polymerases vary throughout the sporulation process. Changes in template specificity and results from chromatography on phosphocellulose of purified enzymes suggest an early modification of σ factor for the two forms. Subunit composition of spore polymerase is also given: the disappearance of α subunits is reported, while two new subunits of lower molecular weights are present. Results are summarized in a scheme giving the kinetics of these modifications.
Purification and Some Properties of ATP Citrate Lyase from Penicillium spiculisporum
Authors: Måhlén Anders
Published: March 2005

ATP citrate lyase occurs in large quantities in Penicillium spiculisporum. It has been purified 25 fold to a specific activity of 3.6 U/mg. At optimal concentrations of the other substrates apparent Km values are: citrate, 180μM; ATP, 90 μM and CoASH 1–3 μM. The enzyme is Mg2+ dependent, but Mn2+ and Co2+ can also be used. 1,N6‐Etheno‐adenosine triphosphate is active as a substrate with a Km of 180 μM, but 1,N6‐etheno‐coenzyme A is not. 2‐Decylcitric acid is not a substrate for the enzyme. ATP citrate lyase has a molecular weight of above 200000. The amount of ATP citrate lyase in Penicillium spiculisporum is such as to support the hypothesis that citrate is a precursor of fatty acids in the organism.
Transcription in vitro Using Mixtures of Ethylene Glycol and Water
Authors: Brody Edward N. , Leautey Josette
Published: March 2005

We have analyzed transcription in vitro in mixtures of ethylene glycol and water. We find two major effects of this solvent on T4 transcription, both of which are reversible on dilution out of the solvent: (a) only very short (10% normal length) T4 chains are formed; (b) a small percentage of initiation errors are made; these include initiation on the “wrong” strand and initiation on the “wrong” site of the correct strand. The ratio of GTP to ATP initiation is also increased in this solvent and it may be that all faulty initiation is done with GTP. This faulty initiation seems to derive, Like normal initiation, from a σ‐dependent reaction on native T4 DNA templates, and is thus distinguished from that faulty initiation which derives from core synthesis on damaged or denatured templates. Analysis of the products of T4 transcription in vitro in ethylene glycol suggests that this reagent has a different effect on RNA chains synthesized from promoter sequences than it does on chains initiated from non‐promoter sequences. Those chains which start at promoters show a very strong asymmetry, while those chains which start from non‐promoter sequences are completely symmetrical despite their σ‐dependence.

Transcription of poly(dG) · poly(dC) and poly(dA) · poly(dT) is relatively little changed in 30% ethylene glycol. The synthesis of short poly(G) chains with frequent terminations and re‐initiations takes place in this solvent just as it does in aqueous buffers. The selective synthesis of polypurines from these templates is not perturbed, even in 40% ethylene glycol.

The inherent asymmetry of promoters in ethylene glycol is considered from the point of view of conformational changes undergone by DNA and homopolymers in ethylene glycol solutions.
The Computation of Parameters in Isotopic Tracer Systems not in a Steady‐State
Authors: Ottaway James H. , Mowbray John
Published: March 2005

Methods of computing estimates of the variable parameters of a tracer kinetic experiment in which the chemical pools were not all in a steady‐state, by means of a function minimisation (optimisation) program coupled to a numerical integration procedure, have been discussed. It has been shown that if the transport rates depend non‐linearly on the concentrations of chemicals, or are independent of their concentrations, the rate constants (fractional turnover rates) of both chemical and tracer compartments may vary with the concentrations of the chemicals to which they refer, and this dependence may show up in the experimental record as a time‐dependence. This variation has nothing to do with changes in the values of the underlying enzyme or permease parameters. Empirical methods of searching for such time‐ or substrate‐dependence have been discussed.

It has also been demonstrated that when tracer movements are formulated in terms of the total radioactivities and rate constants (dimensions, min−1), a parallel formulation of the chemical fluxes should be processed, since the formulation of the tracer in terms of total radioactivities does not specify mathematically any constancy of the chemical pools, even where it exists. Failure to take this precaution may lead to very unsatisfactory estimates of the chemical fluxes.
Xanthine Accumulation during Hypoxanthine Oxidation by Milk Xanthine Oxidase
Authors: JeŻEWSKA Maria M.
Published: March 2005

Accumulation of xanthine as well as uric acid, was found as a result of hypoxanthine oxidation by xanthine: oxygen oxidoreductase from milk. The ratio xanthine/uric acid increases with hypoxanthine concentration, and decreases with the enzyme concentration and the reaction time. The methodical consequences of the xanthine accumulation during hypoxanthine oxidation are discussed. The possibility of regulation of the xanthine/uric acid ratio in the cell by changes of enzyme and/or hypoxanthine levels is suggested.
Studies on the Mechanism of Breakdown of Polyribosomes from Rat Liver
Authors: Bont Willy S. , de Vries Martha , Huizinga Anneke , Mekkelholt Koja
Published: March 2005

[14C]Leucine was incorporated into a cell‐free rat liver system without adding the 19 other non‐radioactive amino acids. The incorporation was practically not influenced by low amounts of cycloheximide or RNAase treatment. All the radioactivity could be recovered as peptidyl‐tRNA even after exhaustive pronase treatment.

About 50% of polyribosomes were broken down into material with a sedimentation coefficient of 59 S and 70 S under the following conditions: (a) by incubation in the complete amino‐acid‐incorporation system but at Mg2+ concentrations that were so low that virtually no incorporation of amino acids took place. (b) when incubation was performed with a purified preparation of elongation factors and with GTP as the only form of energy and (c) when ribosomal monomers of the RNAase type were used instead of fresh polyribosomes in the complete amino‐acid‐incorporation system.

Breakdown of polyribosomes practically did not take place at optimal (8 mM) or higher Mg2+ concentration when amino acid incorporation was appreciable.

From these experiments the following conclusions were reached. (a) 50% of the synthetically active ribosomes isolated from rat liver have their peptidyl‐tRNA in the acceptor site and 50% have it in the donor site: (b) The process of run‐off in the cell‐free rat liver system at sub‐optimal Mg2+ concentrations does not require the sequential release of ribosomes from the mRNA strand at the end of the read‐out process. (c) In our cell‐free system, a maximum of three amino acids per ribosome could be incorporated, corresponding to a maximum of two translocations per ribosome; run‐off took place during one of these translocations.
The Duck‐Globin Messenger‐Ribonucleoprotein Complex
Authors: Morel Carlos , Gander Eugen S. , Herzberg Max , Dubochet Jacques , Scherrer Klaus
Published: March 2005

The duck globin messenger ribonucleoprotein complex which contains the biologically active globin mRNA and two major protein components associated with it, has been further characterized.

When analysed on gradients of increasing ionic strength, the mRNA · protein particle showed resistance to 0.25 M LiCl, a condition which fully dissociates artificial RNA · protein complexes, and was partially resistant to 0.75 M LiCl.

By polyacrylamide gel electrophoresis the particle was shown to consist of one major component A and two minor components B and C of increasing mobility. Component A and C were ribonucleoproteins, B consisted of free proteins; no free RNA could be detected.

Component A, when analysed on dodecylsulfate gels contained all the proteins of the complex: the two major bands of molecular weight 49000 and 73000, diffuse minor bands in the 52000 to 64000 molecular weight range and three more sharp bands in the 86000 to 120000 range. Components B and C contained only some of the protein bands. It is concluded that the composition of the mRNA protein particle is more complex than was originally thought.

The protein bands with molecular weights of 73000, 64000 and 49000 were found to be phosphorylated, containing phosphoserine.

Using the method of dark‐field electron miroscopy, we were able to resolve for the first time internal structural details of the mRNA · protein complex and of the mRNA. The proteins seem to be attached all along the mRNA chain in 4 to 7 distinct blobs, probably in regions of high secondary mRNA structure.

Taken together, these results strongly suggest that mRNA · protein particles are not artefacts formed during cell lysis and probably have a biological significance.
Synthesis in vitro of High‐Molecular‐Weight RNA by Isolated Euglena Chloroplasts
Authors: Carritt Ben , Eisenstadt Jerome M.
Published: March 2005

Chloroplasts isolated from photoheterotrophically grown Euglena gracilis incorporate [3H]UTP into RNA. This incorporation is sensitive to DNAase, RNAase and actinomycin D and requires the presence of magnesium and the other three nucleoside triphosphates. Analysis of the products of the reaction revealed that this incorporation is due to elongation of RNA molecules, resulting in peaks of newly synthesized RNA coincident with the 23 S and 16 S chloroplast ribosomal RNAs. These findings are consistent with chloroplast ribosomal RNA being synthesized in vitro using chloroplast DNA as a template.
Hydrodynamic Properties of the Sucrase Isomaltase Complex from Rabbit Small Intestine
Authors: Mosimann Hugo , Semenza Giorgio , Sund Horst
Published: March 2005

From the hydrodynamic properties of the sucrase · isomaltase complex at low ionic strength, a molecular weight of 221000 was calculated. The complex dimerizes at high ionic strength.

Under denaturing conditions the complex dissociates into two subunits of identical or almost identical molecular weight (112000) which are presumably composed of one polypeptide chain each.

The isomaltase subunit, isolated in enzymatically active form, has a molecular weight of 113000.
Application of the Induced‐Transport Test to the Mechanism of Pig‐Kidney Phosphoglyceromutase
Authors: Britton Hubert G. , Carrears José , Grisolia Santiago
Published: March 2005

The induced transport test has been used to study the mechanism of pig‐kidney phosphoglyceromutase (2,3‐bisphospho‐d‐glycerate: 2‐phospho‐d‐glycerate phosphotransferase). At intermediate substrate concentration (0.5 mM) there was cotransport of 32P‐labelled substrates and no induced transport of 14C‐labelled substrates. These findings, excluded an intramolecular transfer of phosphate or an exchange of phosphate between two or more molecules of substrate, but were quantitatively in accord with a phosphoenzyme (ping pong) mechanism with a rapid isomerisation of the “phosphoenzyme”; or with an intermolecular transfer of phosphate from 2,3‐bisphosphoglycerate to the substrates (the sequential mechanism) if there were a slow isomerisation of the enzyme · bisphosphoglycerate complex. Similar results at low substrate concentrations (9.5 μM) however excluded the latter possibility since it would have required a Km for 3‐phosphoglycerate of less than 1.9 μM compared with a measured value of about 270 μM. The enzyme therefore has a phosphoenzyme mechanism. A lack of induced transport with 14C‐labelled substrates at high substrate concentrations indicated a rate constant for isomerisation of the “phosphoenzyme” in excess of ∼ 106s−1 and the absence of induced transport with 14C‐labelled substrates over a wide concentration range excluded certain types of interaction between active centres. To explain the extremely rapid isomerisation of the phosphoenzyme a modified mechanism is proposed in which there is no formal isomerisation of the phosphoenzyme. The nature of the “phosphoenzyme” is discussed and it is suggested that it is probably a true phosphoenzyme with the phosphate covalently bound to the protein. The possible evolutionary relationship of this mechanism to that of a 2,3‐bisphosphoglycerate phosphatase is discussed.
The Suitability of Bovine‐Lens Leucine Aminopeptidase for Sequence Analysis and Limited Hydrolysis of Polypeptides
Authors: Wiederanders Bernd , Lasch Jürgen , Kirschke Heidrun , Bohley Peter , Ansorge Siegfried , Hanson Horst
Published: March 2005

In the present paper we report findings showing the suitability of recrystallized bovine‐lens leucine aminopeptidase as a tool for peptide chemistry.

The enzyme is homogeneous with respect to hydrodynamic, chromatographic and electrophoretic separation criteria. It is not contaminated by endopeptidases.

The insulin B‐chain (S‐sulfonate) served as model substrate for sequence studies. The initial progress curves of the liberation of the first six amino acids resulted in a sequence expected from the known primary structure. The complete resistance of imido bonds towards the action of leucine aminopeptidase has been proved.

Many commercially available protein preparations are found to be contaminated by endopeptidases. We developed an endopeptidase‐detection method in which the leucine aminopeptidase serves as a sensitivity amplifier.
Formation of the Yeast Mitochondrial Membrane
Authors: Kim In‐Cheol , Beattie Diana S.
Published: March 2005

The kinetics of formation of different enzyme complexes of the inner mitochondrial membrane were studied in yeast undergoing derepression during growth in 5% glucose. Each different enzymic activity started to increase at a unique time in the growth curve, increased at a characteristic rate and reached a maximum at a different time. For example, oligomycin‐sensitive ATPase activity increased from the earliest time studied (7 h) reaching a maximum at 17 h which corresponds to early stationary phase. Succinate dehydrogenase and succinate‐cytochrome c reductase activities increased throughout the 24‐h growth period with no appreciable lag. In contrast, a lag of 13 h occurred before NADH dehydrogenase and NADH‐cytochrome c reductase activities started to increase. NADH‐dehydrogenase activity reached a maximum at 17 h, while the activity of NADH‐cytochrome c reductase increased throughout the growth period. The last enzymic activity to be derepressed was cytochrome oxidase which did not start to increase until about 14 h of growth and then continued to increase throughout the growth period. These results suggest that the various enzyme complexes of the mitochondrial membrane are assembled into the membrane in an asynchronous process.

The inhibitors of protein synthesis, cycloheximide and chloramphenicol, were added at different times during the growth cycle. The appearance of succinate and NADH dehydrogenases, as well as succinate‐cytochrome c reductase was completely blocked by the addition of cycloheximide at all times but unaffected by chloramphenicol suggesting that these enzymes do not contain products of mitochondrial protein synthesis. The increase in ATPase activity was completely inhibited by the addition of both inhibitors at all times indicating that formation of a functional ATPase complex requires the concomitant synthesis of proteins on both sites. Addition of chloramphenicol to repressed cells blocked the appearance of both cytochrome oxidase and NADH‐cytochrome c reductase but activity of these enzymes continued to increase for several hours after addition of cycloheximide at this time. These results suggest that synthesis of mitochondrial proteins on cytoplasmic ribosomes may precede that on mitochondrial ribosomes. When either inhibitor was added to partially derepressed cells, activity of both cytochrome oxidase and NADH‐cytochrome c reductase increased for several hours and then ceased suggesting that mitochondrial proteins synthesized both in the cytoplasm and in the mitochondria accumulate to a limited extent prior to integration into a functional unit in the membrane.
Ultracentrifuge Studies on Binding of Aliphatic Amino Acids to Isoleucyl‐tRNA Synthetase from Escherichia coli MRE 600
Authors: Flossdorf Josef , Kula Maria‐Regina
Published: March 2005

An ultracentrifuge technique has been used to investigate the binding of aliphatic C1 to C6α‐amino acids to isoleucyl‐tRNA synthetase from Escherichia coli MRE 600. The synthetase was found to have an absolute requirement for l‐isomers, but would accept a range of side‐chain structures. l‐Isoleucine was bound best, followed by the lower homologues l‐valine and l‐nor‐valine; the isomers of l‐isoleucine are poorly bound, as is α‐amino‐n‐butyric acid, glycine, l‐alanine, and d,l‐tert.‐leucine are inert. The method is applicable for investigating interactions of small ligands with macromolecules generally.
ATP‐Controlled Fatty‐Acid Synthesis in Cultured Arterial Fibroblasts
Authors: FillipoviĆ Ivan , Kresse Hans , Buddecke Eckhart
Published: March 2005

Cultured arterial fibroblasts synthesize fatty acids from [1‐14C]acetate, mainly by de novo synthesis, when incubated under a gas phase of air/CO2. In hypoxia (gas phase N2/CO2) or aerobically in the presence of 2,4‐dinitrophenol the mode of fatty acid synthesis changes to the chain elongation mechanism as indicated by an increased 14C‐radioactivity in the carboxyl carbon of fatty acids.

The ATP/ADP ratio of aerobically incubated fibroblasts is diminished in anaerobiosis as well as in dinitrophenol experiments, whereas the NADH/NAD ratio rises only in anaerobiosis, thus suggesting that the mode of fatty acid synthesis in fibroblasts is controlled by the ATP/ADP ratio rather than by the redox state of NAD.
Magnetic‐Resonance Studies of the Interactions between Bovine‐Serum Albumin and Surfactants
Authors: Oakes John
Published: March 2005

The interactions between bovine serum albumin and surfactants at pH 7 have been studied by nuclear magnetic resonance and spin‐labelling techniques. When a surfactant binds to a protein the molecular motion of all the alkyl groups of the surfactant is restricted, this effect being greater for the CH2 near the head groups than for the CH3 group and the intermediate CH2 groups. The CH2 group adjacent to the head group is in a more polar environment than the other CH2 groups. Hence although hydrophobic bonding may account for a significant portion of the binding free energy, electrostatic forces are also important and may determine the site of the interaction.
Magnetic‐Resonance Studies of the Interactions between Bovine‐Serum Albumin and Surfactants
Authors: Oakes John , Cafe Michael C.
Published: March 2005

The interactions between bovine serum albumin and surfactants at pH 7 have been studied by electron spin resonance using bovine serum albumin spin labelled at specific sites. The spectrum consists of a superposition of two spectra, one sharp and the other broad, due to labelled amino groups at different sites on the protein, the former being free to tumble quite rapidly, whereas the latter have much less freedom of motion. On denaturation of bovine serum albumin by addition of surfactants, urea or at high pH there is a marked increase in intensity and change in the line‐width of the sharp spectrum which indicates an unfolding of the protein molecule. The decrease in signal intensity and increase in line‐broadening observed at low pH and ionic strength 0.1 supports the evidence that protein aggregation occurs under these conditions.
Amino‐Acid Sequences of Red‐Deer and Roe‐Deer Pancreatic Ribonucleases
Authors: Zwiers Henk , Scheffer Albert Jan , Beintema Jaap J.
Published: March 2005

Pancreatic ribonucleases from red deer (Cervus elaphus) and roe deer (Capreolus capreoluus) have been isolated and the amino acid sequences of both enzymes have been determined. Red deer ribonuclease exists only in a carbohydrate‐free form. Roe deer ribonuclease has been isolated both in a carbohydrate‐free form and in a glycosidated form. The difference in glycosidation between the enzymes from both deer species could be explained by the substitution of an asparagine at position 34 (the carbohydrate attachment site in the glycosidated form of roe deer ribonuclease) by lysine in the red deer enzyme. Both enzymes also differ at position 35 (methionine in red deer, leucine in roe deer). Roe deer ribonuclease exhibits heterogeneity in having both alanine and isoleucine at position 64. All other amino acid positions are occupied by identical residues in both enzymes. Red deer ribonuclease differs at 19 positions from bovine ribonuclease.

A value of 1% substitution per 3.106 year has been calculated for the rate of molecular evolution of mammalian pancreatic ribonucleases from 7 amino acid sequences determined to date.
Properties of a Yeast Cytochrome P‐450‐Containing Enzyme System which Catalyzes the Hydroxylation of Fatty Acids, Alkanes, and Drugs
Authors: Duppel Wilfried , Lebeault Jean‐Michel , Coon Minor J.
Published: March 2005

The growth of Candida tropicalis on tetradecane causes the induction of a cytochrome P‐450‐containing enzyme system which catalyzes the hydroxylation of fatty acids, hydrocarbons and drugs. When the cells are broken by treatment with a French pressure cell, the cytochrome P‐450 is obtained in an apparently soluble form. The enzyme system was resolved into three components: cytochrome P‐450, NADPH‐cytochrome P‐450 reductase, and a heat‐stable lipid fraction, all of which are necessary, along with NADPH and molecular oxygen, for the conversion of laurate to ω‐hydroxylaurate. NADH alone is almost completely inactive but causes a doubling of the activity when present along with a saturating level of NADPH. The yeast reductase and lipid fractions may be replaced by corresponding fractions obtained from rat liver microsomes. The effect of various phospholipids on the hydroxylation activity was investigated, and a yeast lysophosphati‐dylethanolamine fraction was shown to be most effective as judged by laurate hydroxylation. The yeast cytochrome P‐450 is not readily autoxidizable, as shown by experiments in which it was reduced extensively by NADPH in the presence of phospholipid and the reductase under aerobic conditions.
The Mechanism of Enzymatic Cellulose Degradation
Authors: Berghem Lars E. R. , Pettersson L. Göran
Published: March 2005

A cellulolytic enzyme (“C1” enzyme) has been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus Trichoderma viride.

The purification method is a four‐step procedure including chromatography on Bio‐Gel P‐10, DEAE‐Sephadex chromatography, isoelectric focusing and chromatography on Bio‐Gel P‐60.

A yield of 144 mg enzyme was obtained per 100 g commercial cellulase.

The isolated enzyme was homogeneous in polyacrylamide gel electrophoresis at pH 5.0 and at pH 8.0 by isoelectric focusing in a polyacrylamide gel and also in the ultracentrifuge.

No enzyme activity towards carboxymethylcellulose could be detected in the purified material under the assay conditions used. Similarly, there was no β‐glucosidase activity.

The purified enzyme was associated with 3.3% carbohydrate and is assumed to be a glycoprotein. The enzyme was isoelectric at pH 3.79 (10°C). A molecular weight of 46000 was determined by chromatography of the reduced and alkylated enzyme on a calibrated column of Sepharose 6B in 6 M guanidine‐HCl.

Crystalline cellulose (Avicel), phosphoric acid‐swollen Avicel and cellotetraose were degraded by the enzyme and in each case the principle reaction product was cellobiose.

Evidence indicates that the purified enzyme is a β‐1,4‐glucan cellobiohydrolase.
Purification and Characterization of Adenosine Triphosphate: Ribonucleic Acid Adenyltransferase from Escherichia coli
Authors: Sippel Albrecht E.
Published: March 2005

A primer‐dependent poly (A)‐polymerizing activity using ATP as substrate (ATP: RNA adenyltransferase) was isolated in high yield from Escherichia coli and purified to apparent homogeneity. For this purpose an assay system had to be used which restricted a variety of infering enzyme activities. Since the enzyme aggregates to macromolecular cell components or precipitates when kept in low salt conditions (<0.4 M NaCl), it was necessary to perform the entire purification procedure in high salt conditions. This was accomplished by using a high salt ribosomal supernatant, a polyethylenglycol‐dextran‐NaCl phase partition step and a very efficient final high salt phosphocellulose chromatography. At 0.5 M NaCl the enzyme has a molecular weight of approximately 58000. Dodecylsulfate gel electrophoresis shows a single polypeptide chain of molecular weight × 50000. The enzyme has a high preference for ATP as substrate. Manganese ions show higher activity as cofactors than magnesium ions. All classes of natural RNAs are used as primers. The free 3′terminal hydroxyl group of the RNA is required. The enzyme is unable to catalyze a pyrophosphorolysis or a phosphorolysis reaction. It is shown that ATP: RNA adenyl‐transferase preferentially synthesizes rather long chains of poly(A) attached to the RNA primers. No relation between the enzyme protein and subunits of the DNA‐dependent RNA polymerase could be detected.
Lipid Peroxidation of Rat‐Liver Microsomes
Authors: Högberg Johan , Bergstrand Anders , Jakobsson Sten V.
Published: March 2005

Rat liver microsomes were peroxidized in vitro and chemical, physical and morphological changes in the membrane were related to the effects on certain membrane‐bound microsomal enzymes.

With increasing peroxidation, microsomal phospholipids revealed decreasing concentrations of 20:4 and 22:6 fatty acids. At a high level of peroxidation, where alterations in permeability and ultrastructural changes such as breakage and deformation of microsomal vesicles were apparent, there was also a decrease in the concentration of 18:2 fatty acids.

Glucose‐6‐phosphatase, cytochrome P‐450 and uridine‐5′‐diphosphate glucuronyltransferase (UDP glucuronyltransferase) (three microsomal enzymes known to be highly dependent on the integrity of the membrane for their function) responded differently to lipid peroxidation. Glucose‐6‐phosphatase showed an initial decrease in activity as well as in Km for glucose 6‐phosphate, followed by a restoration to control values at an intermediate level of lipid peroxidation. At a high level of peroxidation, both kinetic parameters were again markedly decreased. Cytochrome P‐450 concentration, on the other hand, revealed a continuous decrease with proceeding lipid peroxidation, which was only partly accounted for by a conversion to cytochrome P‐420. The loss of cytochrome P‐450 was paralleled by decreased rates of aminopyrine demethylation and 3,4‐benzpyrene hydroxylation. Glucuronyltransferase activity, finally, was activated at low, but again normalized at high, levels of lipid peroxidation.

It thus seems that although electron‐microscopically observable changes in the microsomal membrane occurs only late in lipid peroxidation, chemical, physical and enzyme alterations are early phenomena during this process. The continuous loss of cytochrome P‐450 and the final decrease in glucose‐6‐phosphatase late during peroxidation probably reflect damage to membrane lipids of vital importance for the integrity and function of these enzymes whereas the activation of UDP glucuronyltransferase early during peroxidation most likely can be ascribed to increased membrane permeability.
Pancreatic Lipase and Co‐Lipase
Authors: Borgström Bengt , Erlanson Charlotte
Published: March 2005

The effect of conjugated bile salts on the activity of pancreatic lipase depends on their concentration in relation to their critical micellar concentration.

Below the critical micellar concentration, conjugated bile salts slightly stimulate the initial rate of hydrolysis of tributyrine, an effect that may be caused by a protection of the enzyme from inactivation at the substrate‐water interface.

Above the critical micellar concentration, conjugated bile salts almost completely inhibit lipase. The inhibition is more marked in alkaline reactions resulting in a pH optimum shift with increasing bile salt concentration. Bile salt inhibition of lipase is related both to the concentration of bile salt and to the substrate concentration or rather to substrate surface area, and most probably is complete when the interface is saturated with detergent.

Co‐lipase, in the absence of bile salts, stimulates the activity of lipase, 1.3 to 1.4‐fold in the whole pH range of its activity. Co‐lipase overcomes the inhibition of lipase caused by bile salts with a shift in the pH optimum to 6–7 compared to 8–9 for lipase alone.

The different conjugated bile salts have similar effects, consideration being taken to differences in their critical micellar concentration; free bile salts have a less inhibitory effect on lipase and the stimulation by co‐lipase shows no pH optimum shift.

Detergents of the acyltaurine type such as decanoyltaurine and dodecanoylsarcosyltaurine inhibit lipase in a similar manner to the conjugated bile salts and this inhibition is also released by co‐lipase. Detergents such as dodecylsulphate above the micellar concentration, irreversibly inhibit lipase. The simultaneous presence of bile salts protects the enzyme from being irreversibly inactivated.

Lipase and co‐lipase interact in a stoichiometrical relationship and it appears justified to classify co‐lipase as a co‐enzyme for lipase.