bluuebunny/crossref_metadata_2025 huggingface dataset:
Light Absorption of Flavosemiquinone Metal Chelates
Authors: Müller F. , Hemmerich P. , Ehrenberg A.
Published: March 2005

The light absorption of flavosemiquinone metal chelates (Zn2+, Cd2+, Mn2+, Co2+, Ni2+, Fe2+, Mo5+, and Cu+) in aprotic polar solvents has been studied. The light absorption of flavosemiquinone metal chelates, the uncomplexed cation and anion free radicals, and the corresponding, very unstable, flavoquinone metal chelates are compared. The visible spectra of the flavosemiquinone metal chelates, irrespective of the metal, are very similar to that of the free radical anion. In addition, they show a rather unique, sharp transition in the near infrared range (650 to 850 nm). The extinction coefficients for d1, d5, d6, d7, d8, and d10 metal centres are between 10 and 2 × 103 M−1cm−1. Solvents that act as quaternary ligands (pyridine, acetonitrile, but not 2,6‐lutidine) strongly affect this near infrared transition, but not the visible spectrum. We therefore conclude that the near infrared absorption reflects the symmetry of the flavosemiquinone‐metal‐H2O‐solvent complex.

The molar extinction coeffcients in the near infrared are independent of chelate concentration, solvent polarity, and ionic strength, and the optical densites are proportional to total spin concentration as monitored by electron spin resonance (for systems of total spin 1/2, i. e. with d10 central ions). This excludes association phenomena. Altering the flavin moiety, even slightly, effects the light absorption spectrum in the visible and near infrared regions.

This near infrared absorption may be a useful tool for detecting flavin‐metal chelation in enzyme systems.
The Interaction of α‐Actinin with F‐Actin and its Abolition by Tropomyosin
Authors: Drabikowski W. , Nowak E.
Published: March 2005

The interaction of α‐actinin with F‐actin was examined with the use of the viscometric technique under various conditions.

α‐Actinin causes the gelation of F‐actin when added both before and after polymerization.

The interaction of α‐actinin with F‐actin seems to be much stronger at 0° than at 21°. In the latter case it takes place only when actin is polymerized with 0.1 M KCl, but not with 1 mM MgCl2 or CaCl2.

Tropomyosin, when added before α‐actinin, prevents the formation of F‐actin gel, and when added to already formed gel, causes its disappearance.

This abolishing effect of tropomyosin is much stronger at 21° than at 0°.

The results are discussed in view of the possible competition between tropomyosin and α‐actinin for the binding to F‐actin.
Mechanism of Thyroxine Formation from Diiodotyrosine and p‐Hydroxydiiodophenylpyruvic Acid
Authors: Blasi F. , Fragomele F. , Covelli I.
Published: March 2005

The mechanisms of the non‐enzymic synthesis of thyroxine from diiodotyrosine and its keto acid derivative 4‐hydroxy‐3,5‐diiodophenylpyruvic acid has been investigated.

The time course of the overall coupling process has been studied by following the formation of thyroxine and the disappearance of the reactants from the incubation medium. Thyroxine formation has a lag phase during which diiodotyrosine remains unchanged, whereas 4‐hydroxy‐3,5‐diiodophenylpyruvic acid disappears very rapidly.

Disappearance of 4‐hydroxy‐3,5‐diiodophenylpyruvic acid from the reaction mixture is due to the formation, besides of a degradation product, of an oxidized form of the keto acid which reacts with diiodotyrosine without a lag phase even in the absence of oxygen. This oxidized intermediate is formed by the uptake of 2 atoms of oxygen per mole of the keto acid. Its spectrum in the ultraviolet region is very similar to that of the unmodified keto acid; furthermore it is stabilized by borate ions and is able to oxidize iodide. The most likely structure of the oxidized intermediate of the 4‐hydroxy‐3,5‐diiodophenylpyruvic acid, therefore, is that of a peroxide.

Propylgallate, a radical scavenger known to block free radicals originated from 4‐hydroxy‐3,5‐diiodophenylpyruvic acid, increases the lag phase, inhibits the oxygen uptake 4‐hydroxy‐3,5‐diiodophenylpyruvic acid and decreases the overall yield of thyroxine if added during the lag phase; it is uneffective when added during the anaerobic period of the coupling reaction.

The following mechanism can be envisaged: (a) in a first step 4‐hydroxy‐3,5‐diiodophenyl‐pyruvic acid looses an electron thus originating a free radical; (b) in a second step 2 atoms of oxygen are taken up by the free radical and a peroxide‐like derivative is formed; (c) in the third step, not requiring the presence of oxygen, the peroxide derivative of the keto acid instantly couples with diiodotyrosine to form thyroxine.
Korrelation des unspezifisch permeablen mitochondrialen Raumes mit dem „Intermembran‐Raum”
Authors: Pfaff E. , Klingenberg M. , Ritt E. , Vogell W.
Published: March 2005

The term “unspecific permeability” in mitochondria is defined for describing a rapid equilibration of solutes between the extraamitochondrial space and a portion of the intramitochondrial space. It appears to be a property of the outer membrane and is restricted only by the size of the molecule (maximal molecular weight of 5,000–12,000).

The unspecifically permeable space varies considerably. It ranges from 20–80% in different preparations of mitochondria. For a single mitochondrial preparation it is shown to be of equal volume for various small molecules such as sucrose, adenine nucleotides, NAD+and aspartate.

The total aqueous phase of rat liver mitochondria in isotonic sucrose medium ranges from 2.5–3.4 μl/mg protein; the aqueous part of the impermeable space from 0.8–1.8 μl/mg protein. Only about 1 μl of this portion expands osmotically and represents the solvent for endogenous mitochondrial solutes. The major part (about 0.4–0.6 μl/mg protein) of the residual water of the impermeable space may be attributed to the hydrate shell of mitochondrial protein.

The unspecifically permeable and the impermeable space of the mitochondria are compared with the morphologically defined intermembrane and matrix space. The tonicity of the incubation medium was varied over a large range for rat liver and rat kidney mitochondria in order to facilitate this comparison. Large variations in the ratio of sucrose permeable to impermeable space (ranging from 1:3 in liver mitochondria to 3:1 in kidney mitochondria) correspond to changes in the ratio of the areas covered by the intermembrane and matrix space in the sections seen in the electron‐micrographs. Mitochondria with low unspecific permeability are homogenous, mitochondria with high unspecific permeability show greater individual variability at all tonicities.
Isolation and Properties of a Mouse Serum Prealbumin Excreted in Urine
Authors: Reuter A. M. , Hamoir G. , Marchand R. , Kennes F.
Published: March 2005

A method is described for the isolation of the fastest prealbumin fraction PA1 from mouse serum.

This fraction appears to behave identically to the urinary prealbumin fraction in electrophoretic migration, strain and sex dependency and in immunological reactions.

Molecular weight is determined by diffusion and sedimentation as well as by sedimentation‐diffusion equilibrium.

The apparent lower molecular weight of serum prealbumin is due to its contamination by polysaccharides extracted from starch gel in the course of its isolation.
Study of the Groups Controlling the Activity and Conformation of Chymotrypsin at Alkaline pH: N‐Terminal Isoleucine and Tyrosines
Authors: Karibian D. , Laurent C. , Labouesse J. , Labouesse B.
Published: March 2005

It is shown that the change in activity and structure of acetyl‐δ‐chymotrypsin in the pH 7–11 range is not correlated with the ionizations of tyrosine residues.

The pKs of the two titratable tyrosines of acetyl‐δ‐chymotrypsin are found to be 10.2 and 11.5, at 15° (9.6 and 10.8 after correction for electrostatic interactions), and those of the starting acetylated zymogen are 10.5 and 12.1 (10.0 and 11.5 after identical corrections). The plot of log rate of deacetylation of the titratable tyrosines versus pH is linear between pH 9 and pH 12. At a given pH there is a linear relation between the log deacetylation rate and the pKs of the residues. The pH rate profile of acylation of acetyl‐δ‐chymotrypsin by 4‐carboxy‐3‐nitrophenyl‐N,N‐diphenylcarbamate is bell‐shaped, with a maximum at pH 7.9 at 15°, in 0.14 M KCl. The right side of the curve shows a pKapp of 8.9, whether the exposed titrable tyrosines of the enzyme are acetylated or not. A similar pKapp (9.1) is found for the pH‐dependent change in optical rotation of the acetylated enzyme, when the exposed tyrosines are acetylated. After correction for electrostatic interactions, this apparent pK becomes 8.4 and can be assigned only to the α‐aminogroup of the N‐terminal isoleucine.
Induction and Repression of Mitochondrial ATPase in Yeast
Authors: Somlo M.
Published: March 2005

A simple mechanical procedure for the preparation of particle‐bound Mg‐ATPase is described. It represented at least 80% of total ATPase activity, both in the case of aerobically and of anaerobically grown yeast. When analyzed in a sucrose gradient, ATPase from aerobic yeast sedimented with NADH‐oxidase. ATPase from aerobic or anaerobic yeast showed 2 pH optima, as had been described for ATPase localized in carefully prepared and purified yeast mitochondria. Particle‐bound ATPase from aerobic or anaerobic yeast showed pronounced oligomycin sensitivity.

Anaerobically grown yeast synthesized particle‐bound Mg‐ATPase at the low basal rate of 7–10 μmoles P/10 min/mg protein. During O2‐induction without growth, respiratory enzymes and ATPase were induced simultaneously. Both inductions were inhibited by acti‐dione.

Aerobically grown, glucose‐repressed yeast synthesized ATPase at a rate similar to anaerobic yeast. Progressive derepression during growth produced a simultaneous increase of the rate of cell respiration (30–270) and of ATPase activity (10–60).

Ethanol‐grown cells had a constant high Qo2 and synthesized ATPase at the high constant rate of 60.

Regulation of ATPase synthesis was strictly identical when activity was measured at the two pH optima, suggesting the presence of a single mitochondrial ATPase. The possibility of separate phosphorylating and hydrolytic sites on the enzyme is discussed.
Regulation of the Enzymes of the β‐Ketoadipate Pathway in Moraxella calcoacetica
Authors: Cánovas J. L. , Johnson B. F. , Wheelis M. L.
Published: March 2005

Further evidence for coordinacy of enzymes operative in the protocatechuate branch of the β‐ketoadipate pathway in Moraxella calcoacetica has been obtained through studies with an analog of protocatechuate, 3‐hydroxy‐4‐methylbenzoate. This compound, which is not metabolizable by M. calcoacetica, inhibits the induced synthesis of shikimate dehydrogenase, protocatechuate oxygenase, carboxymuconate lactonizing enzyme, carboxymuconolactone decarboxylase, β‐ketoadipate enol‐lactone hydrolase I and β‐ketoadipate succinyl‐CoA transferase I, by the wild type. It also effectively abolishes constitutive synthesis of these enzymes by mutants with impaired protocatechuate oxygenase function.

Mutants in which the regulatory control of these enzymes is modified have been isolated. Such mutants are still inducible, but are very poorly induced by protocatechuate, the metabolite which is the normal inducer of these enzymes in the wild type. However, 3‐hydroxy‐4‐methylbenzoate, which interferes with induction in the wild type, has become an effective inducer for the mutants.
Excess of α‐Globin Synthesis in Homozygous β‐Thalassemia and its Removal from the Red Blood Cell Cytoplasm
Authors: Bargellesi A. , Pontremoli S. , Menini C. , Conconi F.
Published: March 2005

The intracellular behaviour of the excess newly synthesized α‐globin has been studied by incubating peripheral blood and bone marrow samples from 9 homozygous β‐thalassemic subjects with labeled amino acids. The globin chains present in the cytoplasm and in association with the stromal material have been separated by column chromatography, and the incorporation of radioactivity has been determined for each globin at various incubation times.

The results obtained show that the excess of α‐globin is removed from the cell cytoplasm and becomes associated with the stromal material.
Correlation between Anion Uptake and the Movement of K+ and H+ across the Mitochondrial Membrane
Authors: Palmieri F. , Quagliariello E.
Published: March 2005

The relationship between the H+ and K+ movement across the mitochondrial membrane and the distribution of anions between the intra‐ and extramitochondrial space has been analyzed in the presence of uncouplers, nigericin and nonactin.

Mitochondria accumulate added anions in the presence of respiratory inhibitors and oligomycin. This accumulation is accompanied by the exit of endogenous anions. Respiration or ATP hydrolysis does not, in itself, increase anion accumulation, but only in as much as it gives rise to proton ejection and cation uptake.

In sucrose medium anion uptake, found in the presence of respiratory inhibitors and oligomycin, is inhibited by uncouplers, nigericin and nonactin, all of which cause H+ uptake and K+ release. The ratio of the intra‐ to the extramitochondrial concentration of endogenous anions is also strongly diminished by uncouplers, nigericin and nonactin.

With nonactin‐pretreated mitochondria, uncouplers, even at very high concentrations, do not inhibit anion uptake.

With nonactin, as the external K+ concentration is increased, the uptake of H+ is progressively decreased and similarly the anion uptake inhibition is diminished.

The 2,4‐dinitrophenol‐induced uptake of H+ is unaffected by the presence of K+ in the medium up to a concentration of 125 mM. The inhibition of anion uptake and the efflux of endogenous anions, induced by dinitrophenol, are equal in the presence or absence of 100 mM KCl.

In the presence of an external concentration of 100 mM KCl, with nigericin the uptake of H+ is strongly diminished and similarly the anion uptake inhibition and the efflux of endogenous anions are greatly decreased. With nonactin, H+ ejection occurs accompanied by stimulation of anion uptake.

The ratio of the intra‐ to the extramitochondrial concentration of succinate increases as the external pH decreases. This increase of anion uptake by lowering the pH is inhibited by dinitrophenol.

It is concluded that inhibition of anion uptake and efflux of endogenous anions are dependent upon the uptake of H+ by the mitochondria and vice versa H+ ejection is responsible for stimulation of anion uptake.
Synthèse des messagers précoces phagiques chez Escherichia coli infecté par T4 durant une carence spécifique en aminoacide
Authors: Legault‐Démare L. , Malhié A. , Gros F.
Published: March 2005

It has been known for many years that the rate of ribosomal and transfer RNAs synthesis in bacteria is determined by the internal amino acid concentration but the existence of the same mechanism of regulation for messenger RNA has not been clearly demonstrated yet.

This work was done to determine if the amino acid regulation also governs phage early messenger RNA synthesis in T4 infected Escherichia coli cells, in which the synthesis of bacterial RNA is known to be turned off rapidly after infection.

The determination of the rate of messenger synthesis in the presence and in the absence of the required amino acid was done (a) by measurement of the amount of radioactivity from labelled RNA which is hybridizable to phage DNA; (b) by estimation in each sample of the amount of messenger RNA which can compete with a messenger RNA labelled in the presence or in the absence of the required amino acid for the complementary sites of phage DNA.

By direct hybridization it was found that in the absence of the required amino acid the rate of labelling of phage messenger RNA is reduced in a stringent strain, but not in a relaxed strain.

By hybridization competition it was found that the molecular species of phage messenger RNA which can be synthesized in the absence of the required amino acid are present at the same concentration in infected cells either in the presence or in the absence of the amino acid.

A study of the kinetics of formation of the messenger pool showed that, as early as the first minute of infection, all the molecular species of messenger are present in the same proportions in the stringent cells whether amino acid‐starved or not.

It is concluded that in a stringent strain those molecular species of messenger which do not require the presence of specific viral protein for their formation are synthesized at the same rate in the presence or in the absence of a required amino acid.

The implications of these results concerning the control of transcription by amino acids are discussed.
Isolation of an Acyl Carrier Protein Component from the Multienzyme Complex of Yeast Fatty Acid Synthetase
Authors: Willecke K. , Ritter E. , Lynen F.
Published: March 2005

[14C]Pantetheine fatty acid synthetase from baker's yeast was denatured in guanidine hydrochloride. Then the protein components of the multienzyme complex were subjected to a Sephadex filtration in guanidine hydrochloride and a preparative polyacrylamide gel electrophoresis in a phenol containing medium. It could be demonstrated that the [14C]pantetheine label was co‐valently bound to one of the smallest polypeptide chains of the multienzyme complex. In analogy to the soluble acyl carrier protein which was first isolated from Escherichia coli and contained 4′‐phosphopantetheine as a prosthetic group, the pantetheine‐protein component of the yeast multienzyme complex was denoted as yeast acyl carrier protein. Its molecular weight seemed to be higher and its amino acid composition different from the E. coli acyl carrier protein.
The Binding of Extra Acidic Proteins to Deoxyribonucleoprotein during the Preparation of Nuclear Proteins
Authors: Johns E. W. , Forrester S.
Published: March 2005

Acidic proteins can be removed from calf thymus deoxyribonucleoprotein, previously washed with 0.14 M NaCl, using 0.35 M NaCl. After the removal of these acidic proteins, the yield of acidic nuclear proteins as extracted by other methods dropped to less than one third. Calf thymus deoxyribonucleoprotein and reconstituted deoxyribonucleohistone adsorb acidic proteins from the cytoplasm and nuclear sap in 0.14 M NaCl. These proteins which can be released in 0.35 M NaCl are similar in amino acid analysis to the acidic nuclear proteins prepared by other methods.
Studies on a Possible Precursor in the Self Assembly of the Bacteriophage fr.
Authors: Hohn T.
Published: March 2005

Complex I, a complex consisting of 1 RNA strand and 6 protein subunits of the icosahedral RNA‐phage fr was formed. The protein subunits bind on more than one site to the RNA. Complex I is very specific in respect to the RNA partner and its formation renders the RNA neither more nor less advantageous in forming phage‐like particles with additional protein.
Magnetic Resonance and Kinetic Studies on the Manganese Activated Arginine Kinase Reaction
Authors: O'sullivan W. J. , Virden R. , Blethen S.
Published: March 2005

Proton relaxation rate studies have been used to study the interaction between arginine kinase from two sources, Homarus vulgaris and Homarus americanus, and manganous ion, MnADP‐ and MnATP2‐, respectively. There was negligible interaction between Mn2+ and the H. vulgaris enzyme but a small effect with the H. americanus enzyme. In both cases, a large enhancement of the proton relaxation rate was seen on the addition of ADP or ATP to a solution containing Mn2+ and enzyme. It was concluded that the enzyme combines with MnADP− or MnATP2‐ rather than with Mn2+.

Enhancements of the proton relaxation rate of the ternary complexes, MnADP‐E (45), Mn2′‐dADP‐E (28) and MnATP‐E (17), were obtained with the H. vulgaris arginine kinase and Mn3′‐dADP− and Mn2′‐dATP2‐ were also demonstrated to form ternary complexes with this enzyme. For arginine kinase from H. americanus, values for MnADP‐E (17), Mn2′‐dADP‐E (approx. 12) and MnATP‐E (approx. 8) were determined. A comparative kinetic study demonstrated reasonable agreement between the kinetically determined Km values and dissociation constants from the proton relaxation rate data. It was also found that for both enzymes, there was a correlation between the enhancement and maximum velocity values, ADP > 2′‐dADP and ATP > 2′‐dATP which could be interpreted as indicative of a graded degree of substrate induced conformational change. The proton relaxation rate and kinetic studies indicated small but probably significant differences in the properties of the two enzymes.

Proton relaxation rate studies provided evidence for a second substrate induced conformational change on the addition of l‐arginine to the ternary MnADP‐enzyme complex. No significant effect was observed with d‐arginine, which is a competitive inhibitor with respect to l‐arginine.
Differential Inhibition of Protein Synthesis in the Nucleus and Cytoplasm of HeLa Cells in the Presence of Actinomycin, Puromycin and Hydroxyurea
Authors: Zampetti‐Bosseler F. , Malpoix P. , Fievez M.
Published: March 2005

A comparison has been made between the synthesis of histones, other nuclear proteins and cytoplasmic proteins after treatment of HeLa cells with substances known to alter their metabolic state: actinomycin, puromycin and hydroxyurea. The use of sensitive methods involving amino acid analysis and measurement of specific radioactivity has revealed differential responses to the action of the different inhibitors. Analysis of these findings as a function of the effects of these inhibitors on other metabolic pathways, suggests that control mechanisms or basic mode of synthesis of some of the histones may differ from that of the other proteins, and that DNA and histone synthesis are probably coordinated in some way yet to be elucidated.
The Role of Heme in the Regulation of δ‐Aminolevulinic Acid and Heme Synthesis in Rabbit Reticulocytes
Authors: Neuwirt J. , Poňka P. , Borová J.
Published: March 2005

The problem of the feedback inhibitory effect of heme on its own synthesis in erythroid cells was studied using different methods.

The activity of δ‐aminolevulinic acid synthetase was determined in the insoluble particles containing mitochondria and stroma prepared from rabbit reticulocytes and in the lysate from these reticulocytes.

The effect of hemin on the incorporation of [2‐14C]glycine into heme was measured in the lysate from rabbit reticulocytes.

The effect of hemin on the incorporation of 59Fe into heme was studied in reticulocytes with artificially elevated labeled non‐heme iron pool.

All the three different methods used produced evidence that hemin does not directly inhibit its own synthesis.

No feedback control by hemin of heme synthesis in the rabbit reticulocytes at the level of δ‐aminolevulinic acid synthetase could be proved.

The principal site of inhibition by hemin in intact reticulocytes appears to be the transfer of iron into the cells.
Reaction of Amino Acid‐tRNA Synthetases with 2′‐Deoxyadenosine Triphosphate
Authors: Mitra S. K. , Mehler A. H.
Published: March 2005

All of the amino acid activating enzymes studied are capable of reacting with dATP in place of ATP. None of the other available nucleotides was found to be used by these enzymes. The Km of dATP is generally higher than that of ATP but the maximum velocities are similar in the complete reaction, esterification of transfer RNA. The rates of pyrophosphate exchange are much lower with dATP than with ATP in the cases studied.
The Status of C‐6, C‐7, C‐15 and C‐16 Hydrogen Atoms in Cholesterol Biosynthesis
Authors: Akhtar M. , Rahimtula A. D. , Watkinson I. A. , Wilton D. C. , Munday K. A.
Published: March 2005

Studies carried out with stereospecifically labelled mevalonic acids have confirmed that in the biosynthesis of cholesterol the 6α, 7β and 15α hydrogen atoms are removed. It is also shown that the C‐16 hydrogen atoms remain undisturbed in cholesterol biosynthesis. The non‐involvement of the C‐16 hydrogen atoms in the biosynthesis has provided further support for the previously proposed mechanism of the removal of the 14α methyl group in sterol biosynthesis.
NMR‐Studies on the Structure of the Active Site of Pancreatic Ribonuclease A
Authors: Rüterjans H. , Witzel H.
Published: March 2005

The chemical shift of the C‐2 imidazole protons of the histidine residues of pancreatic ribo‐nuclease has been studied as a function of pH in 2H2O. Upon protonation of the imidazole ring the C‐2 proton magnetic resonance signal shifts about 1.0 ppm to a lower field. Thus titration curves and approximate pK values of the histidine residues are obtained. The pK values of histidine 119 and histidine 12 of the active site are unusually low and shift by 1.0 and 0.7 units to higher values when the NaCl concentration is changed from 0.1 M to 0.4 M, whereas the pK value of histidine 105 shifts only by about 0.2 units. This indicates the presence of a positively charged group in the immediate environment of histidine 119 and histidine 12. The abnormal chemical shift of the histidine 48 C‐2 proton at low pH is explained by means of an interaction of histidine 48 with the carboxylate anion of aspartic acid 14. From an analysis of the titration curves of histidine 119 and histidine 12 the existence of an enzyme species in which both imidazoles are connected by a hydrogen bond is derived. The C‐2 proton magnetic resonance signals of histidine 119 and histidine 12 shift to a lower field when inhibitors bind to the enzyme. Both histidines are then kept in a protonated form up to the pH range of 7–8. The apparent pK values are higher in the cytidine 2′‐phosphate‐ribonuclease complex than in the cytidine 3′‐phosphate‐ribonuclease complex. A similar shift of the histidine 119 and histidine 12 signals is observed with binding of pyrophosphate to ribonuclease indicating that only the phosphate group causes the behaviour of the histidines. Shifts of the C‐6 proton magnetic resonance signal of the pyrimidine ring indicate an additional binding of the base to an aromatic side chain of the enzyme.

The mechanism for the binding process proposed in previous papers by Witzel and coworkers in which histidine 119 and lysine 41 are the binding groups of the enzyme, whereas histidine 12 takes up a proton when being released from its binding to histidine 119, is in agreement with the results reported in this communication.
Effect of Monovalent Cations on the Activity of the DNA Polymerase of Escherichia coli B
Authors: Klenow H. , Henningsen I.
Published: March 2005

The effect of a number of inorganic salts containing monovalent cations on the activity of the DNA polymerase from E. coli B has been investigated under a variety of conditions. The following results were obtained.

In the presence of buffers prepared from organic amines and HCl the enzyme activity is stimulated 3‐ to 15‐fold by chlorides containing K+, Rb+, Cs+ or NH+4. The buffer concentration influenced the degree of stimulation and the shape of the activation curves. The activation constant for KCl decreased with increasing buffer concentration and with increasing pH. NaCl and LiCl had little or no stimulating effect. The latter two salts counteracted the stimulating effect of the four former salts.

In the absence of amine‐HCl buffers it was found that below certain concentrations sodium, potassium and lithium glycinate buffers and potassium and sodium phosphate buffers stimulated the enzyme activity. Data were obtained in favour of the possibility that the monovalent cations under these conditions are indispensible activators for the DNA polymerase reaction.

The pattern of stimulation of the enzyme activity by KCl at various amine‐HCl buffer concentrations was similar at different pH values in the pH range from 6.5 to 9.3. At pH 10.5, however, KCl stimulated at low buffer concentrations while all concentrations of this salt inhibited at higher buffer concentrations. In the presence of optimal concentrations of KCl and amine‐HCl buffers the pH optimum curve had a plateau reaching from pH 7.7 to about pH 10.5.

The stimulating effect on the enzyme activity observed for potassium salts seems not to be due to an effect on the binding of the substrates to the enzyme but rather to an effect on the catalytic activity of the enzyme.

The DNA degrading activity of the enzyme preparation was stimulated by KCl and inhibited by LiCl.
Effect of Hydroxylamine on Respiration and Oxidative Phosphorylation
Authors: WikstrÖM M. K. F. , Saris N.‐E. L.
Published: March 2005

Rat liver mitochondria were used to study the effect of hydroxylamine on respiration and oxidative phosphorylation with succinate or tetramethyl‐p‐phenylenediamine plus ascorbate as substrate, and ATP phosphohydrolase activities.

With these substrates hydroxylamine inhibited respiration in state 3. Inhibition was released by various uncouplers of oxidative phosphorylation, including gramicidin and calcium ions. Respiration in state 4 was affected much less. Arsenate‐induced respiratory increase was effectively inhibited by hydroxylamine. Succinate oxidase activity in state 3 with ferricyanide as the electron acceptor was not inhibited by hydroxylamine.

Oxidative phosphorylation, with succinate as substrate, was inhibited more than respiration by hydroxylamine, with a concomitant decrease in the P/O ratio. Oxidative phosphorylation during succinate oxidation by ferricyanide was not affected by hydroxylamine.

2,4‐Dinitrophenol‐ and carbonylcyanide p‐trifluoromethoxy phenylhydrazone‐induced ATPase activities were not affected by hydroxylamine. The magnesium‐stimulated ATPase of detergent‐disrupted mitochondria was strongly inhibited but the gramicidin‐induced ATPase of intact mitochondria was only inhibited by about 30%. Under non‐respiring conditions hydroxylamine induced no ATPase by itself.

Our results indicate that the inhibition of oxidative phosphorylation by hydroxylamine is specific for coupling site 3, and in some ways resembles the effects of oligomycin. It is suggested that site specificity exists up to the level of X∼P in the partial reactions of oxidative phosphorylation.
Studies on Microbial RNA
Authors: BjÖRK G. R. , Svensson I.
Published: March 2005

Fractionation of enzymes, which catalyze the transfer of methyl groups from S‐adenosyl‐l‐methionine to transfer RNA, from extracts of Saccharomyces cerevisiae has yielded eight separate methylating fractions. The products of methylation were identified by thin‐layer chromatography of acid hydrolyzed tRNA with known reference compounds. Enzyme activities for synthesis of four methylated guanines, 1‐methyladenine, 5‐methylcytosine and thymine have been demonstrated. Three of the enzyme fractions produced thymine, apparently free of other methylating activity. Some fractions also contained enzymatic activity for formation of a heterogeneous group of compounds resistent to acid hydrolysis, probably related to ribose‐methylated dinucleotides.
Studies on Microbial RNA
Authors: Svensson I. , Björk G. R. , Lundahl P.
Published: March 2005

Partial purification of transfer RNA‐methylating enzymes from Saccharomyces cerevisiae D84 yielded eight different enzyme fractions, the properties of which have been studied. All of these enzymes also showed some activity with ribosomal RNA from Escherichia coli. The enzymes were found to differ in their species specificity. One of the enzyme fractions is highly active with Aerobacter aerogenes tRNA, forming N2‐dimethylguanine, but inactive with wheat germ tRNA. Other fractions are active with wheat germ tRNA, synthesizing 5‐methyluracil only. Chromatography on hydroxylapatite resulted in three different fractions containing activity for the formation of 5‐methyluracil in tRNA from E. coli. Although the same methylated base is formed, these enzymes differ in their specificities.
Double Conditional Lethality: Temperature‐Sensitive and Amber Mutations in the Glucosyl Transferase Gene of Bacteriophage T2
Authors: Molholt B. , de Groot B.
Published: March 2005

The isolation and characterization of two types of conditional lethal mutations in the α‐glucosyl transferase gene of bacteriophage T2 are described.

T2gt7 is temperature‐sensitive for glucosyl transferase by the following criteria:

Glucosylation of T2gt7 DNA is temperature‐sensitive in vivo. Compared to T2+ DNA, DNA of the gt7 mutant is two‐thirds glucosylated at 30° and less than 10% glucosylated at 37°.

The glucosyl transferase induced by T2gt7 is temperature‐sensitive in vitro. Catalytic activity of the mutant enzyme is rapidly and irreversibly lost at temperature above 37° both in the presence and absence of substrates.

The gt7 mutation maps in the T2 gene for glucosyl transferase.

T2gt3 is an amber mutation in the T2 gene for glucosyl transferase. DNA of the gt3 mutant is glucosylated in E. coli K704 (supE+) and once glucosylated is not restricted upon growth in E. coli B carrying an amber suppressor. The gt3 mutation also maps in the T2 gene for glucosyl transferase.
A New Method for the Extraction of R Lipopolysaccharides
Authors: Galanos C. , Lüderitz O. , Westphal O.
Published: March 2005

A method is described which is specific for the extraction of lipopolysaccharides from R form bacteria. The extraction mixture which is monophasic, consists of aqueous phenol, chloroform and petroleum ether. R form lipopolysaccharides (glycolipids). due to their lipophilic nature, are completely soluble in the mixture. S and T form lipopolysaccharides as well as proteins, nucleic acids, and polysaccharides, are insoluble, and they are excluded from the extracts. The method is mild, as it can be carried out at below 10°. The yields are generally higher than those obtained by phenol‐water extraction, and the products are usually water‐soluble. Lipopolysaccharides have been successfully extracted from all R form bacteria so far attempted.
Characterization of a Glutamine Synthetase b Activating (Deadenylylating) Enzyme System in Escherichia coli
Authors: Heilmeyer L. , Battig F. , Holzer H.
Published: March 2005

Biosynthetically inactive labelled glutamine synthetase b was produced by enzymic adenyly‐lation of biosynthetically active glutamine synthetase a with [14C]ATP. Upon incubation of the glutamine synthetase b with a crude extract from E. coli B a reactivation, i. e. an increase of biosynthetic activity, and a release of protein bound radioactivity takes place wth identical kinetics. Thus, E. coli B contains a glutamine synthetase b activating (deadenylylating) enzyme system. The radioactive material released was identified as AMP. Fluoride (2.5 mM) and 2.5 mM ATP stimulate the enzymic activation of glutamine synthetase b multi‐fold. dATP, ITP, GTP, CTP, and UTP also stimulate but AMP inhibits the activation. These effects point to a control of the glutamine synthetase interconverting enzyme system by the ATP/AMP ratio, i. e. by the energy situation of the cell.
On the Mechanism of Uncoupling in Chloroplasts by Ion‐Permeability Inducing Agents
Authors: Karlish S. J. D. , Shavit N. , Avron M.
Published: March 2005

A general mechanism is presented for uncoupling of chloroplasts by compounds or combination of compounds which increase membrane permeability to both protons and alkali metal cations.

Agents which supposedly increase the membrane permeability to protons only, such as 2,4‐dinitrophenol and carbonyl cyanide p‐trifluoromethoxy phenylhydrazone (FCCP) (at low concentrations), did not uncouple chloroplasts. Similarly, the antibiotics valinomycin, and nonactin and the synthetic compound LR6‐125, which apparently increase only the cation permeability of membranes, did not uncouple. However, combinations of dinitrophenol or of low concentrations of FCCP with anyone of the cation permeability inducing agents, led to full uncoupling of the chloroplasts. Studies on the passive permeability properties of the grana membrane, support the assumption that combinations of dinitrophenol or FCCP (at low concentrations), with valino‐mycin or nonactin or LR6‐125, increase the membrane permeability to both proton and cations. High concentrations of FCCP (10 μM) which alone uncoupled chloroplasts, also appeared to increase the permeability of the membrane to both protons and to cations, while low concentrations of FCCP, which did not uncouple alone, sufficed to increase the membrane permeability to protons. It is suggested that the uncoupling produced by the compounds used in this work is of the same type as that induced by the antibiotics, gramicidin and nigericin, both of which were suggested to increase the membrane permeability to both protons and cations.
Kinetics of the Alteration and Modification of DNA‐Dependent RNA‐Polymerase in T4‐Infected E. coli Cells
Authors: Seifert W. , Qasba P. , Walter G. , Palm P. , Schachner M. , Zillig W.
Published: March 2005

The decrease in the activity of DNA‐dependent RNA polymerase in Escherichia coli after infection with bacteriophage T4 is the consequence of a structural change of the polymerase and not of the appearance of an inhibitor. The kinetics of this change were followed using polymerases highly purified at different times after infection (a) by measuring the specific enzymatic activity with T4‐ and calf thymus DNA as templates and (b) by following the appearance of the modified polymerase‐subunit in disc‐electrophoresis.

The results allow one to differentiate between two consecutive processes:

A very rapid alteration which inactivates the RNA polymerase for T4‐DNA, but not for other DNA templates, and which does not require protein synthesis.

A structural modification which depends on protein synthesis and leads to a change in one subunit of the polymerase.

As shown by the use of the T4‐mutants am BL 292 and am N.134 neither gene 55 nor gene 33, both involved in the transcription of late genes, are participating in the modification reaction.
Specificity and Function of Exonuclease II in the Synthesis of Alternating Copolymers by DNA Polymerase
Authors: Lezius A. G. , Metz E.
Published: March 2005

The poly d(A‐T) dependent synthesis of the analog alternating copolymer poly d(A‐4thioT) (in which thymidine is replaced by 4‐thiothymidine) by DNA polymerase of Escherichia coli is accompanied by hydrolysis of at least one mole of primer d(A‐T) per mole of product d(A‐4thioT) formed. Exonuclease II, the deoxyribonuclease associated with DNA polymerase, is responsible for the hydrolysis of the primer. Since the growing 3′ end of the primer is protected from hydrolysis during the enzymatic polymerisation the hydrolysis must occur at the free 5′ end. The enzymatic synthesis of poly d(A‐4thioT) on a poly d(A‐T) template proceeds in a two phase time course. The ratio of polymerisation versus hydrolysis is close to unity for the first rapid phase of synthesis. The temperature‐dependence of poly d(A‐T) and poly d(A‐4thioT) synthesis, represented by usual Arrhenius plots has a discontinuity at 15° and 25°, respectively. Exonuclease II exhibits a similar discontinuity at 26° under the same conditions. Above 25°, the synthesis of poly d(A‐4thioT) has a smaller apparent activation energy than the poly d(A‐T) synthesis, but the latter is twice to three times as fast. The obligatory hydrolysis of the primer offers a plausible explanation of why the synthesis of poly d(A‐4thioT) depends on the amount of poly d(A‐T) primer in a stoichiometric manner since the former copolymer is not a primer for extensive synthesis of poly d(A‐4thioT). A modified enzyme model for DNA polymerase is suggested, implicating the function of exonuclease II in providing an additional driving force for the unidirectional movement of the enzyme along the template. Emphasis is put on the energy contribution arising from formation of new base pairs during synthesis.

The specificity of exonuclease II was also studied using as substrates such polymers which consist of poly d(A‐T) and poly d(A‐4thioT) in one half each. The polymer bearing the d(A‐4thioT)n at the 3′ half is readily hydrolysed by exonuclease II from both the 3′ and 5′ end synchronously at pH 7.2. Upon mercuration of d(A‐4thioT)n with p‐chloromercuribenzoate, preferentially the poly d(A‐T) part is hydrolysed from the 5′ end at a reduced rate. In contrast, no synchronism of nuclease reaction is observed with the polymer bearing d(A‐4thioT)n at the 5′ end. the latter being generally more resistant against hydrolytic attack by the enzyme.
Ligand Induced Conformational Changes in Various Normal and Modified Hemoglobins as Indicated by Changes in Optical Rotatory Dispersion
Authors: Hänisch G. , Engel J. , Brunori M. , Fasold H.
Published: March 2005

A decrease of the optical rotation in the wavelength region from 200 to 250 mμ is observed when ligands bind to hemoglobin. The change at 233 mμ amounts to about 8% of the total rotation at this wavelength, independently of the nature of the ligand (O2, CO, NO or ethyl‐isocyanide). The effect is interpreted as being caused by a small conformational change, which influences the electronic transitions of a number of peptide bonds or (and) a few aromatic side chains. The change is observed with αβ‐dimers, but not with the isolated single chains. The full effect is present however, in chemically modified hemoglobins which no longer show cooperative interactions in the ligand equilibrium curve. It is speculated that the conformational change takes place within the protomers but only when these are joined in αβ‐dimers (or tetramers). The chemical modifications might not prevent the initial ligand‐induced conformational change, but only abolish the transmission of the conformational change from one subunit to the others.
Attempts at Purifying the Galactose Carrier from Galactose‐Induced Baker's Yeast
Authors: Haškovec C. , Kotyk A.
Published: March 2005

An attempt was made to isolate the protein associated with the formation of the inducible d‐galactose‐transporting system in baker's yeast. Of the several fractions obtained by centrifugation of homogenized yeast cells at different speeds only the fraction sedimenting at 40000 x g and containing a predominance of membranes was found to bind d‐galactose, the dissociation constant being 0.2–0.6 mM. No galactokinase activity was found in this fraction.

The membrane proteins were solubilized using various technique but the amount of sugar bound by the solubilized proteins was lower than that bound by the intact membranes, the most promising appearing to be treatment with 1 M NaI.

Membrane proteins labelled by a double‐labelling technique were solubilized with 1 M NaI and separated on DEAE‐cellulose in a linear gradient of NH4HCO3. A peak of unequal labelling with 14C and 3H was identified both in normal baker's yeast and in a galactokinase‐less mutant and assumed to represent the inducible galactose carrier or a part of its transport system.
Preparation and Properties of Cyanoethylated Insulin
Authors: Bosshard H. R. , Jørgensen K. H. , Humbel R. E.
Published: March 2005

Acrylonitrile has been shown to react not only with α‐ and ɛ‐amino groups, but also with imidazole‐nitrogens in beef insulin.

Experimental methods are described for the preparation and analysis of insulin derivatives with cyanoethylated amino groups and various amounts of cyanoethylated histidine residues.

Cyanoethylated derivatives of insulin can be separated from insulin by electrophoresis.

Cyanoethylated insulin represents a mixture of different molecular species as shown by ion exchange chromatography.

Residual histidine and carboxyethyl‐histidine residues were determined in acid hydrolysates of the isolated peptides B 1–7 and B 8–19. The amounts of blocked histidine B 5 were found to be greater than those of histidine B 10 by a factor of 2.7–3.6.

Under the applied experimental conditions immunological properties of cyanoethylated insulin with a residual histidine content of 50% are similar to those of the native hormone.

Depending on the degree of cyanoethylation, cyanoethylated insulin has a biological activity of 50–20% of that of insulin.

Evidence is presented for the important role of histidine residues for biological activity of insulin. The data presented suggest that histidine B 5 be particularly important.
An Improved Method for Isolation and Identification of Hydroxylated Testosterone Metabolites as Trimethylsilyl Ethers from Biological Material
Authors: Demisch K. , Staib W.
Published: March 2005

The chromatographic behaviour of 21 testosterone metabolites and their derivatives hydroxylated at various positions is investigated on thin layer plates and on SE‐30 3.8% and XE‐60 1% columns during gas‐liquid chromatography.

Distinct retention times of these compounds and their derivatives can give good evidence of identification. An improved method for the isolation and identification of hydroxylated testosterone metabolites from biological material is reported.
The Reduction of Flavins by Borohydride: 3,4‐Dihydroflavin
Authors: Müller F. , Massey V. , Heizmann C. , Hemmerich P. , Lhoste J.‐M. , Gould D. C.
Published: March 2005

The reduction of flavins by BH4− is slow, but strongly catalyzed by light. The main product of the reaction is 1,5‐dihydroflavin, i. e. normal leucoflavin, which is rapidly reoxidized upon admission of air. Under continuous aeration, however, a second reaction, irreversible by oxygen in the dark, becomes dominant yielding a tetrahydroflavin which, upon exhaustive reaction of BH4−, is autoxidized to give a new dihydroflavin of structure I. This compound is stable toward further oxidation in the crystalline state and in solution with organic solvents. Compound I has to be considered as a formal 3,4‐dihydroflavin. It is photooxidizable to give starting flavin, reacts very slowly with S2O4= compared to flavoquinone (II), but is easily reduced by EDTA in the presence of light. The compound was also synthesized by an unequivocal route and found identical with the product obtained by BH4−‐reduction of lumiflavin. The light absorption spectrum of I shows absorption maxima at about 405, 320, 270, and 220 nm and is practically identical with that of the BH4−‐reduction product of the flavoproteins d‐ and l‐amino acid oxidase. The BH4−‐reduction products of the amino acid oxidases are catalytically active. From this it follows that position 4 of the flavocoenzyme is not involved in enzymatic dehydrogenation.

The first photo‐excited triplet state of I has been investigated by optical and electron paramagnetic resonance spectroscopy. The structure of I was elucidated by means of infrared and nuclear magnetic resonance spectroscopy.
Fatty Acid Interaction with the Hydroxylating Enzyme System of Rat Liver Microsomes
Authors: Orrenius S. , Thor H.
Published: March 2005

Addition of a low concentration of laurate (Km= 6 μM) to rat liver microsomes in the presence of NADPH was found to stimulate the rate of NADPH disappearance parallel to the formation of oxidized metabolites, mainly ω‐hydroxy laurate. When added to a suspension of liver microsomes laurate gave rise to the formation of a “type I” spectral change indicating an interaction of the fatty acid with the microsomal mixed function oxidase. Whereas laurate present in an optimal concentration for ω‐oxidation caused a competitive inhibition of the oxidative demethylation of drugs, this activity as well as the ω‐oxidation activity was almost completely extinguished in the presence of a high fatty acid concentration (approx. 3 mM). At this concentration laurate had a solubilizing effect on the microsomes which was associated with a conversion of cytochrome P‐450 to the enzymically inactive P‐420 form.