bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation, Subunit Structure and Properties of the ATP‐Dependent Deoxyribonuclease of Bacillus subtilis
Authors: DOLY Janine , ANAGNOSTOPOULOS Constantine
Published: March 2005

A procedure was developed for the purification of the ATP‐dependent deoxyribonuclease of Bacillus subtilis 168. It comprises ammonium sulphate fractionation, Sephadex gel filtration, DFAE‐cellulose chromatography and gel electrophoresis on a discontinuous polyacrylamide gradient. The enzyme has been obtained in a homogeneous state. Its molecular weight was estimated to be 270000 by disc electrophoresis. Dodecylsulfate‐polyacrylamide gel electrophoresis showed the presence of five nonidentical subunits of the following molecular weights: 81 000, 70 000, 62000, 52 500 and 42 500. These values give 308 000 as the molecular weight of the native enzyme. The pH optimum of the purified enzyme is 9.6. The optimal concentrations of Mg2+ and ATP for exonuclease activity on native B. subtilis DNA were determined. ATP‐requirement for hydrolysis of single‐stranded DNA is less stringent. The enzyme also possesses high DNA‐dependent ATPase activity. The purification procedure was applied to extracts of a mutant devoid of activity for this enzyme (strain GSY 1290). A protein was isolated which is very similar to the active DNAase as regards electrophoretic mobility, reaction with specific antisera and size of four of the subunits. One subunit is missing (Mr 70 000) and is replaced by a smaller polypeptide (Mr 56 500). The latter results suggest that the mutant is affected in the genetic locus coding for the 70 000‐Mr subunit.
Chorismate Mutase/Prephenate Dehydratase from Escherichia coli K12
Authors: GETHING Mary‐Jane H. , DAVIDSON Barrie E. , DOPHEIDE Theo A. A.
Published: March 2005

A new simplified procedure for the purification of chorismate mutase/prephenate dehydratase, based on affinity chromatography on Sepharosyl‐phenylalanine, has been developed. The method utilizes the effect of NaCl on the binding properties of the enzyme.

NaCl inhibits both the mutase and dehydratase activities of the enzyme. In each case this inhibition is cooperative indicating homotropic interactions between NaCl binding sites on the enzyme. In addition NaCl induces homotropic cooperative effects between chorismate binding sites and between prephenate binding sites. NaCl also increases the sensitivity of the enzyme to inhibition by phenylalanine.
A pH‐Dependent Interaction between Histones H2A and H2B Involving Secondary and Tertiary Folding
Authors: MOSS Tom , CARY Peter D. , ABERCROMBIE Barry D. , CRANE‐ROBINSON Colyn , BRADBURY E. Morton
Published: March 2005

It has been shown by high‐resolution proton magnetic resonance (PMR) spectroscopy and circular dichroism (CD) that an H2A/H2B histone complex exists after salt extraction of these histones from chromatin and that this complex can be fully renatured from both urea‐denatured acid‐extracted and from urea‐denatured salt‐extracted histones. The histone complex is shown to involve specific secondary and tertiary structure. Formation of this complex is observed to be critically dependent on pH, occurring at and above pH 5. It cannot be induced below pH 5 by increase in ionic strength.

From CD spectra the H2A/H2B complex is shown to contain about 37%α helix but no β structure, the latter being confirmed by infrared spectroscopy in the 6‐μm region. The PMR spectra show that the structured region includes most of the aromatic residues of both histones, at least two histidine residues of H2B and probably histidines 31 and 82 of histone H2A.

The secondary structure of histones H2A and H2B is predicted using the Chou and Fasman procedure and comparisons are made between the predictions for histones of different species. These results in conjunction with the experimental evidence lead to the conclusion that at least residues 31–95 of H2A and residues 37–114 of H2B, i.e. the more apolar regions of the molecules, are involved in the tertiary structure of the H2A/H2B complex.
Stopped‐Flow Studies of Changes in Fluorescence of 8‐Anilino‐1‐naphthalene Sulfonic Acid Caused by Magnesium and Salt Binding to Yeast Enolase
Authors: BREWER John M.
Published: March 2005

Stopped‐flow studies of magnesium and salt (potassium chloride and acetate) effects on yeast enolase were carried out by following 8‐anilino‐1‐naphthalenesulfonic acid fluorescence changes. The fluorescence changes appear to be largely caused by subunit association and dissociation, though there is evidence in some reactions for large changes in fluorescence occurring within the dead time of the stopped‐flow measurements. These data are combined with measurements of initial enzyme activity after incubation in various solvents with or without magnesium to obtain subunit association and dissociation rates. From these, it is concluded that magnesium and the salts act by directly changing the affinities of the subunits for each other, apparently by producing a rapid change in protein conformation.
Interaction of DNA with DNA‐Binding Proteins
Authors: BERTHOLD Verena , GEIDER Klaus
Published: March 2005

DNA‐binding protein HD with a monomer molecular weight of 9000 was isolated from Escherichia coli cells. The protein occurs as a tetramer under native conditions and binds to single and double‐stranded DNA and also to RNA. DNA complexed with protein HD is a poor template for DNA synthesis by E. coli polymerase I, II or III holoenzyme. Exonuclease III is hindered in degrading HD‐protein‐covered double‐stranded DNA, whereas exonuclease I can digest complexed single‐stranded DNA. Transcription is slightly stimulated in the presence of protein HD.
Characterization and Site of Action of a Soluble Protein that Stimulates Peptide‐Bond Synthesis
Authors: GLICK Bernard R. , GANOZA M. Clelia
Published: March 2005

A recently identified soluble protein, named EF‐P, stimulates peptide bond synthesis from ribosomal‐bound N‐formylmethionyl‐tRNA and the aminoacyl‐tRNA analog, puromycin. Using this model of peptide bond formation we have purified this activity approximately 100‐fold from ribosome‐free extracts of Escherichia coli.

In order to study the mechanism by which the EF‐P factor stimulates peptide bond formation, we examined and compared the requirements and site of action of the spontaneous and the EF‐P‐mediated synthesis of peptide bonds. We find that ‘enzymic’ peptide bond synthesis (+EF‐P) is characterized by relatively broad temperature and NH4Cl optima, a sharp Mg2+ optimum at 12 mM, and an apparent pKa of approximately 8.5. The characteristics of enzymic peptide bond synthesis closely resemble those reported for native peptidyl‐puromycin formation rather than other models of peptide synthesis.

Factor EF‐P requires both 30‐S and 50‐S subunits for activity. The 30‐S particle is inactive by itself and may function in the reaction merely to bind the fMet‐tRNA substrate. Both the peptidyl transferase and the EF‐P binding site may be part of the 50‐S subunit.

Unlike all other propagation factors, EF‐P does not require the 50‐S ribosomal proteins L7 and L12 and may therefore occupy a different ribosomal site.
Levansucrase of Bacillus subtilis
Authors: CHAMBERT Régis , GONZY‐TREBOUL Geneviève
Published: March 2005

A covalently linked fructosyl‐enzyme complex was isolated from a reaction mixture of enzyme and sucrose submitted to the quenching effect of a large decrease of the pH. The fructosyl‐enzyme bond was shown to be stable under acidic and neutral conditions in the presence of high concentration of urea and of sodium dodecyl sulfate. This intermediate did not transfer at a measurable rate its fructosyl group to the usual fructosyl acceptors of the enzyme reaction under the usual conditions of enzyme activity. However stability measurements of the fructosyl‐enzyme bond indicated a marked lability at pH values above 8.5. The apparent rate constant of the hydrolytic reaction of this bond evaluated under the standard state of molar concentration of hydroxide ion was of the same order of magnitude as the apparent rate constant of the hydrolytic reaction of the transient fructosyl‐enzyme postulated from the kinetic analysis of levansucrase. Furthermore, nucleophilic agents like imidazole enhanced the hydrolytic reaction of the fructosyl‐enzyme bond. Identification of the fructosyl binding site on the enzyme was accomplished by proteolytic hydrolysis of the trapped complex. Peptic digestion followed by pronase digestion released a fructosyl‐aspartate compound that we have isolated in a high state of purity. The lability of the fructosyl‐aspartate bond under mild alkaline conditions suggested that the fructosyl was linked through an ester bond involving the β‐carboxyl of the aspartate residue. Treatment of the trapped complex with cyanogen bromide released only one fructosylated peptide. The apparent molecular weight of this peptide was estimated to be lower than 10000.
Symmetry and Asymmetry of the Pyruvate Dehydrogenase Complexes from Azotobacter vinelandii and Escherichia coli as Reflected by Fluorescence and Spin‐Label Studies
Authors: GRANDE Hans J. , VAN TELGEN Hendrik J. , VEEGER Cees
Published: March 2005

Fluorescence‐lifetime measurements of FAD bound to lipoamide dehydrogenase from Azotobacter vinelandii and Escherichia coli were performed. It is shown from these results that the two FAD groups in the isolated dimeric enzyme, as well as in the enzyme in the intact complex of E. coli, are in non‐equivalent surroundings. This contrasts with the near equivalence of the FAD groups of both the enzyme and complex isolated from A. vinelandii. Reduction of the complex with Mg2+, thiamine pyrophosphate and pyruvate or with NADH enables the attachment of a maleimide analogue specifically to the lipoyl moieties of the transacetylase(s). Spin label [N‐(1‐oxyl‐2,2,5,5‐tetramethyl‐3‐pyrrolidinyl)maleimide] introduced in such a way proves the existence of at least two different micro‐environments around the lipoyl moieties in complex isolated from A. vinelandii. Electron paramagnetic resonance spectra of the specifically spin‐labelled complexes from E. coli and A. vinelandii, when dissolved in tricine [N‐tris(hydroxymethyl)‐methylglycine] buffer, show interactions of at least two electron spins with each other, which indicate that the lipoyl moieties are rather close together.

Fluorescent label [N‐(1‐anilinonaphthyl‐4)maleimide] is specifically attached to the lipoyl moiety of the high‐Mr transacetylase of the freshly isolated complex from A. vinelandii. From the large differences in the apparent lifetimes τp and τm, as detected by phase fluorimetry, it is shown that this fluorescent label is distributed in different micro‐environments. The differences observed in energy transfer between fluorescent label, attached to the lipoyl moiety of the high‐Mr transacetylase, indicate different conformations of the complex from A. vinelandii. Upon introduction of the label after reduction with NADH a much larger energy transfer, thus a shorter distance, is observed between the label and FAD than when reduction is performed with Mg2+, thiamine pyrophosphate and pyruvate. A similar conformation dependence upon reduction is found for the pyruvate dehydrogenase complex from E. coli. It is thus proposed that the transacetylase of E. coli and the high‐Mr transacetylase of A. vinelandii are both non‐symmetrically distributed within the complex.
Conformational Analysis of a DNA Triplet in Aqueous Solution
Authors: ALTONA Cornelis , VAN BOOM Jacques H. , HAASNOOT Cornelis A. G.
Published: March 2005

The proton signals of a 50 mM solution of thymidylyl‐(3′‐5′)‐thymidylyl‐(3′‐5′)‐2′‐deoxyadenosine, d(T‐T‐A), in 2H2O at 65 °C and p2H = 7.4, were detected by nuclear magnetic resonance at 360 MHz. Complete assignment of the 21 ribose proton signals was achieved by extensive decoupling experiments and computer simulations. The coupling constant data show that the ribose rings prefer to adopt the S (2′‐endo) conformation. The presence of a substantial amount of a right handed helical structure of the d(‐T‐A) fragment is indicated from chemical shift considerations. The molecule displays a high degree of conformational purity along the sugar‐phosphate backbone.
Lipid Requirement of Membrane‐Bound 3‐Oxosteroid δ4—δ5‐Isomerase
Authors: GEYNET Philippe , DE PAILLERETS Christine , ALFSEN Annette
Published: March 2005

The role of phospholipid in the beef adrenal microsomal 3‐oxosteroid δ4—δ5‐isomerase (EC 5.3.1.1) has been investigated with the use of phospholipase A to alter the microsomal phospholipids. The byproducts of phospholipase A digestion have been removed with a wash solution containing bovine serum albumin. Removal of 80–85% of the phospholipid leads to loss of 80–90% of the 3‐oxosteroid δ4—δ5‐isomerase activity. Reconstitution experiments have been performed by introduction of lipid aqueous dispersions in the enzymatic assay. Asolectin, a commercially available preparation of soy phosphatides, is able to stimulate the enzymatic activity but does not restore the 3‐oxosteroid δ4—δ5‐isomerase activity in phospholipase‐A‐treated membranes. In contrast, the introduction of aqueous dispersions of microsomal total lipid mixtures in the enzymatic assay brings about a complete restoration of the 3‐oxosteroid δ4—δ5‐isomerase activity in the lipid‐depleted membranes. It is concluded that the bovine adrenal microsomal 3‐oxosteroid δ4—δ5‐isomerase requires phospholipid(s) to exhibit its full catalytic activity.
Biochemistry of Development in Insects
Authors: MEGIAS Alicia , MUNICIO Angel M. , PEREZ‐ALBARSANZ Miguel A.
Published: March 2005

A different biochemical behaviour has been previously shown during several stages of the development of the insect Ceratitis capitata; thus, the acyl transferase activity has a different participation in the pathways of synthesis of triacylglycerols and phosphoglycerides by either larval or pharate adult stages of development. The results reported here are concerned with the behaviour of mitochondria and microsomes from larvae and pharate adults for the synthesis of triacylglycerols and phosphoglycerides. In all preparations of either mitochondrial or microsomal fractions the optimum protein amount, previously determined, has been used.

The highest incorporation of [14C]palmitate into triacylglycerols and phosphoglycerides was achieved by the microsomal fraction from the larval stage of development of the insect. However, mitochondrial fractions from pharate adults utilized higher levels of [14C]palmitate than the microsomal preparation. These differences were not correlated with the palmitoyl‐CoA synthetase activity.

Glycerol 3‐phosphate influenced the fatty acid incorporation in a different manner depending on the stage of development of the insect. Increasing concentrations of glycerol 3‐phosphate stimulated the synthesis of triacylglycerols by microsomal and mitochondrial preparations. This effect was not exhibited by the subcellular preparations of pharate adults.

Double‐label experiments using [14C]glycerol 3‐phosphate and [3H]palmitate showed qualitative differences in the synthesis of triacylglycerols by mitochondrial preparations from either larvae or pharate adults. Incorporation of the labelled fatty acids to endogenous triglycerides was preferentially shown by the mitochondrial larval preparations. The pathways of synthesis of triacylglycerols and phosphoglycerides by microsomal preparations showed only quantitative differences depending on the stage of development of the insect.

Acylation of 14C‐labelled lyso‐phosphatidylcholine and 14C‐labelled lyso‐phosphatidylethanolamine by [3H]olate was assayed in the presence of mitochondrial and microsomal preparations from the different stages of development of the insect. Microsomal fractions showed an efficient acyl transferase activity mainly when lyso‐phosphatidyl‐ethanolamine was used as substrate.
Lysozyme from the Insect Ceratitis capitata Eggs
Authors: FERNANDEZ‐SOUSA José M. , GAVILANES José G. , MUNICIO Angel M. , PEREZ‐ARANDA Agustin , RODRIGUEZ Rosalia
Published: March 2005

Lysozyme from eggs of the Dipterous Ceratitis capitata (Wiedeman) has been purified by ionexchange chromatography and gel filtration and its physicochemical properties have been investigated. This is the first insect lysozyme characterized so far and it exhibits some properties different to those described for other animal lysozymes.

Lysozyme from the insect eggs has a molecular weight of about 23200 and a sedimentation coefficient of 2.4 S. Molecular weight determination by sodium dodecylsulphate gel electrophoresis indicates that the molecule consists of a single polypeptide chain.

This lysozyme preparation shows notable stability at acidic pH values and lability at alkaline pH values. It shows a single optimum pH at about 6.5.

Chitinase/muramidase specific activity ratio is around 350 times higher for the insect lysozyme than for the hen egg‐white enzyme.

The amino acid composition shows the presence of one tryptophan residue per molecule of enzyme. This fact differentiates the lysozyme from insect eggs from other animal and plant lysozymes. From the amino acid composition, the absorption coefficient and the partial specific volume are calculated.

Glycine is the N‐terminal residue.
Proton Translocation and ATP Formation Coupled to Electron Transport from H2O to the Primary Acceptor of Photosystem 2
Authors: BEN‐HAYYIM Gozal , NEUMANN Joseph
Published: March 2005

The rate of electron transport from H2O to silicomolybdate in the presence of 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (diuron) (which involves the oxygen‐evolving enzyme, the photochemistry of photosystem 2 and the primary electron acceptor of photosystem 2) is controlled by internal pH. This is based on the shift of the pH profile of the rate of electron transport upon addition of uncouplers, or by using EDTA‐treated chloroplasts. Both stimulation and inhibition of electron transport by addition of uncouplers (depending on external pH) could be observed. These effects are obtained in the diuron‐insensitive photoreductions of either silicomolybdate or ferricyanide. These experiments provide strong evidence that a proton translocating site exists in the sequence of the electron transport H2O→ Q (the primary acceptor of photosystem 2).

The photoreduction of silicomolybdate in the presence of diuron causes the formation of Δφ. The value of Δφ depends on the external pH and its maximal value was shown to be 2.4. The calculated internal pH at different external pH values was found to be rather constant, namely between 5.1‐5.2.

Electron transport from H2O to silicomolybdate (in the presence of diuron) does not support ATP formation.

It is suggested that this is due to the fact that the Δφ formed is below the “threshold”Δφ required for the synthesis of ATP. By adding an additional source of energy in the form of a dark diffusion potential created in the presence of K+ and valinomycin, significant amounts of ATP are formed in this system.
Structural Studies on the Coat Protein of Alfalfa Mosaic Virus
Authors: VAN BEYNUM Gerard M. A. , DE GRAAF J. Martien , CASTEL Ad , KRAAL Barend , BOSCH Leendert
Published: March 2005

The complete amino acid sequence of the coat protein of alfalfa mosaic virus (strain 425) is reported. Sequence determinations were mainly performed on peptides obtained from fragmentation by cyanogen bromide and trypsin. Both manual and automatic sequence methods were used. Some refinements of the solid‐phase Edman degradation were introduced. The final alignment of the peptides was established by means of alternative cleavage methods, such as limited tryptic digestion of intact virus particles, tryptic digestion after blockage of lysine residues and chymotryptic digestion. The coat protein consists of 220 amino acid residues corresponding to a molecular weight of 24252. A remarkable clustering of basic residues occurs in the N‐terminal part of the protein chain. Several internal hydrophobic clusters and a strongly acidic site at the C‐terminus can be observed. Two regions of sequence homology (12 residues) were found. Some features of the secondary structure are predicted.
Calorimetric Studies on Melting of tRNAPhe (Yeast)
Authors: HINZ Hans‐Jürgen , FILIMONOV Vladimir V. , PRIVALOV Peter L.
Published: March 2005

The heat effects involved in thermal unfolding of tRNAPhe from yeast have been determined in various buffer systems by direct differential scanning calorimetry. Perfect reversibility of the melting process has been demonstrated for measurements in the absence of Mg2+ ions. The overall molar transition enthalpy, ΔHt= 298 ± 15 kcal mol−1 (1247 ± 63 kJ mol−1), has been shown to be independent of the NaCl concentration and the nature of the buffers used in this study. ΔHt is identical in the presence and in the absence of Mg2+ ions within the margin of experimental error. This experimental result implies a vanishing or very small heat capacity change to be associated with melting. Decomposition of the calorimetrically determined complex transition curves, on the assumption that the experimental melting profile represents the sum of independent two‐state transitions, results in five transitions which have been assigned to melting of different structural domains of the tRNA.
myo‐Inositol Transport in Klebsiella aerogenes
Authors: DESHUSSES Jacques , REBER Guido
Published: March 2005

scyllo‐Inositol is transported against a concentration gradient by Klebsiella aerogenes, but is not metabolized.

The apparent KT of scyllo‐inositol is 0.05 × 10−3 M while that of myo‐inositol is 0.23 × 10−3 M. The V values are respectively 32 and 12 nmol min−1 mg cells−1 (dry weight).

Both cyclitols bind to the same carrier, since typical competitive inhibition kinetics are observed.

Neither phosphoenolpyruvate phosphotransferase nor a periplasmic binding protein seems to be involved in the scyllo‐inositol transport system.

The induction of the transport system is not repressed by malate, fumarate, citrate, succinate or pyruvate; however, acetate represses the induction. The activity of the transport system is increased when the growth medium is supplemented with adenosine 3′:5′‐monophosphate.
Transport of Cyclitols by a Proton Symport in Klebsiella aerogenes
Authors: REBER Guido , MERMOD Marion , DESHUSSES Jacques
Published: March 2005

The respiration and the ATP content of Klebsiella aerogenes in the presence of various inhibitors were compared to the transport of scyllo‐inositol. The ATPase was found to be inhibited by dicyclohexyl carbodiimide. The transport has been tested in anaerobiosis and aerobiosis. From the results obtained it is concluded that either ATP or respiration can sustain the transport activity in independent manner.

The energy derived from the respiratory chain reactions or the ATP hydrolysis results in electrogenic extrusion of protons. The electrochemical potential created drives the accumulation of scyllo‐inositol, as shown by an increase of pH of the medium on addition of the substrate to cells in anaerobiosis. With non‐induced cells no change in pH occurs, which demonstrates that proton flow is really linked to the transport. No H+/Na+ or K+ exchange is observed and the proton conductor carbonylcyanide m‐chlorophenylhydrazone abolishes the pH shift caused by substrate addition. The stoichiometry of the symport H+/cyclitol is 1 and the half‐maximum value of the pH variation as a function of the amount of scyllo‐inositol added corresponds to a concentration of scyllo‐inositol very close to the KT of influx.
Kinetic Studies on a 4‐Methoxybenzoate O‐Demethylase from Pseudomonas putida
Authors: BERNHARDT Frithjof‐Hans , NASTAINCZYK Wolfgang , SEYDEWITZ Vera
Published: March 2005

A direct, sensitive and reliable photometric assay procedure for monitoring the activity of nonspecific 4‐methoxybenzoate O‐demethylases of microorganisms is described. The assay is based on the O‐demethylation of 3‐nitro‐4‐methoxybenzoate to the yellow‐coloured product 3‐nitro‐4‐hydroxybenzoate. Using this assay and by monitoring the oxidation rate of reduced pyridine nucleotides, the kinetic properties of a purified, reconstituted enzyme system composed of 4‐methoxybenzoate monooxygenase (O‐demethylating) and a reductase from Pseudomonas putida have been investigated.

It has been found that the Km value of the monooxygenase of this enzyme system towards different substrates (i.e. tight couplers, uncouplers and partial uncouplers) rises from the extremely low value of 0.07 μM for the tight couplers to about 55 μM for the uncouplers.

The effect of possible inhibitors and metal ions on the reconstituted enzyme system was investigated. The inhibition pattern was almost identical to that found for the purified reductase, only batho‐phenanthrolinedisulfonate showing a greater inhibition of the reconstituted enzyme system.

The affinity of the reductase towards NADH was found to be approximately 200‐fold greater than that towards NADPH. Furthermore, the affinity of this reductase to NADH depended on the nature of the electron acceptor. The affinity to NADH was more than 10 times higher when the monooxygenase. substrate complex was used as the electron acceptor, than when cytochrome c or 2,6‐dichloroindophenol was used. These differences are discussed on the basis of enzyme‐enzyme interactions between the reductase and the monooxygenase.
Cyclic Nucleotide Hydrolysis in the Thyroid Gland
Authors: ERNEUX Christophe , VAN SANDE Jacqueline , DUMONT Jacques E. , BOEYNAEMS Jean‐Marie
Published: March 2005

Phosphodiesterase activities of horse (and dog) thyroid soluble fraction were compared with either cyclic AMP (adenosine 3′: 3′‐monophosphate) or cyclic GMP (guanosine 3′: 5′‐monophosphate) as substrate. Optimal activity for cyclic AMP hydrolysis was observed at pH 8, and at pH 7.6 for cyclic GMP. Increasing concentrations of ethyleneglycol bis(2‐aminoethyl)‐N, N‐tetraacetic acid inhibited both phosphodiesterase activities; in the presence of exogenous Ca2+, this effect was shifted to higher concentrations of the chelator. In a dialysed supernatant preparation, Ca2+ had no significant stimulatory effect, but both Mg2+ and Mn2+ increased cyclic nucleotides breakdown. Mn2+ promoted the hydrolysis of cyclic AMP more effectively than that of cyclic GMP. For both substrates, substrate velocity curves exhibited a two‐slope pattern in a Hofstee plot. Cyclic GMP stimulated cyclic AMP hydrolysis, both nucleotides being at micromolar concentrations. Conversely, at no concentration had cyclic AMP any stimulatory effect on cyclic GMP hydrolysis. 1‐Methyl‐3‐isobutylxanthine and theophylline blocked the activation by cyclic GMP of cyclic AMP hydrolysis, whereas Ro 20‐1724 (4‐(3‐butoxy‐4‐methoxybenzyl)‐2‐imidazolidinone), a non‐methylxanthine inhibitor of phosphodiesterases, did not alter this effect. In dog thyroid slices, carbamoylcholine, which promotes an accumulation of cyclic GMP, inhibits the thyrotropin‐induced increase in cyclic AMP. This inhibitory effect of carbamoylcholine was blocked by theophylline and 1‐methyl‐3‐iso‐butylxanthine, but not by Ro 20–1724. It is suggested that the cholinergic inhibitory effect on cyclic AMP accumulation is mediated by cyclic GMP, through a direct activation of phosphodiesterase activity.
Formate Hydrogenlyase System in Salmonella typhimurium LT2
Authors: CHIPPAUX Marc , PASCAL Marie‐Claire , CASSE Francine
Published: March 2005

Isolation from Salmonella typhimurium of mutants unable to reduce benzyl viologen under anaerobic conditions has allowed the study of the factors involved in the multienzymic formate hydrogenylase system.

Depending on the affected activities, different classes of mutants were found: FHL‐A mutants have lost formate dehydrogenase 1 and formate dehydrogenase 2 activities; mutations in fdhA (117 min) or fdhB (33 min) lead to such a phenotype. FHL‐B and FHL‐C mutants have lost formate dehydrogenase 2 activity and part or all of hydrogenase activity, respectively; both phenotypes correspond to mutations in the hyd gene (approximately 90 min). FHL‐D mutants have lost only formate dehydrogenase 2 activity; fhlD gene maps at 120 min.

In some cases, mixtures of extracts from two mutants display formate dehydrogenase 2 and formate hydrogenylase activities. Restoration studies suggest the existence of one factor sensitive to growth conditions and inactivated by oxygen or heating. This factor which is present and active in FHL‐C mutants, is probably the one missing in FHL‐D mutants.

A new scheme for the formate hydrogenylase system is proposed, in which hydrogenase transfers electrons directly to benzyl viologen.
On the Optical Activity of Ionized Tyrosyl Residues in Ovine Lutropin
Authors: ASCOLI Mario , WARD Darrell N. , JIRGENSONS Bruno
Published: March 2005

The effect of alkali on the circular dichroic (CD) spectra of ovine lutropin and its subunits has been studied. Mild alkaline pH induces the appearance of a new optically active band in the 250‐nm region of the spectra of lutropin without any detectable alteration in the secondary structure of the protein. This change is reversible and can be correlated with ionization of 2–3 exposed tyrosyl residues in the intact hormone. In a previous report from this laboratory it was concluded that the three exposed tyrosyl residues are located in the α subunit, in positions 21, 92 and 93 [Burleigh, B. D., Liu, W.‐K. and Ward, D. N. (1976) J. Biol. Chem. 251, 308–315]. Nitration of these residues lowers the pH at which the intensity of the 250‐nm band is maximal.

The importance of the tyrosyl residues of lutropin α (as opposed to those of lutropin β) is also supported by the similarity of the effect of alkali on the CD spectra of lutropin and lutropin α. Further evidence for this involvement was also obtained by a comparison of the alkali‐induced changes of refolded lutropin (α+β recombinant) and the product obtained by recombination of des‐(92–96)‐lutropin α (obtained from carboxypeptidase treatment of the α‐subunit) and lutropin β. The results indicate that removal of tyrosines α92 and α93 results in a decrease of the intensity of the 235‐nm band of ovine lutropin (at pH 7.5) as well as that of the 250‐nm band observed under alkaline conditions. It is therefore concluded that the 250‐nm band observed in alkaline solutions of lutropin arises (at least partially) from the red shift produced in the short‐wavelength optically active band of tyrosines α21, α92, and α93 upon ionization.
Phosphorylation in vitro and in vivo of Ribosomal Proteins from Saccharomyces cerevisiae
Authors: HÉBERT Josiane , PIERRE Michel , LOEB Jacques E.
Published: March 2005

Crude ribosomes from Saccharomyces cerevisiae cultures were phosphorylated in vitro when incubated in the presence of [γ‐32P]ATP. Analysis of the ribosomal proteins with two‐dimensional electrophoresis revealed that of the 29 proteins identified in the small subunit, only protein S6 was phosphorylated. Of the 37 proteins identified in the large subunit, one was highly phosphorylated (L3) and two only slightly phosphorylated (L11 and L14). The protein kinase activity associated with the ribosomes was extracted with 1 MKC1 and was not dependent on adenosine 3′:5′‐monophosphate; it preferentially phosphorylated casein and phosvitin, but was less active on histones.

Structural ribosomal proteins were also phosphorylated in vivo when the yeast cultures were incubated with [32P]orthophosphate; the radioactivity resistant to hydrolysis by hot perchloric acid was incorporated into the proteins of the two subunits. Radioactive phosphoserine was found by subjecting hydrolysates of ribosomal proteins to high‐voltage electrophoresis. After two‐dimensional electrophoresis, one poorly phosphorylated protein (S10) was identified in the small subunit. In the large subunit, one protein (L3) was highly labelled, and two proteins (L11 and L24) only slightly labelled.
Binding Studies of a Spin‐Labelled Oxidized Coenzyme to Bovine‐Liver Glutamate Dehydrogenase
Authors: ZANTEMA Alt , TROMMER Wolfgang E. , WENZEL Herbert , ROBILLARD George T.
Published: March 2005

NAD+ with a nitroxide piperidine ring linked to the NH2 group of the adenine possesses full coenzymatic activity with glutamate dehydrogenase. Electron spin resonance spectra in the presence of glutamate dehydrogenase show mixtures of free and strongly immobilized spin‐label. Binding studies in phosphate buffer demonstrate: (a) weak binary binding to the enzyme with a dissociation constant in the order of 2mM; (b) an indication for negative cooperativity or different sites for binding to enzyme. 2‐oxoglutarate, with dissociation constants in the order of 20–250 μM; (c) similar but much weaker binding to enzyme. 2‐oxoglutarate. ADP; (d) a strong positive cooperative binding to enzyme. 2‐oxoglutarate. GTP, dependent on the enzyme concentration.

Binding of phosphate to the enzyme with a Kd of about 20 mM or binding of pyrophosphate or tripolyphosphate with a Kd of about 2.5 mM enhances the binding of spin‐labelled NAD+ in the presence of 2‐oxoglutarate. There is evidence that the binding sites for these phosphates coincide with phosphate binding subsites of GTP.
The Stereochemical Basis of Template Function
Authors: RACKWITZ Hans Richard , SCHEIT Karl Heinz
Published: March 2005

The behaviour of nucleotides with thioketo‐substituted pyrimidine bases (4‐thiouracil, 2‐thiouracil and 2‐thiocytosine) or amino‐analogue purine bases (2‐aminopurine and 2,6‐diaminopurine) in transcription and translation was investigated. The experimental results obtained led to the following conclusions.

The stereochemical basis of substrate selection in transcription is the geometry of Watson‐Crick base pairs A · U (or A · T) and G · C between substrate and template bases.

The topology of the active site of Escherichia coli RNA polymerase is precisely adopted to the geometry of Watson‐Crick base pairs.

The enzyme active site discriminates between A·U (A · T) and G · C base pairs. An essential feature in this discrimination is the 6‐NH2 group of the A · U (A · T) base pair and the 2‐keto group of cytosine in the G · C base pair.

The codon properties of a nucleic acid base in messenger RNA can be predicted on the basis of its specificity in polynucleotide interactions. There seems to be no evidence for the participation of protein topological sites in the control of the specificity of codon‐anticodon interactions in translation.
Purification and Control of Bovine Adrenal Cortical Cholesterol Ester Hydrolase and Evidence for the Activation of the Enzyme by a Phosphorylation
Authors: BECKETT Geoffrey J. , BOYD George S.
Published: March 2005

A procedure for the purification of cholesterol ester hydrolase from bovine adrenal cortical 105000 ×g supernatant is described. Preincubation of a crude enzyme extract with [γ‐32P]ATP followed by purification resulted in the isolation of a phosphorylated preparation of cholesterol ester hydrolase. The phosphorylated cholesterol ester hydrolase appeared to be composed of 4 subunits, each having a molecular weight of 41000 ± 280 only one of which may be phosphorylated. Preincubation of the crude enzyme preparation with [α‐32P]ATP followed by purification did not produce a phosphorylated preparation of cholesterol ester hydrolase.

Cyclic‐AMP‐dependent protein kinase, cyclic AMP, ATP and magnesium ions were required for activation of purified cholesterol ester hydrolase in vitro and the time course of activation closely paralleled the time course of phosphorylation of the enzyme. The addition of ATP, cyclic AMP and magnesium ions to the bovine adrenal cortical 105000 ×gsupernatant produced a 2.5‐fold stimulation in cholesterol ester hydrolase activity. This stimulation was abolished if protein kinase inhibitor was added prior to the addition of ATP, cyclic AMP and magnesium ions.

The addition of magnesium ions or calcium ions to a crude preparation of cholesterol ester hydrolase was found to inhibit activity; however the same additions made to a purified preparation of cholesterol ester hydrolase were not inhibitory. The decrease in cholesterol ester hydrolase activity on incubation with magnesium ions was accompanied by a loss of 32P radioactivity from the protein. Preincubation of a crude preparation of cholesterol ester hydrolase with alkaline phosphatase resulted in a deactivation of cholesterol ester hydrolase.

It is suggested that bovine adrenal cortex cholesterol ester hydrolase is activated by a phosphorylation catalysed by a cyclic‐AMP‐dependent protein kinase. Deactivation of cholesterol ester hydrolase is accomplished by dephosphorylation catalysed by a phosphoprotein phosphatase, dependent on magnesium or calcium ions.
Effect of 2‐Deoxy‐d‐glucose on the Cell‐Surface Glycoproteins of Hamster Fibroblasts
Authors: HUGHES R. Colin , MEAGER Anthony , NAIRN Roderick
Published: March 2005

Growth of baby hamster kidney (BHK) cells in medium containing 2‐deoxy‐d‐glucose is retarded in direct proportion to the 2‐deoxyglucose concentration. The severity of the effect is reduced in medium containing high relative concentrations of glucose.

2‐Deoxyglucose inhibits the incorporation of radioactivity from mannose, galactose, glucosamine, fucose and N‐acetylmannosamine precursors into acid‐insoluble cellular material. Incorporation of radioactively labelled leucine into protein is not affected by 2‐deoxyglucose.

BHK cells grown in the presence of 2‐deoxyglucose become less sensitive to the toxic action of certain plant lectins, ricin of Ricinus communis and Phaseolus vulgaris phytohaemagglutinin, which bind specifically to cell surface galactose and N‐acetyl‐galactosamine residues. By contrast, 2‐deoxyglucose increased the sensitivity of BHK cells to the weak toxicity of concanavalin A which binds to surface mannosides. Treated cells also become more agglutinable with concanavalin A.

Cell surface glycoproteins labelled by lactoperoxidase‐catalysed iodination have been examined by dodecylsulphate‐polyacrylamide gel electrophoresis. The radio‐iodinated glycoproteins prepared from cells grown in medium containing 2‐deoxyglucose migrate more rapidly than glycoproteins from cells grown in the absence of inhibitor.
Properties of a Baby‐Hamster‐Kidney Cell Line with Increased Resistance to 2‐Deoxy‐d‐glucose
Authors: MEAGER Anthony , NAIRN Roderick , HUGHES R. Colin
Published: March 2005

A cultured cell line with increased resistance to 2‐deoxy‐d‐glucose was obtained from cloned baby hamster kidney fibroblasts, BHK 21/C13, after repeated exposure to high concentrations of 2‐deoxyglucose. The increased resistance could not be attributed to a decreased permeability of deoxysugar. The resistant cell line incorporated radioactive 2‐deoxy‐d‐glucose into glycoproteins at a similar rate as parental BHK 21/C13 cells. Incorporation of radioactive glucosamine, galactose and to lesser extent mannose into cellular glycoproteins was inhibited by 2‐deoxyglucose to similar extents in the resistant cells and parental BHK 21/C13 cells. Changes induced by 2‐deoxyglucose in cell surface carbohydrate structure of the resistant cells were detected by altered sensitivities to toxic plant lectins and by surface labelling as described for parental cells in the preceding paper. It is suggested that the toxicity of 2‐deoxy‐d‐glucose to normal fibroblasts is not mediated through effects on glycosylation of cellular glycoproteins.
On the Role of Ferredoxin and Ferredoxin‐NADP+Reductase in Cyclic Electron Transport of Spinach Chloroplasts
Authors: BÖHME Herbert
Published: March 2005

Antibodies prepared against purified spinach ferredoxin and ferredoxin‐NADP+reductase were used as specific inhibitors of electron‐transfer reactions dependent on either ferredoxin or ferredoxin‐NADP+reductase. The possible role of both electron carriers in cyclic electron transport was checked using cytochromeb6photoreactions as indicator. It could be demonstrated that the ferredoxin antibody inhibits cytochromeb6photoreduction. Ferredoxin‐NADP+reductase, however, appears not to be involved in this pathway: reductase antibody in concentrations sufficient to completely inhibit electron transport to NADP+had no effect on cytochromeb6photoreduction.

Quantitative treatment of the immunoassay data showed that osmotically shocked chloroplasts contain both bound ferredoxin and ferredoxin‐NADP+reductase in concentrations approximately equal to that of cytochromeb6.
Biophysical and Enzymological Studies upon the Interaction of trans‐Cinnamic Acid with Higher Plant Microsomal Cytochromes P‐450
Authors: RICH Peter R. , LAMB Christopher J.
Published: March 2005

The interaction of trans‐cinnamic acid with the cytochrome P‐450 of microsomes derived from washed potato slices has been studied. The washing process increased the specific content of microsomal electron transport components and hence provided a useful material in which to study the interaction.

Evidence is presented that the trans‐cinnamic acid interacts with the cytochrome P‐450, and that this interaction is analogous to ‘type I’ interactions of other cytochrome P‐450 systems. This evidence includes the formation of a ‘type I’ substrate binding spectrum, an increased rate of reduction of cytochrome P‐450 by NADPH in the presence of trans‐cinnamic acid, an increased oxygen uptake and NADPH oxidation when trans‐cinnamic acid is added to the microsomes in the presence of NADPH, and a close correlation between biophysical parameters of electron transport in the cytochrome P‐450 system and enzymological parameters of the trans‐cinnamic acid 4‐hydroxylation reaction.

The investigation has been extended to cytochrome P‐450 systems of other tissues and it has been found that the trans‐cinnamic acid 4‐hydroxylation reaction cannot account for the presence of most of the cytochrome P‐450 in several tissues. This suggests that other functions of higher plant cytochrome P‐450 chains exist, and that the substrate specificity of the hemoprotein may vary in different plant tissues.
Progesterone Receptors in the Chick Oviduct.
Authors: MESTER Jan , BAULIEU Etienne‐Emile
Published: March 2005

Exchange techniques were developed for measurement of total progesterone receptor binding sites concentration in the cytosol and nuclei of chicken oviduct.

The level of progesterone receptor in the cytosol was under both oestrogen and progesterone control. Primary stimulation by oestradiol benzoate increased the receptor concentration from approximately 10000 sites/cell to approximately 40000 sites/cell in the magnum cytosol; subsequent withdrawal from oestrogen treatment led to a decrease to approximately 14000 sites/cell after 6 weeks. After progesterone administration (3 mg/kg) to oestrogen‐stimulated, withdrawn chicken, the receptor concentration decreased to approximately 40% of the initial level within the first 4 h; afterwards the receptor level rose again and by 40 h exceeded slightly the initial one.

The nuclear levels of the receptor reached a maximum at 1 h after the progesterone injection; however, the gain of the binding sites by the nuclei (∼ 900/nucleus) did not account for their loss from the cytosol. The maximum nuclear receptor level was not influenced by the dose of progesterone within range of 1‐10 mg/kg.

The extractibility of the nuclear progesterone receptor by 0.5 M NaCl was strongly influenced by preincubation of the nuclei at 30 C; irrespective of this preincubation, approximately 12% of the nuclear receptor resisted extraction with 2 M NaCl/5 M urea.
Differences between Alcohol Dehydeogenases
Authors: JÖRNVALL Hans
Published: March 2005

Comparisons of the primary structures of yeast and horse liver alcohol dehydrogenases reveal that the enzymes are homologous but distantly related. The overall positional identity is 25% between common regions, and several deletions/insertions occur in either enzyme, the longest apparently corresponding to 21 residues, showing that the different subunit sizes are largely explained by internal differences.

Variabilities in the structural similarities can be coupled with functional requirements but not directly with whole domains in the previously known tertiary structure of the horse protein. The two most similar regions of the enzymes affect active‐site segments and the two most dissimilar regions seem to affect a loop structure without known function, and a segment participating in subunit interactions. The dissimilarities may probably be correlated with changes in zinc‐binding properties and quaternary structures.

The extra region corresponding to the large internal chain‐length difference shows an apparent coincidence in sequence to a following segment of the horse enzyme, and additional elements of internal coincidences, or superficial similarities with other dehydrogenases, are noticed. These characteristics are not fully distinguishable from chance distributions but in view of the extensive species variations in alcohol dehydrogenases some evolutionary considerations may not be excluded, in which case a model relating all regions of these and associated enzymes to a common ancestor is shown to be compatible with all known observations.
Nucleotide Sequence (n= 159) of the Amino‐Acid‐Accepting 3′‐OH Extremity of Turnip‐Yellow‐Mosaic‐Virus RNA and the Last Portion of Its Coat‐Protein Cistron
Authors: BRIAND Jean‐Paul , JONARD Gérard , GUILLEY Hubert , RICHARDS Kenneth , HIRTH Léon
Published: March 2005

The experiments described in this paper and the following one establish the sequence of the 3′‐OH terminal 159 nucleotides of turnip yellow mosaic virus RNA. Uniformly 32P‐labeled turnip yellow mosaic virus RNA was partially digested with T1 ribonuclease and the fragments were fractionated by polyacrylamide gel electrophoresis. Fragments originating from the 3′‐OH end of the RNA molecule were identified by testing for the 3′‐terminal oligonucleotide, C‐COH, after total U2 ribonuclease hydrolysis. Once identified, the 3′‐OH terminal fragments were sequenced by the methods of Sanger et al. The first 51 nucleotides of the longest of the sequenced fragments (158 nucleotides) extends into the 3′‐terminal part of the coat protein cistron. The coat protein cistron is followed by a stretch of 108 untranslated nucleotides whose function, though still unknown, is probably linked to the tRNA‐like properties which have been attributed to the 3′‐OH extremity of this viral RNA. Two possible secondary structures are proposed for the sequence and the implications of the findings with regard to the tRNA‐like properties of the extremity are discussed.
Studies on the Sequence of the 3′‐Terminal Region of Turnip‐Yellow‐Mosaic‐Virus RNA
Authors: SILBERKLANG Melvin , PROCHIANTZ Alain , HAENNI Anne‐Lise , RAJBHANDARY Uttam L.
Published: March 2005

A fragment representing the 3′‐terminal ‘tRNA‐like’ region of turnip yellow mosaic (TYM) virus RNA has been purified following incubation of intact TYM virus RNA with Escherichia coli‘RNase P’. This fragment, which is 112 ± 3‐nucleotides long has been completely digested with T1 RNase and pancreatic RNase and all the oligonucleotides present in such digests have been sequenced using 32P‐end labelling techniques in vitro. The TYM virus RNA fragment is free of modified nucleosides and does not contain a G‐U‐U‐C‐R sequence. Using nuclease P1 from Penicillium citrinum, the sequence of 26 nucleotides from the 5′ end and 16 nucleotides from the 3′ end of this fragment has been deduced. The nucleotide sequence at the 5′ end of the TYM virus RNA fragment indicates that this fragment includes the end of the TYM virus coat protein gene.
Selective Inhibition of tRNATyr Transcription by Guanosine 3′‐Diphosphate 5′‐Diphosphate
Authors: DEBENHAM Paul , TRAVERS Andrew
Published: March 2005

Guanosine 3′‐diphosphate 5′‐diphosphate (ppGpp) selectively reduces the synthesis of su+III tRNA from θ80 psu+III DNA relative to the synthesis of θ80 RNA in a system in vitro containing DNA and Escherichia coli RNA polymerase holoenzyme as the sole macromolecular components. The response of su+III tRNA synthesis to increasing salt and to temperature in the presence of ppGpp suggests that the nucleotide may reduce the affinity of the enzyme for su+III promoters. The Ki for the selective inhibition of tRNA synthesis by ppGpp is 4μM in contrast to the value of 150 μM for the inhibition of rRNA synthesis.