bluuebunny/crossref_metadata_2025 huggingface dataset:
tRNA Conformation and Magnesium Binding
Authors: RÖMER Roland , HACH Renate
Published: March 2005

The binding of Mg2+ to tRNAPhe (yeast) in three conformational states was studied at 10, 30, 45, and 70°C by the fluorescence indicator 8‐hydroxyquinoline 5‐sulphonic acid in the presence of 0.032 M monovalent cations (Na+). At temperatures below those characteristic for early melting (completely folded tRNA) the Scatchard plots are biphasic. They are well fitted by two classes of noninteracting binding sites with stability constants independent of temperature (KA= 9 × 104, KB= 6 × 103 M−1). In partially unfolded tRNA the strong binding process is co‐operative. A single class of weak sites was found in the statistically coiled conformation at 70°C (KB= 3.3 × 103 M−1). The total number of binding sites is 23 ± 5; differences for the folded and unfolded conformations are smaller than 1. The influence of Mg2+ on the stability of the conformational elements of tRNAPhe (yeast) and its CCA‐half (i.e. nucleotides 38–76) was determined by differential ultraviolet absorbance and depolarisation melting curves using the fluorescence of the Y base. Tertiary structure corresponding to early melting is stabilized by strongly bound Mg2+, whereas all other melting transitions are only influenced by Mg2+ bound at weak sites. The stability constants of tertiary structure obtained from the melting experiments can quantitatively be described by assuming that 5 ± 1 non‐interacting strong sites as characterized by the fluorescence titrations are converted to weak sites upon unfolding of the tertiary structure. Co‐operative interaction of Mg2+ with the 5 strong sites in the folded conformation of tRNA can be ruled out. Strong binding of Mg2+ to completely folded tRNA does not produce a conformational transition changing ultraviolet absorbance, circular dichroism and sedimentation coefficient.
Two Ribose‐5‐Phosphate Isomerases from Escherichia coli K12: Partial Characterisation of the Enzymes and Consideration of Their Possible Physiological Roles
Authors: ESSENBERG Margaret K. , COOPER Ronald A.
Published: March 2005

Two physically and genetically distinct forms of ribosephosphate isomerase have been identified in Escherichia coli K12. The constitutive ribosephosphate isomerase A has a Km for ribose 5‐phosphate (4.4 ± 0.5 mM) six times greater than that of the inducible ribosephosphate isomerase B (0.83 ± 0.13 mM). Treatment of the enzymes with 1.25 mM iodoacetate resulted in 100% loss of activity for ribosephosphate isomerase B, whereas ribosephosphate isomerase A was unaffected. Various cellular metabolites were tested and found to be without significant effect on either enzyme. The two enzymes could be separated by filtration on Sephadex G75 superfine and their apparent molecular weights were 45000 for ribosephosphate isomerase A and 32000–34000 for ribosephosphate isomerase B. Under certain conditions the two enzymes showed different patterns of heat inactivation but the results with ribosephosphate isomerase A varied in an unusual way with the protein concentration. Ribosephosphate isomerase B was formed inducibly in a mutant lacking ribosephosphate isomerase A but there was no evidence for the production of ribosephosphate isomerase B in wild‐type cells. The formation of ribosephosphate isomerase B was not a consequence of the ribosephosphate isomerase A mutation, since strains could be constructed which formed both enzymes constitutively in the anticipated amounts. The ribosephosphate isomerase formed by a secondary mutant obtained from a ribosephosphate‐isomerase‐A‐negative strain was identified as ribosephosphate isomerase B on the basis of its Km, elution profile from Sephadex G75, inhibition by iodoacetate, and heat inactivation. The ribosephosphate isomerases of another Escherichia coli K12 strain, X289, were investigated, since their properties were reported to be different from many of these described here for ribosephosphate isomerases A and B. In our hands strain X289 contained two ribosephosphate isomerases apparently identical to ribosephosphate isomerases A and B. The evidence to date suggests that ribosephosphate isomerase A catalyses the formation of ribose 5‐phosphate from ribulose 5‐phosphate and also participates in the reverse reaction during ribose and adenosine catabolism. The normal physiological role of the inducible ribosephosphate isomerase B is still uncertain.
The Energy‐Coupling Controlled Efflux of 2‐Keto‐3‐deoxy‐d‐gluconate in Escherichia coli K 12
Authors: LAGARDE Alain E. , STOEBER François R.
Published: March 2005

Experiments were devised to test the plausibility and the predictions of an efflux rate equation which was previously derived [10].

2‐Keto‐3‐deoxy‐d‐gluconate transport system conforms with universal laws relating zero‐trans influx, influx at steady‐state, steady‐state levels of accumulation to external and internal substrate concentrations.

Full‐time‐course uptake kinetics fit the linearized graphical representation that can be inferred from the integrated rate equation.

Influx does not depend upon internal substrate concentration nor upon energy‐coupling.

Zero‐trans outflux (leak into empty medium) is a first‐order process (rate constant:0.02 min−1) and not mediated by the carrier. Absence of cis‐competition with d‐glucuronate is in agreement with a simple diffusion mechanism.

Outflux increases when external substrate concentration is raised (counterflow). Outflux at steady‐state equilibrates influx, and is a first‐order process (rate constant: 0.15 min−1).

Uncoupling with azide leads to accelerate zero‐trans outflux by a factor of 2–3. No further acceleration is obtained when other classical uncouplers are used. The process remains first‐order, independent of the amount of carrier, and is accelerated by the presence of internal d‐glucuronate as a result from trans‐inhibition of the recapture.

Exchange outflux is all the more accelerated by azide as the carrier is less saturated. The process is clearly carrier‐mediated and the outflux rate obeys a Michaelis law with respect to internal concentration. V is equal to V for influx.

Homo and hetero‐overshoot experiments are in agreement with the participation of the carrier for mediating influx as well as outflux.

The kinetics of d‐glucuronate outflux in a strain lacking the specific hexuronate permease but carrying the 2‐keto‐3‐deoxy‐d‐gluconate permease are similar to those obtained with 2‐keto‐3‐deoxy‐d‐gluconate.

We draw the conclusion that energy‐coupling promotes the adjustment of outflux without interfering with influx rate. It apparently acts by reducing, in a continuous range, the affinity of the carrier facing inwards. The discussion is focused on the comparison with previously published models and on possible molecular mechanisms.
The 3′‐Amido and 5′‐Amido Analogues of Adenosine 3′:5′‐Monophosphate; Interaction with cAMP‐Specific Proteins
Authors: PANITZ Norbert , RIEKE Erwin , MORR Michael , WAGNER Karl G. , ROESLER Georg , JASTORFF Bernd
Published: March 2005

The sensitivity for recognition of adenosine 3′:5′‐monophosphate (cAMP) by its coordinate proteins towards chemical changes in the six‐membered cyclic phosphate ring has been investigated. A comparison of the interaction parameters of the 3′ and 5′‐amido analogues (I,II) and of unsubstituted cAMP has been made using two different protein kinases and the phosphodiesterase from bovine heart.

Binding affinity and the capacity of the amido analogues to stimulate the phosphotransferase activity of the kinases is greatly reduced relative to cAMP, the 3′‐position being more sensitive towards the modification than the 5′‐position. The coordinate noncyclic derivatives, 3′‐deoxy‐3′‐amino‐5′‐AMP (IV) and 5′‐deoxy‐5′‐amino‐3′‐AMP (III), were also tested. Surprisingly activity towards protein kinases was found to be considerable for the 5′‐deoxy‐5′‐amino‐3′‐AMP (III), while the 3′‐deoxy‐3′‐amino‐5′‐AMP (IV) is practically inactive. A possible reason for this is that the noncyclic 5′‐analogue (III) may be able to assume a cyclic structure maintained by internal salt formation.

The phosphodiesterase splits both cyclic amido analogues but with reduced rates compared to that of natural cAMP. Kinetic data obtained from different methods reveal a stronger affinity for the 5′‐analogue (I) than the 3′‐analogue (II) for the active site, although the reaction rate at saturated substrate concentration is significantly higher with II than with I.

The properties of the amido and the noncyclic amino analogues are discussed with available data from chemotaxis of the cellular slime moulds. Furthermore data of the respective methylene cyclic derivatives are used for a more comprehensive comparison. The above is interpreted in terms of the electronic features of the substitutions and of the changes in bond distances or angles upon replacement of O by NH or CH2 in the cyclic phosphate ring (obtained from X‐ray work).
Interconversion between Different States of Affinity for Acetylcholine of the Cholinergic Receptor Protein from Torpedo marmorata
Authors: SUGIYAMA Hiroyuki , CHANGEUX Jean‐Pierre
Published: March 2005

In receptor‐rich membrane fragments from Torpedo, acetylcholine binds, in the presence of 70 μM Tetram, to a homogeneous population of high‐affinity sites with Kd= (3.4 ± 0.8) × 10−8 M. Dissolution of these membrane fragments by sodium cholate causes a decrease of affinity associated with the appearance of medium‐affinity (Kd∼ 10−7 M) and low‐affinity (Kd≥ 10−6 M) sites. Dissolution by neutral detergents Triton X‐100 or Emulphogene preserves the high affinity of the acetylcholine binding sites. In all the soluble states of the receptor protein, Ca2+ ions and local anaesthetics no longer enhance the affinity for acetylcholine. Elimination of sodium cholate by dilution leads to the reassociation of the receptor protein, the recovery of high‐affinity sites and the control by Ca2+ ions and local anaesthetics. Purification by affinity chromatography of the receptor protein in Triton X‐100 is accompanied by a conversion of a majority of the acetylcholine sites into their state of low affinity. High‐affinity sites can no longer be recovered by detergent dilution from these low‐affinity ones.
The Relation of Conformation and Association of Insulin to Receptor Binding; X‐Ray and Circular‐Dichroism Studies on Bovine and Hystricomorph Insulins
Authors: WOOD Stephen P. , BLUNDELL Thomas L. , WOLLMER Axel , LAZARUS Norman R. , NEVILLE Raymond W. J.
Published: March 2005

Crystal and solution structure studies on insulins of different sequences and of widely different receptor binding affinities are reported. Bovine insulin, studied as a control, has a circular dichroism spectrum which is dependent both on protein concentration and zinc concentration. The spectrum appears to be related to the level of association of the insulin molecules. This implies that when using circular dichroism to compare solution structures of insulin derivatives or species variants one must make the comparison at equivalent levels of association and not merely at the same concentration. Changes in circular dichroism are related to the known crystal structure of zinc insulin hexamers.

The chinchilla insulin spectrum shows a reduced zinc dependence in low‐salt conditions which correlates with the inability to form crystals in similar conditions. This is attributed to an amino acid substitution at position B4. Crystals are obtained in high‐salt conditions and X‐ray diffraction patterns show these to be isomorphous with bovine 4Zn insulin crystals. Guinea pig insulin failed to crystallise under conditions which are normally conducive to the formation of crystals of zinc insulin hexamers and the circular dichroism showed no zinc dependence. This is consistent with a monomeric structure. The significance of the association behaviour of chinchilla and guinea pig insulins may be in the storage of the hormone in vivo. Whereas the monomeric form of chinchilla insulin has a structure closely related to bovine insulin, the circular dichroism indicates a gross structural difference for guinea pig insulin. This may be similar to that in des‐A21, des‐B30‐insulin, as both lack the Arg‐B22–Asn‐A21 carboxylate ion pair. The similarity of structure of chinchilla and bovine insulins is reflected in their receptor binding whereas the low receptor binding of guinea pig insulin probably results from the changes in its conformation rather than an alteration in residues of a receptor binding region.
Zum Mechanismus der Kondensationsreaktion der Fettsäurebiosynthese
Authors: ARNSTADT Klaus‐Ingo , LYNEN Feodor , SCHINDLBECK Gudrun
Published: March 2005

Mechanism for the Condensation Reaction of Fatty‐Acid Biosynthesis

The spontaneous hydrogen‐deuterium exchange of the methylene group of malonyl‐thioesters was investigated by nuclear‐magnetic‐resonance (NMR) spectroscopy using the model compound S‐malonyl‐N‐acetylcysteamine. The half life of the methylene proteins is 12 to 16 min in 0.1 M K‐Phosphate buffer at pH 6.5 to 7.0 at 25°C, the conditions of maximal activity of fatty acid synthetase from yeast. Proton catalysis was used for the quick preparation of deuterium‐ and tritium‐labeled malonylthioesters.

Compared with malonyl‐CoA, dideutero‐malonyl‐CoA had no primary isotope effect on the reaction velocity of the yeast enzyme catalysed fatty acid synthesis, in which the rate limiting step is the condensation reaction. Although deuterium oxide had a solvent isotope effect, there was no difference in reaction velocities between malonyl CoA and dideuteromalonyl CoA in deuterium oxide.

The condensation reaction was investigated separately from the overall fatty acid synthesis using β‐ketoacyl‐acyl‐carrier‐protein (ACP) synthetase (condensing enzyme) of Escherichia coli. The condensation reaction with deuteromalonyl‐ACP had no kinetic isotope effect, in agreement with the observations on the overall reaction. However, in this case no solvent isotope effect was observed with 2H2O.

When the condensation reaction was carried out in the presence of tritiated water, there was no incorporation of label into the reaction product acetoacetyl‐thioester, excluding proton exchange with the solvent.

The results exclude a mechanism for the condensation reaction involving a malonyl carbanion and its acylation as intermediates in the sense of an organic‐chemical malonic ester synthesis, and they indicate that the condensation reaction follows a concerted mechanism: The formation of the new carbon‐carbon bond is coupled with the cleavage of the carboxyl bond of the malonyl group.
Control of Hepatic Protein Synthesis
Authors: AYUSO‐PARRILLA Matilde S. , PARRILLA Roberto
Published: March 2005

The effect of nucleotide energy levels in vivo on the different steps of protein synthesis has been studied. Hepatic anoxia was induced by interrupting the blood portal‐vein flow. At 5 min of anoxia ATP fell to 59% of the control values and the amino acid incorporation into protein was inhibited by more than 70%. This strong inhibition was not paralleled by polyribosomal breakdown. On the contrary, when fasted rats were used, at 5 min of anoxia the ribosomal state of aggregation was found to increase. Longer periods of anoxia resulted in a further decrease in triphosphonucleoside content and polyribosomal breakdown. Based on these results and other reports from the literature it is concluded that the Km for the GTP of the peptide‐chain‐elongation mechanism must be higher than the Km of the initiation step. This finding implies that variations of nucleotide levels in vivo within the physiological range may control protein synthesis at the elongation step without apparent changes in the polyribosomal profiles.
Uptake of Taurocholic Acid into Isolated Rat‐Liver Cells
Authors: SCHWARZ Leslie R. , BURR Reinhold , SCHWENK Michael , PFAFF Erich , GREIM Helmut
Published: March 2005

Binding and transport characteristics for uptake of taurocholic acid by isolated rat liver cells were studied.

An adsorption of taurocholate to the cell surface is terminated in less than 15 s. A Ks of 0.55 mM and a total binding capacity of 3.8 nmol/mg cell protein is determined.

The rate of uptake of taurocholate follows Michaelis‐Menten kinetics with Km= 19 μM and V= 1.7 nmol/mg protein min.

There is a broad pH optimum for uptake between pH 6.5–8.0.

The activation energy amounts to 29 kcal/mol. At high taurocholate concentration an unusual upward bend is observed in the Arrhenius plot.

Taurocholate uptake is competitively inhibited by taurochenodeoxycholate (Ki= 9 μM). It is noncompetitively inhibited by bromosulfophthalein (Ki= 3 μM).

At physiological taurocholate concentrations a 200‐fold intracellular accumulation of taurocholate is observed.

Uptake is inhibited by about 75% by either antimycin A, carbonylcyanide m‐chlorophenyl‐hydrazone, ouabain.

Replacement of extracellular Na+ by either K+ or sucrose results in a 75% decrease of uptake.

It is concluded that taurocholate uptake is a carrier‐mediated process, and suggested that the energy for intracellular accumulation is made available by cotransport of Na+.
The Properties of Cytochrome f and P700 in Chloroplasts Suspended in Fluid Media at Sub‐zero Temperatures
Authors: COX Raymond P.
Published: March 2005

The properties of P700 and cytochrome f have been studied at sub‐zero temperatures in chloroplasts suspended in a medium containing 50% (v/v) ethylene glycol. The dark reduction of these components after a period of illumination provided information about the rate‐limiting step of photosynthetic electron transport under these conditions.

The oxidation of P700 on illumination in the presence of methyl viologen and its subsequent dark reduction can be observed at −35°C. This cycle of reactions could be repeated many times. The rate of reduction was increased by NH4Cl and reduction was inhibited by 3(3,4‐dichloro‐phenyl)‐1,1‐dimethylurea.

The oxidation and reduction of cytochrome f could also be observed under similar conditions. The activation energies for the reduction of cytochrome f and P700 are similar (about 75 kJ mol−1) and the reduction of cytochrome f is also inhibited by dichlorophenyldimethylurea and stimulated by NH4Cl.

The reduction of both cytochrome f and P700 seemed to follow first‐order kinetics, but the t1/2 for the reduction of the cytochrome was at least three times that for the reduction of P700 at the same temperature. It was concluded that the results were only compatible with a model in which the main pathway of electrons from plastoquinone to P700 involved cytochrome f if the equilibrium constant between the cytochrome and P700 was very much less than that expected from their redox potentials.
Role of Ribosomal Protein S1 in Protein Synthesis: Effects of Its Addition to Bacillus stearothermophilus Cell‐Free System
Authors: ISONO Setsuko , ISONO Katsumi
Published: March 2005

Products of the f2 phage RNA‐directed protein synthesizing systems in vitro, derived from Escherichia coli and Bacillus stearothermophilus, were analyzed. In contrast to other reports, it was found that B. stearothermophilus ribosomes synthesized phage coat protein when the high‐speed (100000 x g) supernatant (S100) and ribosomal wash (crude initiation factors) of homologous origin were used. Furthermore, a marked stimulation of coat protein synthesis was observed with B. stearothermophilus ribosomes when either S100 or ribosomal wash or both from E. coli cells were used instead of the respective homologous components. The principle responsible for this stimulation was identified as the 30‐S ribosomal protein S1 present in S100 and ribosomal wash. Purified S1 from E. coli at roughly one‐to‐one molar ratio to ribosomes resulted in about 10‐fold stimulation of the incorporation of [14C]valine when added to the B. stearothermophilus system. This stimulation was observed mainly in the synthesis of coat protein and replicase.
Cell‐Wall Lipopolysaccharide from Escherichia coli B
Authors: PREHM Peter , STIRM Stephan , JANN Barbara , JANN Klaus
Published: March 2005

The lipopolysaccharide of Escherichia coli BB and a number of R‐phage selected (e.g. T3, T4) cell‐wall‐defective mutants were analyzed. From their lipopolysaccharides the respective core oligosaccharides were obtained. Following dephosphorylation, the oligosaccharides were methylated and analyzed by gas chromatography/mass spectrometry. This revealed the sugar sequence in the hexose‐heptose region of the core. The linkage of heptose (Hep) to 2‐keto‐3‐deoxyoctonate (KDO) was established as … Hep KDO … by methylation analysis. The substituted derivative of KDO was identified by gas chromatography and mass spectrometry. The KDO region contains three KDO units. Its structure was elaborated by (a) selective removal and identification of 7‐phosphoryl ethanolamine‐KDO (KDO‐PN), (b) periodate oxidation and thiobarbituric acid reaction in conjunction with mild hydrolysis, (c) a modified methylation analysis. Phosphate substitution of the E. coli BB core was studied by β‐elimination and using the information obtained with KDO‐PN. The structures of the cell wall lipopolysaccharides from E. coli BB and cell‐wall‐defective mutants are given.
The Catalytic Activity and Penicillin Sensitivity in the Liquid and Frozen States of Membrane‐Bound and Detergent‐Solubilised Transpeptidase of Streptomyces R61
Authors: DUSART Jean , GHUYSEN Jean‐Marie , MARQUET Alberto , PERKINS Harold R.
Published: March 2005

The Km, app. values of the membrane‐bound transpeptidase of Streptomyces R61 for the donor Ac2‐l‐Lys‐d‐Ala‐d‐Ala and the acceptor Gly‐Gly are not affected by temperature variations when the reaction mixtures are incubated in liquid suspensions. At – 5°C, the incubation can be carried out either in the liquid or in the frozen state. The enzyme is active in the latter state. In the frozen state, the Km, app. value for the acceptor remains unchanged but there is a 3‐fold increase in the maximum velocity, a 10‐fold decrease of the Km, app. value for the donor and a 10‐fold increase of the benzylpenicillin concentration required to inhibit the enzyme activity by 50% (ID50 value). Temperatures of −35°C or below are required to completely inhibit the membranebound enzyme in the frozen state. Cetyltrimethylammonium bromide extracts the transpeptidase both from the isolated membranes and, with a much higher yield, from the intact mycelium. The extracted enzyme is not active in the frozen state, requires detergent for activity, has decreased Km, app. values for both donor and acceptor, exhibits the same sensitivity to benzylpenicillin and cephalosporin C as the membrane‐bound transpeptidase (in liquid suspensions) and, like this latter enzyme, has no dd‐carboxypeptidase activity. The detergent‐extracted transpeptidase penetrates gels of Sephadex‐100 and is not sedimented at 200000 ×g.
Purification of Arginases from Human‐Leukemic Lymphocytes and Granulocytes: Study of Their Physicochemical and Kinetic Properties
Authors: REYERO Cristina , DORNER Friedrich
Published: March 2005

Arginase has been isolated from granulocytes of a patient with chronic myelocytic leukemia and from lymphocytes of a patient with chronic lymphocytic leukemia and both enzymes have been purified to apparent homogeneity. The purification procedure employed acetone extraction, ammonium sulfate precipitation, DEAE‐cellulose and CM‐Sephadex chromatography and gel filtration on Bio‐Gel A 1.5 m. Both enzymes appear to be metalloenzymes, and to have molecular weights of about 120000. Studies with the dissociated enzymes suggest that the subunit molecular weight is about 37000, in agreement with a tetrameric aggregate structure of the native enzymes. Human leukemic granulocyte and lymphocyte arginases are strongly basic proteins with pI values between 9.25 and 9.35. Their free – SH groups enabled them to be linked to organomercurial‐agarose. The kinetic properties estimated for both enzymes showed an optimum pH of 8.5, and an optimal MnCl2 concentration of 0.01 M. The Km for l‐arginine is 2.7–3.1 mM and l‐ornithine exhibits a mixed type of inhibition, with a Ki of 15.5–15.7 mM.
Enzymic Synthesis of an Aromatic Ring from Acetate Units
Authors: KREUZALER Fritz , HAHLBROCK Klaus
Published: March 2005

Flavanone synthase was isolated and purified about 300‐fold from fermenter‐grown, light‐induced cell suspension cultures of Petroselinum hortense. The enzyme catalyzed the formation of the flavanone naringenin from p‐coumaroyl‐CoA and malonyl‐CoA. Trapping experiments with an enzyme preparation, which was free of chalcone isomerase activity, revealed that in fact the flavanone and not the isomeric chalcone was the immediate product of the synthase reaction. Thus the enzyme is not a chalcone synthase as previously assumed.

No cofactors were required for flavanone synthase activity. The enzyme was strongly inhibited by the two reaction products naringenin and CoASH, by the antibiotic cerulenin, by acetyl‐CoA, and by several compounds reacting with sulfhydryl groups. Optimal enzyme activity was found at pH 8.0, at 30°C, and at an ionic strength of 0.1–0.3 M potassium phosphate. EDTA, Mg2+, Ca2+, or Fe2+ at concentrations of about 0.7 μM did not affect the enzyme activity. Apparent molecular weights of approx. 120000, 50000, and 70000, respectively, were determined for flavanone synthase and two metabolically related enzymes, chalcone isomerase and malonyl‐CoA : flavonoid glycoside malonyl transferase.

The partially purified flavanone synthase efficiently catalyzed the formation of malonyl pantetheine from malonyl‐CoA and pantetheine. This malonyl transferase activity, and a general similarity with the condensation steps involved in the mechanisms of fatty acid and 6‐methyl‐salicylic acid synthesis from “acetate units”, are the basis for a hypothetical scheme which is proposed for the sequence of reactions catalyzed by the multifunctional flavanone synthase.
Transport of Messenger RNA into Different Classes of Membrane‐Associated Polyribosomes in Ehrlich‐Ascites‐Tumor Cells
Authors: VAN VENROOIJ Walther J. , GIELKENS Arno L. J. , JANSSEN Albert P. M. , BLOEMENDAL Hans
Published: March 2005

The membrane‐bound polyribosomes in Ehrlich ascites tumor cells can be separated into a loosely bound and a tightly bound fraction by means of a high salt treatment. Both membrane fractions as well as the free polyribosomes in the supernatant synthesize about the same set of proteins, suggesting a close relationship between these polyribosome fractions in the Ehrlich cell.

Relatively high concentrations of cycloheximide do not prevent newly synthesized poly(A)‐containing mRNA from entering the tightly bound polyribosome fraction. Nor had treatment of the cells with puromycin in the presence of cycloheximide, which released about 70% of the nascent chains, any significant effect on the entrance of newly synthesized mRNA into tightly bound polyribosomes.

These results suggest that in Ehrlich ascites tumor cells nascent polypeptide chains are not involved in the binding of polyribosomes to membranes.
Ribonucleotide Reductase from Escherichia coli: Demonstration of a Highly Active Form of the Enzyme
Authors: ERIKSSON Staffan
Published: March 2005

Ribonucleotide reductase from Escherichia coli consists of two nonidentical subunits, proteins B1 and B2. The activity of the enzyme in crude extracts prepared from mechanically disrupted bacteria is very low. Enzyme activity is stimulated 5 to 10‐fold by addition of an excess of either subunit. Concentrated extracts from cells lysed gently on Cellophane discs (Schaller et al.) contained 10 to 20‐fold higher activity than extracts from mechanically disrupted cells. This activity was not further stimulated by either B1 or B2. The system is suitable for complementation tests for the analysis of temperature‐sensitive mutants affecting the ribonucleotide reductase system.

Concentrated high‐spced supernatants from E. coli treated with lysozyme (Wickner et al.) also contained a high ribonucleotide reductase activity, which was stimulated slightly or not at all by addition of B1 and B2. This active form of the enzyme was unstable and could not be purified.

The results suggest that the intracellular form of the enzyme consists of a tight complex of proteins B1 and B2, possibly stabilized by other intracellular structures.
Human Luteinizing Hormone
Authors: CLOSSET Jean , VANDALEM Jean‐Louis , HENNEN Georges , LEQUIN Rudy M.
Published: March 2005

A new procedure is described for the isolation of the α and β chains of the hormone. In this method, the native hormone is incubated in acidic urea and the chains are then separated by ion‐exchange chromatography.

The amino‐terminal residue of the α subunit is valine. The carboxy‐terminal end of the α subunit is of variable length. No amino‐terminal residue was detected for the β chain; glycine was found at its carboxy‐terminal end by the selective titration method.

The amino acid and carbohydrate compositions of the hormone and both subunits are presented. The β chain contains sialic acid and is devoid of galactosamine in contrast to the β subunits of other species.

Contamination of our human lutenizing hormone preparation by other pituitary glycoprotein hormones such as thyroid‐stimulating hormone and follicle‐stimulating hormone amounted to 0.5 and 0.25 percent by weight respectively.

Cross‐contamination of the initial α and β subunit preparations was measured by specific radioimmunoassays and amounted to 4.1 and 2 percent by weight respectively.

Further extensive purification of these subunit preparations was then performed by means of affinity chromatography using immunosorbants.

The final preparations exhibited a residual cross‐contamination amounting to 0.2 and 0.02 percent by weight for the α and β subunits respectively.
Binding Studies of NADPH to NADP‐Specific l‐Glutamate Dehydrogenase from Saccharomyces cerevisiae
Authors: VENARD Renée , JALLON Jean Marc , FOURCADE Anne , IWATSUBO Motohiro
Published: March 2005

Optical characteristics of enzyme‐reduced coenzyme complexes of yeast NADP‐specific glutamate dehydrogenase have been investigated in the presence and absence of product (l‐glutamate) and in the presence or absence of phosphate.

The phosphate effect, pointed out in a previous work, is found again: inorganic phosphate (Pi) destabilizes the binary complex (E · NADPH), the dissociation constant of which is equal to 14 μM, a value much higher than that determined in Tris‐HCl buffer: Kd= 0.9 μM. Concerning the role of phosphate some assumptions are drawn up with respect to a similar behaviour of Pi toward yeast glutamate dehydrogenase and ADP toward the beef liver enzyme.

In the same way, l‐glutamate induces a stabilization of the binary complex; this latter effect is unchanged in the presence of phosphate, yet it is less marked than in the case of beef liver glutamate dehydrogenase.

Protein fluorescence, nucleotide fluorescence and circular dichroism measurements allowed the determination of three identical and independent NADPH binding sites per hexameric active unit. In analogy with beef liver enzyme, it seems that yeast glutamate dehydrogenase is a good model to study anticooperativity in ligand binding.
DNA Replication in Methotrexate‐Treated Human Lymphoblasts
Authors: FRIDLAND Arnold , BRENT Thomas P.
Published: March 2005

The rate of DNA synthesis in cultures of human lymphoblasts decreased more than 80% within 30 min after the cells were exposed to methotrexate, a potent inhibitor of dihydrofolate reductase. Despite this rapid initial inhibition, DNA continued to be synthesized for at least an additional 6 h. The mode of this subsequent replication appeared to be semiconservative, as indicated by the buoyant density of 5‐bromodeoxyuridine‐substituted DNA in alkaline CsCl gradients. The growth rates of DNA chains in cells exposed to methotrexate were determined by sedimentation rate analysis in alkaline sucrose gradients. DNA synthesized during 2‐min or 10‐min pulses with labeled deoxycitidine in the presence of methotrexate had about the same sedimentation coefficient, 35 S, as controls. When methotrexate‐treated cultures were pulse‐labeled for 10 min and then chased for various times, DNA fragments of about 80 S accumulated. DNA synthesized in the presence of methotrexate was stable and elongated to bulk‐size DNA after methotrexate inhibition of growth was removed by addition of thymidine and deoxycytidine. The data suggest that methotrexate reduces the rate of DNA replication by inhibiting chain initiation independently of chain elongation.
Immunologically Active and Structurally Similar Fragments of Protein A from Staphylococcus aureus
Authors: HJELM Hans , SJÖDAHL Jörgen , SJÖQUIST John
Published: March 2005

To study the active site(s) in protein A, partial tryptic digestions of the protein and of intact Staphylococcus aureus were performed. Fragments which bind to the Fc‐part of human IgG were isolated by affinity chromatography on IgG‐Sepharose 4B and purified by ion‐exchange chromatography on phosphocellulose.

From a partial tryptic digest of pure protein A at 30°C, pH 8.2 for 30 min we have isolated and characterized six active fragments with molecular weights ranging from 6000 to 8000. Two active fragments, obtained in high yields by digestion at pH 7.2 of intact protein‐A‐containing bacteria, were shown to be similar to two of the six characterized fragments from the digest of pure protein A.

All fragments appeared to have similar amino acid sequences, judged by peptide mapping, specific staining and amino acid analysis; some are very possibly overlapping peptides. Each fragment probably contains only one active site region since all are monovalent in the Fc‐reaction when studied with a hemagglutination technique. The maximal molar yield of active fragments obtained from the digestion of pure protein A accounts for about 210% of the amount of protein A used. Thus protein A, suggested to consist of repeating units, should exhibit at least three similar if not identical active regions.
Similarities and Differences of Five Peroxidases from Turnip and Horseradish
Authors: WELINDER Karen Gjesing , MAZZA Gilbert
Published: March 2005

Four isoperoxidases of turnip root and isoperoxidase C of horseradish root were digested with trypsin. and their peptide maps, prepared by high‐voltage paper electrophoresis, were compared. All five tryptic digests were completely soluble at pH 8. The maps were developed with a variety of general and specific reagents: ninhydrin, histidine, tyrosine, tryptophan and arginine reagents. Cystine peptides and cysteic acid derivatives have also been characterized. All detected half‐cystine residues seemed engaged in disulfide bridges. For each individual peroxidase the number of specifically staining peptides agreed very well with the amino acid composition.

The two most acidic peroxidases of turnip, P1 and P2, only differ significantly in one peptide. The P2 gene is tentatively proposed to have developed from the P1 gene by a single base mutation, changing an asparagine residue to a lysine residue. A less acidic turnip peroxidase, P3, is distinct, although related to peroxidases P1 and P2. Horseradish isoperoxidase C also belongs to this group which appears to be closely related in the amino acid sequences around four disulfide bridges. Peroxidase P7 differs from this group, at least around two of its disulfide bridges, and therefore, may differ from the other four in parts of its three dimensional structure.

Sequences of particular importance to peroxidase function must be present in all peroxidases. From the peptide mapping studies we only find two highly homologous sequences present in all five examined peroxidases. Both contain histidine. This finding corroborates previous suggestions of two histidine sequences near the peroxidase heme prosthetic group.

The rules applied in relating peptides of different proteins are outlined, and the sources of errors in mapping of glycoproteins of high carbohydrate content (about 20%) are discussed in detail.
The Interaction of Fusidic Acid with Peptidyl‐Transfer‐Ribonucleic‐Acid · Ribosome Complexes
Authors: SAN MILLAN María José , VAZQUEZ David , MODOLELI Juan
Published: March 2005

The inhibitory action of fusidic acid on peptide‐chain elongation was studied with systems in vitro directed by either polyuridylic acid or endogenous messenger (Escherichia coli polysomes washed with 1 M NH4Cl) or R 17 RNA, and supplemented with either crude or purified elongation factors. In all cases strong inhibition of synthesis required high concentrations of the antibiotic (approx. 1 mM), while a similar inhibition of the EF‐G‐plus‐ribosome‐dependent GTP hydrolysis required between 10 and 100 times less antibiotic. Since most of the GTP hydrolysis observed was presumably due to free ribosomes (without aminoacyl‐tRNA or peptidyl‐tRNA), fusidic acid seemed to interact far more easily with these ribosomes than with ribosomes engaged in peptide‐chain elongation.

The role of the GDP · EF‐G · ribosome · fusidic acid complex in the inhibition of polypeptide synthesis was assessed by measuring formation of this complex on polysomes engaged in peptide‐chain elongation. Using purified elongation factors the complex formed on only 25–35% of ribosomes, as measured either by retention of [3H]GDP or by hydrolysis of [3H, γ‐32P]GTP. In contrast, with crude factors (S100 extract) it formed on more than 70% of ribosomes. The results are compatible with the postulated role of the complex in polypeptide synthesis inhibition (blockade of the ribosomal acceptor site and subsequent inhibition of aminoacyl‐tRNA binding) and indicate that formation of the complex takes place by overriding the control that prevents interaction of EF‐G when the donor site is occupied by peptidyl‐tRNA.

In the polyuridylic‐acid‐directed system for synthesis of oligophenylalanine the antibiotic inhibits every round of peptide elongation, including dipeptide formation, to roughly the same extent.
Analysis of the Pancreatic‐Ribonuclease‐Digestion Products of Alfalfa‐Mosaic‐Virus Ribonucleic Acid
Authors: PINCK Lothaire , FAUQUET Claude
Published: March 2005

Alfalfa mosaic virus (AMV) genome consists of three pieces of RNA (24‐S, 20‐S and 17‐S RNA). For infectivity these three RNAs and the coat protein are required. In the absence of coat protein, infectivity is obtained by adding the 12‐S RNA also normally present in the virus. This 12‐S RNA represents the message for coat protein. Thus a redundancy of the gene for coat protein exists between 12‐S RNA and one of the other RNAs. Sequence analysis of the oligonucleotides resulting from pancreatic ribonuclease digestion of the AMV RNAs indicates that the nucleotide sequence of 12‐S RNA occurs in 17‐S RNA.

Analysis of the pancreatic ribonuclease digestion products of the two larger alfalfa mosaic virus RNAs (20‐S and 24‐S RNA) shows some oligonucleotides containing seven, eight and nine nucle‐otides with the same structure present in both RNAs. The possibility of a limited nucleotide sequence homology between these two RNAs is discussed. The comparison of the RNase digestion products of 20‐S and 24‐S RNA with those of 12‐S or 17‐S RNA revealed no homologous oligonucleotides, thus the origin of 12‐S RNA appears to be 17‐S RNA.
Identification of NADH‐Specific and NADPH‐Specific FMN Reductases in Beneckea harveyi
Authors: GERLO Erik , CHARLIER Josée
Published: March 2005

Distinct FMN reductases specific for NADH and NADPH were identified in extracts of Beneckea harveyi. These enzymes differ in their physical (molecular weight, thermostability) as well as in their chemical properties (binding constants for NADH and NADPH). The NADH‐specific enzyme is more efficient then the NADPH‐specific one with respect to the bioluminescent reaction.
Studies on the Role and Mode of Operation of the Very‐Lysine‐Rich Histone H1 (F1) in Eukaryote Chromatin
Authors: BRADBURY E. Morton , CHAPMAN George E. , DANBY Shirley E. , HARTMAN Peter G. , RICHES Paul L.
Published: March 2005

Restricted chymotrypsin digestion of calf thymus H1 histone gives two fragments, residues 1–106 and 107–C‐terminal. These were studied by proton magnetic resonance and circular dichroism. The N‐terminal fragment exhibited some salt‐induced structure in aqueous solution, but this did not parallel the globular structure of the intact H1 molecule. Comparison of circular dichroism results with helix predictions for this portion of the molecule suggests that the secondary structure may be the same in this fragment as it is in the corresponding region of the whole molecule. The C‐terminal fragments show very little salt‐induced structure. The N‐terminal fragments binds to DNA very weakly, but the C‐terminal fragment binds as strongly as the whole molecule. In the C‐terminal fragment, about one quarter of the lysine residues are not bound to the DNA in water, but initial increase of salt concentration causes them to become bound. This increasing binding occurs under the same ionic conditions that cause chromatin condensation and condensation of H1 · DNA complexes, and it is suggested that there may be a connection between these phenomena.
Interaction of RNA Polymerase from Escherichia coli with DNA
Authors: DAUSSE Jean‐Pierre , SENTENAC André , FROMAGEOT Pierre
Published: March 2005

A method was devised for directing RNA polymerase on a single promoter site on T7 DNA. Initiation complexes were formed on each of the three main promoter sites using one dinucleotide plus one nucleoside triphosphate. The ternary initiation complexes are resistant to rifampicin action, to inhibition by (rI)n at 0 °C and are stable at high salt concentrations. A minimum of a trinucleotide is required to form a stable ternary complex. To determine which promoter site was selected by RNA polymerase during initiation, the (rI)n‐resistant RNA was digested by RNAse III to generate three characteristic initiator RNA fragments, resolved by gel electrophoresis. The three major promoter sites could be selected individually by using different primer and substrate combinations: ApC + ATP selected promoter A3, CpG + CTP selected A2 and CpC + ATP specified preferentially A1. A number of primer‐substrate combinations specified each site at low salt concentration but the substrate requirement became very stringent at high salt concentration, suggesting that the postulated local opening of the promoter site could be more or less extensive, depending on the ionic strength. The minimum opening observed at high salt concentration corresponded to the insertion of a leader trinucleotide sequence. The promoter region melted by RNA polymerase at low salt concentration was (G + C)‐rich and corresponded to about 9 to 11 base pairs. Sequences of the melting recognition regions were tentatively inferred from the results.
GTP Degradation to Guanine Catalyzed by Ribosomal Subunits and Microsomal‐Wash Factors
Authors: GRUMMT Friedrich , SPECKBACHER Monika
Published: March 2005

Ribosomes from stringent strains of bacteria generate (p)ppGpp if incubated with uncharged tRNA, a ribosomal wash fraction, GTP and ATP. By contrast, an analogous system from rat liver does not transform GTP to (p)ppGpp but degrades it to guanine. The reaction requires the ribosomal subunits, a 40000‐Mr and a 60000‐Mr microsomal wash protein factor and is inhibited if the ribosomal A‐site is charged with aminoacyl tRNA. The degradation of GTP to guanine occurs in the following four distinct reaction steps: (a) hydrolysis of GTP to GDP + Pi, (b) hydrolysis of GDP to GMP + Pi, (c) hydrolysis of GMP to guanosine + Pi, (d) hydrolysis of guanosine to guanine + ribose. The reaction step (a) is inhibited by fusidic acid, cycloheximide, emetine, tetracycline and puromycin. The hydrolysis of GDP is inhibited strongly by fusidic acid, emetine and tetracycline. A putative physiological significance of this ribosome‐dependent pathway in the processes of growth control of animal cells under conditions of amino acid deprivation is discussed.
Evidence for Essential Histidine Residues in Bovine‐Liver Mitochondrial Monoamine Oxidase
Authors: HIRAMATSU Akira , TSURUSHIIN Shigeko , YASUNOBU Kerry T.
Published: March 2005

Ethoxyformic acid anhydride, amino‐1 H‐tetrazole, and photooxidation in the presence of rose bengal, which are reagents known to react with histidine residues of proteins, were shown to in‐activate monoamine oxidase. Ethoxyformic acid anhydride reacted with about 6 histidine residues per 100000 g of protein under the experimental conditions adopted and completely inactivated the enzyme. However, NH2OH reactivated the ethoxyformic acid derivative to 100% of the value of the enzyme treated with 0.2 M NH2OH only. Since NH2OH specifically deacylates N‐ethoxyformylimidazole, it was shown that at least some of the histidine residues are essential for activity. In addition, photooxidation experiments in the presence of 0.01% rose bengal confirmed that only histidine residues of bovine hepatic monoamine oxidase are destroyed under the designated experimental conditions. About 9 histidine residues per 100000 g of protein were destroyed during the photo‐oxidation experiments. In the presence of substrate, kynuramine or benzylamine, only 7 histidine residues were destroyed, which indicates that 2 histidine residues per 100000 g of protein are essential for activity.
Mapping of Cleavage Sites for Restriction Endonucleases in λdv Plasmids
Authors: STREECK Rolf E. , HOBOM Gerd
Published: March 2005

Restriction nucleases from Escherichia coli carrying the resistance transfer factor RI, from Hemophilus influenzae, Hemophilus parainfluenzae, and Bacillus subtilis were used for the generation of specific DNA fragments from λdv plasmids. Cleavage maps were established for various plasmids containing different but overlapping parts of bacteriophage λ DNA by analysis of digestion patterns obtained in polyacrylamide gel electrophoresis. The correlation between the plasmid cleavage maps and the genetic map of λ was based upon the location of the EcoRI cleavage site at 81.0% of λ fractional length and the position of promoters pL and pR on the plasmid DNA fragments.
β‐Glucosidases from Cicer arietinum L.
Authors: HÖSEL Wolfgang , BARZ Wolfgang
Published: March 2005

β‐Glucosidases specific for isoflavone 7‐O‐glucosides have been isolated from garbanzo plants, Cicer arietinum L.

These aryl‐β‐glucohydrolases occur in the different organs of the plant as multiple molecular forms. The major isoenzymes of the roots, the leaves and the hypocotyl were purified to electrophoretic homogeneity. When subjected to isoelectric focussing in polyacrylamide gels the electrophoretically homogeneous glucohydrolases were found to consist of one or two major and several minor enzymically active molecular species.

In roots the β‐glucohydrolase isoenzymes constitute a considerable portion of the extractable protein, so that purification to an electrophoretically homogeneous form is easily attainable.

All β‐glucosidases analyzed possess molecular weights in the range of 125000 (ultracentrifugation) to 135000 (Sephadex G‐200) and contain two subunits of molecular weight near 68000. The pH optimum for enzymic activity is 7–7.5 with a second optimum of 4.5–5. The isoelectric points of the various species range between pH 5.9 and 7.1. Staining for glycoprotein was positive.

Kinetic analysis demonstrated a pronounced specificity of the enzymes for aromatic substrates with glucose as the sugar moiety. α‐Glucosides as well as disaccharides were not hydrolyzed at all. Isoflavone 7‐O‐glucosides are the most favoured substrates with a Km of 2 × 10−5 M, while the Km with aromatic glucosides (i.e. salicin, 4‐nitrophenyl glucoside) are 100 times larger. In addition the β‐glucosidases show a pronounced specificity for glucose in the 7‐position of the flavonoid nucleus. Using isoflavone aglycones as substrates glucose transferase activity was also demonstrable.

The β‐glucohydrolase activity is strongly inhibited by Hg2+. This inhibition is partially reversible and preferentially influences the Km values of the enzymes compared to to V. Ag+ glucono‐1,5‐lactone, ethyleneglycol monomethyl ether and glycerol are only weakly inhibitory, while glucose, p‐chloromercuribenzoate and Cu2+ are without effect.