bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Genomic Complexity of Acanthamoeba castellanii Mitochondrial DNA
Authors: BOHNERT Hans J. , HERRMANN Reinhold G. 
Published: March 2005

DNA of DNase‐treated mitochondria of Acanthamoeba castellanii bands at a buoyant density of 1.690 g/cm3. Up to 80% of the molecules were circular, with a circumference of 12.7 μm. Two circular conformations, the closed twisted and the open form, were separated in preparative CsCl–ethidium bromide gradients.
When mtDNA was renatured at a temperature Tm– 23°C, deviations from second‐order kinetics were observed. At Tm– 10°C, however, it renatured as a single kinetic class without indications of sequence repetition. The coincidence of the kinetic complexity (2.57 × 107) and the circle contour length within the limits of error of the methods suggests that each circular molecule represents a chondriome.
The main DNA component, which is of nuclear origin and has a peak density of 1.716 g/cm3 in CsCl, displays heterophasic renaturation kinetics. Its slow‐reassociating fraction shows a kinetic complexity corresponding to a molecular weight of 2.1 × 1010. Approximately 22% of the main component appears to be repetitive at a renaturation temperature of Tm– 23°C.
On the Conformation of Pyridoxal Phosphate Imine in Solution and in Aspartate‐Aminotransferase Active Site
Authors: TUMANYAN Vladymir G. , MAMAEVA Olga K. , BOCHAROV Alexander L. , IVANOV Valery I. , KARPEISKY Marat Y. , YAKOVLEV Gennady I. 
Published: March 2005

The work comprises:

Determination by nuclear magnetic resonance method of the conformation of the imino group of pyridoxal‐P oxime in solution. It has been found to be nearly coplanar with the aromatic ring.
Theoretical calculation by the method of atom‐atom potentials of the permitted conformations of pyridoxal‐P imine and its analogs: 6‐methylpyridoxal‐P and 5′‐methylpyridoxal‐P. There have been found seven favourable conformations for pyridoxal‐P imine, some of which were prohibited in the analogs mentioned.
Experimental estimation of the conformation of pyridoxal‐P imine in the active site of aspartate aminotransferase with the use of the above sterically hindered analogs and 5′‐deoxy‐5′‐carboxy‐methylenepyridoxal analog. In result, the number of the possible conformations for pyridoxal‐P imine in the active site has been reduced to a pair of symmetrical conformations.
Catabolism of Hamamelose
Authors: THANBICHLER Alfred , BECK Erwin 
Published: March 2005

Hamamelose (2‐C‐hydroxymethyl‐d‐ribose) is a common sugar of green land plants. However, until now, all experiments failed to show dissimilation of this sugar by its producers or by soil microorganisms. For investigation of its biological degradation, microorganisms were used which had been isolated from plant material (leaves of Primula clusiana Tausch). These microorganisms belong to the Enterobacteriaceae and are almost identical to those described by Asai et al. (1956, 1962) as Kluyvera citrophila. They differ from the ATCC strains of this organism by their ability to grow on hamamelose.Isotope studies using 14C‐labelled hamamelose and glucose (for comparison) as substrates were carried out to elucidate the biochemical route of hamamelose catabolism. This degradation leads to CO2 under aerobic conditions and to lactate, succinate, formate, acetate and ethanol under anaerobic ones. Glucose dissimilation proceeds via glycolysis and leads to the same end‐products. As intermediates of the biological degradation of hamamelose, hamamelose 21‐phosphate, fructose 1‐phosphate, fructose 1, 6‐bisphosphate and phosphoglycerate were identified. Thus the biochemical route of hamamelose dissimilation appears to be rather similar to glycolysis. The following sequence is proposed: hamamelose → hamamelose 21‐phosphate → fructose 1‐phosphate → fructose 1, 6‐bisphosphate → phosphoglycerate → pyruvate → volatile end‐products. Differences from the glycolytic pathway occur at step 2 in which there is a rearrangement of the carbon skeleton and at step 3, in which fructose 1‐phosphate instead of fructose 6‐phosphate has to be phosphorylated.
Structural Studies of Human‐Liver Alcohol‐Dehydrogenase Isoenzymes
Authors: BERGER Denis , BERGER Marianne , VON WARTBURG Jean‐Pierre 
Published: March 2005

Alcohol dehydrogenase isoenzymes BE were isolated from human livers of both “normal” and “atypical” phenotypes [1]. Their pH‐rate profiles, their behaviour on CM‐cellulose and the number of hybrids formed with horse subunit A show that the “atypical” isoenzymes contain subunits B1 and B2 the “normal” one only subunit B1. These results indicate that most “atypical” individuals genetically represent heterozygotes.
A high degree of homology between horse subunit A and human subunits B is found. However, the human isoenzymes have a molecular weight of 87000 and contain about 410 amino acid residues, 6 tyrosines and 3 tryptophans per subunit.
The sequence of one tryptic peptide from subunit B1 is Phe‐Ala‐Lys and is altered to Phe‐Pro‐Lys in the subunit B2. This substituted residue is located in a region which corresponds to the coenzyme‐binding site of the horse enzyme.
Escherichia coli Ferredoxin, an Iron‐Sulfur Protein of the Adrenodoxin Type
Authors: KNOELL Hans‐Emil , KNAPPE Joachim 
Published: March 2005

Ferredoxin of Escherichia coli is an iron‐sulfur protein of the molecular weight about 12000 which contains two iron and two labile sulfur atoms. The sequence of the amino terminus is HN‐Pro‐Lys‐Val‐Ile‐Val‐Leu‐Tyr‐.The electron paramagnetic resonance spectrum of the reduced form shows a low‐field signal with g∥= 2.02 and a single high‐field signal with g⊥= 1.94. From the circular dichroism spectrum in the peptide absorption region an amount of 10% helically ordered regions is computed. A new peak in the light absorption spectrum at 545 nm appears upon reduction. From the spectral data, which reflect the fine structure of the chromophore and the gross conformation of the polypeptide chain, a close relationship to the mitochondrial adrenodoxin is concluded.The midpoint potential of the protein is –0.38 V. It does not mediate electron transport in the NADP‐photoreduction system of spinach and is incapable of replacing the Pseudomonas putida ferredoxin in the camphor hydroxylation. The physiological role of this ferredoxin, which is not a functional analogue of the Escherichia coli flavodoxin and which is synthesized by aerobically as well as anaerobically growing cells, is unknown.
The Active Species of ‘CO2’ Utilized by Reduced Ferredoxin: CO2 Oxidoreductase from Clostridium pasteurianum
Authors: THAUER Rudolf K. , KÄUFER Barbara , FUCHS Georg 
Published: March 2005

Reduced ferredoxin: CO2 oxidoreductase (CO2 reductase) from Clostridium pasteurianum catalyzes the reduction of ‘CO2’ to formate with reduced ferredoxin, an isotopic exchange between ‘CO2’ and formate in the absence of ferredoxin, and the oxidation of formate to ‘CO2’ with oxidized ferredoxin. The active species of ‘CO2’, i.e. CO2 or HCO3− (H2CO3), utilized by the enzyme was determined. The method employed for the species identification was that of Cooper et al. (1968). Both ‘CO2’ reduction to formate and the exchange reaction were studied. Data were obtained which are compatible with those expected if CO2 is the active species.The V and the dissociation constant Ks of the enzyme · CO2 complex in dependence of pH were determined from initial velocity studies of the exchange reaction. V was found to be only slightly affected by pH between 5.5 and 7.5. Ks was markedly dependent on pH; the constant increased with decreasing pH from 0.2 mM at pH 7.5 to 3 mM at pH 5.5.
Initiation of Transcription within an RNA‐Polymerase Binding Site
Authors: HEYDEN Bertold , NÜSSLEIN Christiane , SCHALLER Heinz 
Published: March 2005

The 5′‐terminal sequence of the RNA transcribed from bacteriophage fd replicative form DNA under the control of promoter region I has been determined to be ppp(Gp)nUpApApApGpApCpCpUpGpApUpUp …
This sequence is complementary to the 5′‐terminal sequence of the minus strand of the corresponding RNA polymerase binding site I, the starting point for RNA synthesis lying approximately in the middle of the binding site.
This initial sequence is also transcribed faithfully from isolated complexes of RNA polymerase and binding site I, obtained by DNase digestion of complexes between RNA polymerase and fd replicative form DNA. These highly stable complexes can not be reconstituted from binding site and enzyme.
It is concluded that RNA polymerase binding site and initiation site are identical parts of a promoter region, and that no “drift” between these sites is required as a step in RNA chain initiation. An additional non‐transcribed outside region is implicated as essential for full promoter function.
Association of Glyceraldehyde‐3‐Phosphate Dehydrogenase with the Particulate Fraction of Chicken Skeletal Muscle
Authors: DAGHER Shawky M. , HULTIN Herbert O. 
Published: March 2005

When chicken breast muscle was homogenized in water, approximately 86% of the glyceraldehyde‐3‐phosphate dehydrogenase was associated with the particulate fraction.The enzyme was solubilized by increasing pH with a very marked increase in the pH range of 6.9 to 7.1. At low ionic strength (about 0.015), approximately 50% of the enzyme is solubilized at pH 7.5 and above.Increasing ionic strength also led to increased solubilization. In addition, there was a specific cation effect with Ca2+ > Mg2+ > K+ > Na+ at a constant ionic strength. Glyceraldehyde 3‐phosphate and 2,3‐bisphosphoglycerate were effective in partially solubilizing the enzyme.Solubilized glyceraldehyde‐3‐phosphate dehydrogenase can rebind to the particulate fraction of the homogenized muscle. The soluble form of the enzyme has a higher V and a higher Km (glyceraldehyde 3‐phosphate) than the enzyme bound to the particulate fraction.
Affinity Chromatography on Immobilised Nucleotides
Authors: COMER Michael J. , ATKINSON Anthony , CRAVEN David B. , HARVEY Michael J. , DEAN Peter D. G. 
Published: March 2005

The effect of pH and temperature on the capacity and binding of Bacillus stearothermophilus, alcohol dehydrogenase and phosphofructokinase to N6‐(6‐aminohexyl)‐5′‐AMP‐Sepharose has been examined. Specific elution from the substituted AMP‐Sepharose was examined using a variety of cofactors, fragments of cofactors and substrates. A purification scheme for each enzyme on the substituted AMP‐Sepharose using nucleotides and gradients of pH and salt is presented. Interestingly, elevated temperature increased the affinity of both enzymes for N6‐(6‐aminohexyl)‐5′‐AMP‐Sepharose, however, the Michaelis constant for nucleotide determined at various temperatures remained constant.The effect of pH and salt concentration on the binding of B. stearothermophilus glyceraldehyde‐3‐phosphate dehydrogenase to 6‐aminohexanoyl‐NAD+‐Sepharose was also examined; raising the pH above 7.5 lowers the capacity of the matrix and the effect of a range of ammonium sulphate concentrations on the adsorption of the enzyme was examined. A specific purification of glyceraldehyde‐3‐phosphate dehydrogenase from partially purified extracts of this organism was achieved.
Broad‐Specificity Proteinase Inhibitors in Scopolia japonica (Solanaceae) Cultured Cells
Authors: SAKATO Kuniaki , TANAKA Hozumi , MISAWA Masanaru 
Published: March 2005

Proteinase inhibitors were isolated from Scopolia japonica cultured cells. Isolation procedures involve concentration by a hydrophobic resin of Diaion HP‐20, decolorization by Duolite A‐7, affinity chromatography on trypsin‐Sepharose, and Bio‐Gel P‐4 chromatography. It was found that the proteinase inhibitors from S. japonica cells are a mixture of at least five components.For the inhibitory components except one, amino acid analyses, measurements of sedimentation equilibrium and optical rotatory dispersion (ORD) were carried out. The inhibitors were shown to be the polypeptides with molecular weights in the range of approximately 4000 to 6000. In addition, one of them was found to have approximately 15%α‐helical conformation by the Moffitt‐Yang analysis of ORD data.The inhibitors were found to have potent inhibitory activity for trypsin, chymotrypsin, plasmin, kallikrein and pepsin but not for papain with synthetic and natural substrates. These inhibitors formed stable complexes with trypsin and chymotrypsin in an equimolar ratio, and their inhibitory mechanisms for both enzymes were of non‐competitive type.
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).
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.
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+.