bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Bohr Proton‐Binding Site in a Monomeric Haemoglobin
Authors: Sick Hinrich , Gersonde Klaus , Thompson John C. , Maurer Wolfgang , Haar Wolfgang , Rüterjans Heinrich 
Published: March 2005

Individual haemoglobin III from Chironomus thummi thummi is a monomeric protein (Mr= 15500) which has recently been shown by us to be characterized by a simple Bohr effect. Three of the four histidines, the position of which in the polypeptide chain is known from sequence analysis, are distinguished by their titration characteristics, namely His‐G2, His‐G19 and His‐E5. The chemical shift of the C‐2 proton resonances of the imidazole groups, which varies upon protonation of the histidine, has been used for the determination of the intrinsic pK values. His‐F8, the proximally bound histidine, is not observable because its C‐2 proton resonance is broadened.One of the three titratable histidines reveals a pK value which is varied in the range from 7.5 to 7.1, depending on the 6th ligand at the haem iron. The relatively high pK value of this histidine and its abnormal value of the chemical shift of the C‐2 proton resonance at low pH indicate an interaction with a carboxylate group. This shift of the pK value caused by the binding of CO to reduced haemoglobin is in agreement with the interaction energy, calculated from the amplitude of the Bohr effect curve, and corresponds to 500 cal.In this simple case the assignment of the C‐2 proton resonances to the known histidines is possible considering the atomic model, described by Huber et al. [4]. The results indicate that His‐G2 is the allosteric binding site; the remaining two detectable histidines are not involved in the allosteric interaction. The molecular mechanism of the Bohr effect in this haemoglobin is discussed.
Structural Relationship of Biotin‐Containing Enzymes
Authors: Sumper Manfred , Riepertinger Christl 
Published: March 2005

Acetyl‐CoA carboxylase from yeast was isolated in homogeneous form and compared in its properties with pyruvate carboxylase from yeast. Both enzymes have very similar sedimentation coefficients and molecular weights. Both enzymes are composed of four protomers. Acetyl‐CoA carboxylase and pyruvate carboxylase split under identical conditions into a variety of aggregates; besides the protomer, dimeric, trimeric and polymeric forms are found. Patterns of the dissociated enzymes obtained by sedimentation in sucrose density gradients and by electrophoresis in polyacrylamide are almost identical.Within the limits of sensitivity of the immunochemical techniques used in this study no cross reaction could be observed between anti‐acetyl‐CoA carboxylase and pyruvate carboxylase. This indicates that the substructures catalyzing the ATP‐dependent carboxylation of biotin, common to both enzymes are not based on identical primary structures. From these results it is proposed that the genes for acetyl‐CoA carboxylase and pyruvate carboxylase may have been derived from a common ancestor.
Effects of Antibiotics on the Development and Stability of Mitochondrial Enzymes in Saccharomyces cerevisiae
Authors: Görts Coen P. M. , Hasilík Andrej 
Published: March 2005

The effects of cycloheximide and chloramphenicol on the development of complex oxidase systems and of several parts of the respiratory chain of yeast mitochondria have been investigated.
In repressed cells addition of cycloheximide has a stronger effect on the development of several mitochondrial activities than has the addition of chloramphenicol. Probably in this phase the derepression is primarily dependent on the synthesis of enzymic proteins on the cytoplasmic ribosomes.
Incubation of partially derepressed cultures with chloramphenicol results in an inactivation of cytochrome oxidase and of the succinate and NADH oxidase systems and in a decrease in the respiratory control. After a lag the development of succinate and NADH dehydrogenase and of NADH‐cytochrome c oxidoreductase is severely inhibited.
There are some remarkable parallels between the effects of incubation of the cells with chloramphenicol and treatment of the mitochondrial fractions with digitonin. This suggests that chloramphenicol may indirectly interfere with the structure or composition of mitochondrial membranes.
The inhibition of the mitochondrial protein synthesis may affect mitochondrial membranes and interfere with some of the functions of the integrated oxidase systems. The inactivation of these systems suggests that their development and stability is dependent on a continual supply of some products of the mitochondrial protein‐synthesizing system.
Spectropolarimetric Studies of Binary and Ternary Complexes of Octopine Dehydrogenase
Authors: Oriol Christine , Olomucki Anna 
Published: March 2005

The optical rotatory dispersion and the circular dichroism of octopine dehydrogenase were studied in the presence and in the absence of NADH and of substrate analogues.Calculation based on the parameter b0 yields for the apoenzyme a helix content of approx. 27%. The presence of β‐structure is suggested by the particular shapes of the spectra and the great difference between the values of the helix content calculated from A193 and A225.In the far‐ultraviolet region there is no change in the optical rotatory dispersion and circular dichroic spectra after addition of NADH alone or NADH +dl‐arginine.The binding of NADH to the apoenzyme induces a positive extrinsic Cotton effect centered around the absorption maximum of the reduced coenzyme. It also produced a broad positive ellipticity band in the same spectral region. This change in the molecular ellipticity was used to calculate the number of binding sites for NADH.The attachment of NADH to the apoenzyme also gives rise to a modification of the circular dichroic spectrum in the region of absorption of aromatic chromophores.The binding of a substrate analogue to the holoenzyme also induces spectral changes which might result from the spatial rearrangement of tyrosine and tryptophan. This fact is discussed in relation to earlier spectrophotometric studies which have also indicated environmental changes of aromatic chromophores due to the formation of ternary complexes.
Glucose Repression of Inducible Enzyme Synthesis in the Yeast Rhodotorula gracilis
Authors: Höfer Milan , Dahle Petra 
Published: March 2005

The mechanism of glucose repression of inducible enzyme synthesis in the obligatory aerobic yeast Rhodotorula gracilis has been investigated. Induction of the d‐xylose catabolizing enzyme system has been used as the test‐tool. It has been found that:

d‐Glucose blocks the uptake of d‐xylose from a mixture of the two aldoses. d‐Xylose enters the cell interior only after all the d‐glucose has been consumed.
d‐Fructose, d‐galactose, d‐ and l‐arabinose do not affect the uptake of d‐xylose.
Accordingly, d‐glucose represses induction of the d‐xylose catabolizing enzyme system, whereas d‐fructose, d‐galactose, d‐ and l‐arabinose do not.
d‐Glucose is without effect in cells which have been preincubated with d‐xylose, whereby the latter is accumulated in the cells. In this case, d‐xylose catabolism proceeds simultaneously with that of d‐glucose.
d‐Glucose does not interfere with enzyme induction if d‐xylose has already been accumulated in the cells.
3‐O‐Methyl‐d‐glucose, an unmetabolizable analogue of d‐glucose, exhibits the same effect on inducible enzyme synthesis started with d‐xylose as d‐glucose does.

From these results the following conclusions can be drawn:

The cell membrane is one of the important regulatory sites of cell metabolism in R. gracilis.
Glucose repression of inducible enzyme synthesis in R. gracilis is regulated at the cell membrane.
On the Mechanism of Carbamoylphosphate Synthetase
Authors: Chabas Amparo , Grisolia Santiago , Silverstein Richard 
Published: March 2005

Studies were undertaken to investigate whether 2‐acetamido glutaric anhydride is an intermediate in the synthesis of carbamoylphosphate. Experiments were carried out with pools of 2‐acetamido‐glutaric anhydride, its hydroxamate and NH2OH to trap the anhydride. The anhydride was also tested as an inhibitor. The experiments show no evidence for the participation of free 2‐acetamido‐glutaric anhydride as an intermediate.The effect of NH2OH on the synthesis of ATP from carbamoylphosphate and ADP was investigated in order to clarify whether this reaction is an integral part of the overall reaction catalyzed by carbamoylphosphate synthetase and whether there is a common intermediate which could react with NH2OH. The unexpected observation was made that NH2OH is an inhibitor of ATP synthesis at low levels of ADP, and that the enzyme has a high affinity for ADP. These results together with other experiments on carbamoylphosphate binding by the enzyme point to a competition between ADP site and NH2OH. No increase in the release of Pi from carbamoylphosphate in the presence of both acetylglutamate and NH2OH was observed; therefore the intermediate responsible for the acetylglutamate NH2OH‐dependent ATPase is not formed extensively from carbamoylphosphate.The activation of carbamoylphosphate synthetase by acetylglutamate for both the forward and back reactions was studied. The enzyme was activated with acetylglutamate and rapidly separated from the cofactor on Sephadex. There was no evidence for retention of activation, indicating a fast return to the non‐activated state. Therefore, enzyme samples were activated, diluted and assayed immediately and after time intervals. A time‐dependent reversibility of the activated enzyme for both the forward and back reactions is illustrated.
The Effect of ATP upon the Oxygen Sensitivity of Nitrogenase from Azotobacter chroococcum
Authors: Yates M. Geoffrey 
Published: March 2005

Crude preparations of nitrogenase (AS1) from Azotobacter chroococcum which were normally oxygen‐tolerant, became oxygen‐sensitive in the presence of either ATP, GTP, ITP, UTP, CTP or sodium pyrophosphate. MgCl2 antagonised this effect of ATP.
ATP‐induced oxygen damage was faster at 4°C than at 30°C.
ATP‐induced oxygen damage to prepn AS1 affected fraction 2 (Ac2) of the nitrogenase; fraction 1 (Ac1) was unaffected. Purified fraction Ac2 became hypersensitive to oxygen in the presence of ATP.
Pre‐treatment of prepn AS1 with “Chelex 100” or EDTA under anaerobic conditions made the nitrogenase oxygen‐sensitive. Oxygen‐tolerance was partly restored by metal ions. Similar anaerobic pre‐treatment of prepn AS1 with ATP followed by removing the ATP by dialysis did not induce oxygen‐sensitivity.
Pure fraction Ac2 bound significantly more [14C]ATP in the presence of MgCl2 than did EDTA‐treated fraction Ac2 in the absence of MgCl2.
Hill plots of the ATP requirement for acetylene reduction, for ATP‐induced oxygen damage, for the MgCl2, requirement for acetylene reduction and for the MgCl2, requirement for protection against oxygen damage to EDTA‐treated prepn AS1 all yielded nvalues between 1 and 2.
These results are discussed in terms of a role for ATP in nitrogen fixation.
The Release of Ribonucleic Acid from the Membranes of Relaxed Escherichia coli Cells Starved for Amino Acids
Authors: Kaplan Ruth , Silman Nitza 
Published: March 2005

Various treatments were tested for their capacity to abolish the preferential accumulation of ribonucleic acid in the pelletable‐membrane fraction of rel− cells of Escherichia coli starved for amino acids. DNAase in the cold increased the extraction of RNA both in exponentially‐growing and in starved cells. The most pronounced increase in DNAase‐dependent RNA extraction was observed with rel− cells starved for short periods. Incubation at 37°C, or a high concentration of ammonium or potassium, abolished completely the preferential accumulation of RNA in the pelletable‐membrane fraction of rel− cells starved for 15 min, but left undisturbed considerable amounts of RNA in the membranes of rel− cells which had undergone prolonged starvation (60 min). No preferential accumulation of ribonucleic acid in the membrane fraction of amino acid‐deprived cells could be detected when extracts were prepared in the presence of 5 mM magnesium.The sedimentation pattern of the RNA released by several methods was studied. The RNA formed during amino acid starvation of rel− cells was found mainly in polysomes.
The Histidines in Liver Alcohol Dehydrogenase
Authors: Morris David L. , Mckinley‐mckee John S. 
Published: March 2005

N‐Carbethoxylation of histidine in liver alcohol dehydrogenase resulted in two irreversible processes, a rapid activation and a slower inactivation.Formation of the binary complex with NADH or the ternary complex with NADH and isobutyramide prevented the slow inactivation occurring but not the rapid activation.Four histidine residues were carbethoxylated per subunit of the native enzyme, enzyme · NADH complex and enzyme · NADH · isobutyramide complex. It is suggested that none of the four histidines carbethoxylated are at the active centre but that one or more may be important for maintaining the native conformation of the enzyme.
Conformational Aspects and Reactivity of DNA
Authors: Luck Gerhard , Zimmer Christoph 
Published: March 2005

The complex formation of manganese(II) and magnesium(II) with DNA has been investigated by ultraviolet absorption and circular dichroism (CD) measurements.G · C‐rich DNA shows a difference spectrum in the presence of manganese(II) but not with magnesium(II). The lowering of the melting temperature observed at Mn2+/DNA‐phosphorus ratios > 1 increases with increasing G · C content but is diminished in the presence of higher ionic strength.The circular dichroism spectra of native DNA are greatly affected by manganese(II). Increasing Mn2+ concentration decrease the CD maximum of G · C‐rich DNA at 270 nm while Mg2+ does not change the CD curve. Contrary to the ordered double helical form the CD spectra of denatured DNA are unaltered. The manganese(II)‐induced perturbation of the circular dichroism of native DNA becomes increased with increasing G · C content and reaches a saturation level around 50 mol % guanine‐plus‐cytosine.The results show that manganese(II) is preferably bound to G · C pairs besides the phosphate groups. The CD spectrum of protonated G · C‐rich DNA is strongly changed by manganese(II) which indicates competition between the divalent metal ion and the proton at the binding site. In agreement with the behavior of methylated DNA the proton displacement by Mn2+ from G · C pairs suggests that Mn2+ coordinates with the N‐7 position of guanine. The spectral changes of the circular dichroism depend also on the shielding of phosphate groups by sodium ions. The results of the circular dichroism which indicate G · C selective conformational changes of the DNA are interpreted by tilting of the bases of locally Mn2+ chelated regions. The possible functional role of the differences observed in the complexing of DNA by Mn2+ and Mg2+ are discussed.
Beef‐Liver 5‐Aminolevulinic Acid Dehydratase
Authors: Wilson Elaine L. , Burger Patricia E. , Dowdle Eugene B. 
Published: March 2005

Beef liver 5‐aminolevulinate dehydratase was purified 700‐fold by salt fractionation, heat treatment, gel filtration and ion‐exchange chromatography. The final product was homogeneous by electrophoretic, ultracentrifugal and immunological criteria and had a molecular weight, by the low‐speed equilibrium method, of 260000. Treatment with sodium dodecylsulfate and 2‐mercaptoethanol dissociated the enzyme into dimeric subunits which could be further dissociated into monomeric subunits by treatment with 7.3 M guanidine hydrochloride and dithioerythreitol. The molecular weight of the monomeric subunit and the results of amino acid analysis suggested the presence of 14 subunits per mole. Carboxymethylation and hydrolysis showed the presence of 56 cysteine residues per mole, approximately half of which were available for titration with p‐chloromercuribenzoate in the urea‐denatured enzyme.The enzyme has a pH optimum of 6.3 and a Km of 0.46 mM in the crude state and 0.15 mM when pure. Thiol activation was required for catalytic activity and some evidence was obtained for a requirement for zinc as a co‐factor. Heavy metals inhibited the enzyme in a complex manner. Zinc acted as a competitive inhibitor, lead acted as a non‐competitive inhibitor, while cadmium acted in a unique manner, causing inhibition at low concentrations of substrate and stimulation at high levels of substrate.
A Simplified Procedure for the Isolation of the Sucrase · Isomaltase Complex from Rabbit Intestine Its Amino‐Acid and Sugar Composition
Authors: Cogoli Augusto , Mosimann Hugo , Vock Claudia , von Balthazar Anna‐Karin , Semenza Giorgio 
Published: March 2005

A simplified procedure for the isolation of the sucrase · isomaltase complex from rabbit small intestine was worked out, which allows the preparation of this protein in a homogeneous form in good yield.The sucrase · isomaltase complex from rabbit small intestine is a glycoprotein. It is rich in acidic amino acids; it has no free thiols in the native state but denaturation unmasks six thiols. The tyrosine/tryptophan ratio is 2. The sugar moiety(ies) (150%) is composed of d‐glucose, d‐galactose, d‐mannose, fucose, glucosamine and galactosamine (the latter two are probably acetylated). The sucrase · isomaltase complex contains no neuraminic, uronic acid, lipids, phosphate, sulfate or zinc.The partial specific volume, calculated from the amino acid and sugar composition, agrees with that experimentally determined. The hydrophobicity coefficient of this membrane protein does not differ from that of other membrane bound or water soluble proteins.
The Cytoplasmic DNAs of Cultured Human Cells
Authors: Koch Jürgen 
Published: March 2005

Ethidium bromide at a concentration of 1 μg × ml−1 in the medium completely inhibits the synthesis of mitochondrial DNA (ϱ= 1.706 g×ml−1) in exponentially growing Chang liver cells (ATCC; CCL 13), as indicated by the fact that labeled thymidine is not incorporated into any of the three forms of mitochondrial DNA (closed circular, open circular and linear DNA). Existing mitochondrial DNA undergoes a structural change under these growth conditions. At first closed circular DNA with a higher super‐helix density is generated. After prolonged exposure of the cells to the drug (48 h) no remaining closed circular DNA is detectable in the mitochondria. The bulk of the remaining mitochondrial DNA behaves during sedimentation like a linear DNA molecule with the length of the circumference of the circular mitochondrial DNA. Removal of the drug from the growth medium reverses this process of interconversion of the different forms of mitochondrial DNA.No such drastic effects of ethidium bromide on the light cytoplasmic DNA (ϱ= 1.688 g × ml−1) have been observed.
Structural and Enzymic Studies on the Process of Dye‐Sensitized Photoinactivation of Yeast Hexokinase
Authors: Menezes Luiz C. , Grouselle Michel , Pudles Julio 
Published: March 2005

In the work presented here, we have examined the effects of methylene‐blue‐sensitized photo‐oxidation on yeast hexokinase (ATP:D‐hexose‐6‐phosphotransferase). This treatment causes the enzyme to lose all activity by a biphasic process, an initial, rapid phase accounts for a 50% loss in activity.The 50% inactivated enzyme had the same Km as the native enzyme for both substrates d‐glucose and Mg‐ATP. Only V was affected. The pH dependency for the fist rate of inactivation corresponds to that expected for the oxidation of the imidazole ring.The initial rate of enzyme inactivation at pH 7.5 in presence or in absence of the substrate (d‐glucose) was accompanied by an initial decrease in the histidine content followed in the second phase of the inactivation rate process by the decrease of cysteine, methionine, tryptophan and tyrosine contents. In the course of photooxidation at pH 6.0, not only the hisitdine but also the methionine residues were oxidized during the initial rate of enzyme inactivation, followed by cysteine, tryptophan and tyrosine. Other amino acid residues were not affected through all the photodynamic process.Kinetic and conformational studies of the photoinactivation process suggest that from the five accessible histidine residues which are destroyed during the initial rate of enzyme inactivation at pH 7.5, one residue is essentially involved in the stabilization of the protein conformation.Neither the tryptophan nor the tyrosine residues modified during the photodynamic process appear to have an effect on the enzyme activity loss. The slow process of photoinactivation must depend then on the oxidation of cysteine and (or) methionine residues.
Métabolisme phospholipidique des hématies nucéées
Authors: Soula Georges , Souillard Cécilia , Douste‐blazy Louis 
Published: March 2005

Incorporation of [32P]orthophosphate into chicken and chick‐erythrocyte and plasma phospholipids in vivo was studied by means of one and two‐dimensional thin‐layer chromatography between 3 and 144 h.Radiophosphate was actively incorporated into plasma phospholipids, more rapidly into chick than chicken (maximum specific activity after 6 and 12 h, respectively) and more slowly into erythrocyte phospholipids.After 12 h, the chicken‐plasma phospholipids were labelled in the order: phosphatidylcholine, phosphatidyléthanolamine, lysophosphatidylcholine, phosphatidylinositol, sphingomyelin and phosphatidylserine; while the chick‐plasma phospholipids, after 6 h, were labelled in the order: phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine and sphingomyelin.In chick‐ and chicken‐erythrocyte phospholipids, higher specific activities, very similar for all the phospholipids studied, were observed at 144 h. These results strongly differ from those observed in in vitro incorporation: phosphatidic acid and phosphatidylglycerol were lowly labeled while phosphatidylserine showed high specific activities in the two experiments.Exchange reactions between plasma and erythrocyte phospholipids are not the same as in mammals; the high specific activities of the different erythrocyte phospholipids were too late for an exchange with plasma phospholipids. De novo biosynthesis was probably effective in the erythrocyte. The turnover of lysophosphatidyllcholine was low and the acylation of plasma phospholipids appeared less active than in mammal erythrocyte.
Enzyme Regulation in Neuroblastoma Cells
Authors: Siman‐Tov Rabi , Sachs Leo 
Published: March 2005

Acetylcholinesterase from mouse neuroblastoma C‐1300 was purified 733 times by affinity chromatography. Antibodies against the purified enzyme were used to select clones with a 100‐times‐lower acetylcholinesterase activity than in an unselected clone. Antibodies against an enzyme located on the surface membrane, can thus be used to select genetic variants with lower enzyme activity. The decrease in acetylcholinesterase activity was not associated with a decrease in choline‐O‐acetyltransferase activity, The two clones that showed a 100‐times difference in acetylcholinesterase activity showed about the same choline‐O‐acetyltransferase activity. This lack of correlation between the activity of these two cholinergic enzymes was also found in clones isolated without using antibodies.The existence of an independent control of these two enzyme activities was further shown by treatment of neuroblastoma cells with adenine, cyclic AMP and dibutyryl cyclic AMP, which increased acetylcholinesterase activity but decreased the activity of choline‐O‐acetyltransferase. There was no effect on either enzyme by treatment with guanine, hypoxanthine, cytidine, or uridine, indicating that adenine plays a specific role in the regulation of these two enzymes.High acetylcholinsterase activity was associated with low saturation density of the cells in tissue culture, and a low affinity to the surface of a tissue‐culture petri dish. It is suggested, that clonal differences in acetylcholinesterase activity and in these two biological properties, can be explained by differences in the structure of the surface membrane.