bluuebunny/crossref_metadata_2025 huggingface dataset:
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 Isolation and Characterization of Nerve‐Growth Factor from the Venom of Vipera russelli
Authors: Pearce Frederick L. , Banks Barbara E. C. , Banthorpe Derek V. , Berry Anthony R. , Davies Hugh ff. S. , Vernon Charles A.
Published: March 2005

Nerve‐growth factor, a substance specifically promoting, in vitro, the growth of nerve fibres from explants of embryonic dorsal root and sympathetic ganglia, has been purified from the venom of Vipera russelli. The purification proved remarkably difficult and presented some unusual features. The final product, which was homogeneous to all the tests available to us, was found to be a glycoprotein of molecular weight 37000 and containing about 20% of carbohydrate. From the values of the sedimentation and diffusion coefficients it may be shown that the axial ratio is approximately 1:7; the molecule has, therefore, an unusual shape. The substance is highly basic (isoelectric point approximately pH 10.5). It is also relatively stable: neither trypsin nor chymotrypsin affect its biological activity. However, activity is completely destroyed on treatment with pepsin. Comparison with nerve‐growth factor from other sources does not, with the data at present available, reveal any striking similarities. The difficulties involved in the isolation and characterization of this factor from snake venoms are briefly discussed.
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 Covalent Protein · Sepharose Complex for the Specific Adsorption and Assay of Adenosine 3′: 5′‐Monophosphate
Authors: Fisch Hans‐Ulrich , Pliška Vladimir , Schwyzer Robert
Published: March 2005

Specific cyclic AMP‐binding proteins were isolated from cow adrenals, partially purified, and covalently linked to Sepharose with BrCN. The semisynthetic material contains the probin in an insoluble, remarkably stable form. It exhibits specific affinity for cyclic AMP (mean association constant, Kass≃ 0.45 nM−1 at pH 7.4 in Tris‐theophyllin buffer), and can be conveniently used for the assay of 0.2 to 10pmol of cyclic AMP by isotope competitition experiments (cold versus3H‐labelled cyclic AMP). The results obtained compare favorably with those of parallel immunochemical assays of rat liver cyclic AMP. Dependence of the maximal binding capacity, P0 the mean association constant, Kass, and the Hill coefficient, n, on pH and NaCl concentration is discussed, and the calculation methods are presented.
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.
Stereospecificity of the Dihydroorotate‐Dehydrogenase Reaction
Authors: Blattmann Paul , Rétey János
Published: March 2005

Dihydroorotate dehydrogenase from Zymobacterium oroticum is shown to catalyse the anti‐elimination of hydrogen from the substrate.

In the presence of a catalytic amount of NAD+ the enzyme catalyses the exchange between the abstractable hydrogen atoms of dihydroorotate and solvent protons, the exchange of (5S)‐H atom being twice as fast as that of the 4‐H atom.

Sodium‐ethoxide‐catalysed exchange affects both diastereotopic protons in the methylene group of dihydroorotate, the (5S)‐H atom being exchanged somewhat faster than the (5R)‐H atom.

These findings are discussed in terms of stereospecificity and mechanism of flavin dependent dehydrogenase reactions.
The Mechanism of the Inhibition of Gramicidin‐S Synthesis by d‐Leucine
Authors: Saxholm Harald , Zimmer Trine‐Lise , Laland Søen G.
Published: March 2005

The results show that enzyme I of gramicidin S synthetase activates d‐leucine in a manner analogous to that of l‐leucine (ATP +d‐leucine ⇌d‐leucyl‐adenylate + PP1) and binds two moles of d‐leucine in thioester linkage. It has therefore two thiol sites for d‐leucine, compared to one for the l‐isomer. One of the sites seems to be the thiol site for L‐leucine which is therefore stereo‐unspecific. The results also indicate that the site for the formation of the aminoacyl adenylates is the same for the two isomers. In contrast to thioester‐bound l‐leucine, enzyme‐bound D‐leucine does not catalyze the ATP‐[14C]AMP exchange reaction.

At a molar ratio of l‐leucine: D‐leucine equal to 4, gramicidin S synthesis is almost completely inhibited and the growth of the peptide chain stops at the tetrapeptide stage (D‐Phe‐l‐Pro‐l‐Val‐L‐Orn). Enzyme‐bound D‐leucine is thus not able to replace L‐leucine in the formation of the pentapeptide.
The Covalent Structure of Collagen
Authors: Fietzek Peter P. , Rexrodt Friedrich W. , Wendt Peter , Stark Marlies , Kühn Klaus
Published: March 2005

Chymotryptic cleavage of α1‐CB6, the C‐terminal CNBr peptide of the α1‐chain from calf skin collagen, yields three peptides, designated C1 (positions 1–113), C2 (positions 114–196) and C3 (positions 197–217), respectively. The present paper describes the amino acid sequence of C2. It was determined exclusively by application of the automated Edman degradation. The entire peptide C2 as well as some of its tryptic and thermolytic peptides were subjected to this procedure. The helical portion of α1‐CB6‐C2 (positions 114–192) was found to consist of tripeptide units containing glycine in every third position, hence being typical of the collagen structure. The α1‐chain of calf skin collagen contains a 3‐hydroxyproline residue which was located in position 164 in the tripeptide unit Gly‐3Hyp‐4Hyp. This is the first case of a vertebrate collagen in which hydroxylation was found of a proline in position X of the tripeptide unit Gly‐X‐Y. The helical part of the α1‐chain terminates in a five‐fold repetition of the tripeptide Gly‐Pro‐Pro. The four C‐terminal amino acids of C2 are already part of the non‐helical area at the end of the α1‐chain which in addition comprises the entire peptide C3.
The Covalent Structure of Collagen
Authors: Wendt Peter , von der Mark Klaus , Rexrodt Friedrich , Küuhn Klaus
Published: March 2005

The peptide α1(78)‐CBC representing the amino‐terminal half of α1‐CB6, was obtained from α1(78) by cleavage with CNBr. The parent peptide α1(78) can be isolated from a mixture of collagen fragments which are products of limited digestion of native collagen with bacterial collagenase. Having the amino acid sequence of the tryptic peptides, it was possible to determine their order by the use of overlapping peptides derived from partial cleavage with thermolysine. The peptide chain is characterized by regions with predominantly polar and regions with mainly apolar amino acids, the distribution of which was found in excellent correlation to the distribution of dark and light cross‐striation bands of this specific part of the molecule, as depicted in long‐spacing segment crystallites. The sequence of nine amino acid residues (Gly‐Asp‐Arg‐Gly‐Glu‐Thr‐GIy‐Pro‐) was found twice in the elucidated peptide. Another sequence including 11 amino acid residues (Gly‐Ile‐Hyl‐Gly‐His‐Arg‐Gly‐Phe‐Ser‐Gly‐Leu) is identical to a sequence found in α1‐CB(4–5) except in one position in which a methionine was replaced by an isoleucine.
α‐l‐FucosyItransferases in Human Serum from Donors of Different ABO, Secretor and Lewis Blood‐Group Phenotypes
Authors: Schenkel‐Brunner Helmut , Chester M. Alan , Watkins Winifred M.
Published: March 2005

The presence of α‐l‐fucosyltransferases in human serum of different ABO, secretor and Lewis blood‐group phenotypes was investigated with guanosine diphosphate fucose as the sugar donor and low‐molecular‐weight oligosaccharides, or a macromolecular glycoprotein, as acceptors. In contrast to the α‐2‐l‐fucosyltransferases in human milk, submaxillary glands and stomach mucosa that occur only in ABH secretors, an enzyme that transfers l‐fucose to the C‐2 position of non‐reducing β‐galactosyl residues was found in the serum of all normal ABO donors irrespective of their ABH secretor‐nonsecretor status. The only sera in which the α‐2‐l‐fucosyltransferase was not demonstrable were three examples of the blood group Bombay Oh phenotype and one example of the Bh phenotype. An a‐3‐l‐fucosyltransferase that conveyed L‐fucose to the C‐3 position in N‐acetyl‐d‐glucosamine or d‐glucose occurred in all the sera examined including those from donors of the Bombay Oh and Bh phenotypes. a‐4‐l‐Fucosyltransferase activity was virtually absent from the sera, irrespective of the Lewis blood group of the donors.
Mouse‐Myeloma RNA Polymerase B Template Specificities and the Role of a Transcription‐Stimulating Factor
Authors: Lentfer Dierck , Lezius Axel G.
Published: March 2005

A DNA‐dependent transcriptase has been purified from mouse myeloma tissue. The system consists of an RNA polymerase of type B and a basic, heat‐labile protein of Mr 20000–30000 (stimulating factor). The RNA polymerase was characterized by its requirement for Mn2+ and medium ionic strength as well as by its susceptibility to inhibition by α‐amanitin and phosphate ions.

The template specificities were studied using native and heat‐denatured DNA as well as synthetic double and single‐stranded polydeoxynucleotides. Alternating polydeoxynucleotides like poly[d(A‐T) · d(A‐T)] and poly[d(I‐C) · d(I‐C)] are excellent templates, the latter exceeding native DNA by two orders of magnitude. With these templates, factor stimulation is less expressed and at most two‐fold. With DNA‐like templates, such as poly[d(A‐C) · d(T‐G)] and poly[d(A‐G) · d(T‐C)], transcription proceeds at a lower rate (though still exceeding native DNA), but factor can stimulate up to 5‐fold. In contrast, the homoduplexes poly(dA) · poly(dT), poly(dG) · poly(dC), and poly(dI) · poly(dC) are transcribed poorly, if at all, irrespective of the presence or absence of stimulating factor. Among the single‐stranded polydeoxynucleotides tested poly[d(A‐C)], poly(dA), poly(dI), and poly(dC) are moderately well transcribed, whereas poly[d(T‐G)] and poly(dI) are completely ineffective.

Initiation of transcription was studied using [γ‐32P]ATP. From the results it is concluded that the stimulating factor is involved in the progress of transcription rather than in initiation.
Energy‐Dependent Changes in Membranes of Rhodospirillum rubrum Chromatophores as Measured by 8‐Anilino‐naphthalene‐1‐sulfonic Acid
Authors: Vainio Harri , Baltscheffsky Margareta , Baltscheffsky Herrick , Azzi Angelo
Published: March 2005

Rhodospirillum rubrum chromatophores enhance the fluorescence of 8‐anilino‐naphthalene‐1‐sulfonic acid (ANS). Energization of the chromatophore membrane by ATP, PPi, or succinate produces a fluorescence increase in ANS; PPi is the most potent in this respect.

It was possible to inhibit the fluorescence increase due to ATP, PPi, and succinate by uncouplers; namely, gramicidin, valinomycin, and tetraphenylboron. The uncouplers did not, however, affect the non‐energy‐dependent ANS fluorescence.

It was possible to inhibit the increase in ANS fluorescence due to ATP by oligomycin, whereas the PPi‐induced ANS fluorescence increase was even slightly stimulated in the presence of oligomycin.

The antibiotic dio‐9 was able to inhibit the fluorescence increase due to both ATP and PPi, without, however, affecting the non‐energy‐dependent ANS fluorescence.

Chaotropic agents such as KSCN and urea, and the detergent deoxycholate all inhibited the PPi‐induced membrane energization, as detected by ANS fluorescence.
Interaction between Pyridoxamine 5′‐Phosphate and Apo‐Aspartate Aminotransferase from Pig Heart
Authors: Arrio‐Dupont Martine
Published: March 2005

Apoenzyme of aspartate aminotransferase prepared according to various techniques can bind the pyridoxamine‐P coenzyme and recover its full activity. An anticooperativity is observed between the two sites of the dimer; the intrinsic dissociation constants are 20 nM and 200 nM for apoenzymes prepared in the presence of phosphate and less than 1.8 and 6 nM for an apoenzyme prepared without phosphate. The phosphate ions can bind to two sites of the apoenzyme with an intrinsic constant of 1.6 μM; a competition is observed between pyridoxamine‐P and phosphate but it is not sufficient to explain the observed differences between the various apoenzymes. At pH 8.3, pyridoxamine‐P can dissociate from the holoenzyme: the dissociation rate is very slow so that it does not occur in enzymatic transamination.

In the presence of succinate, the two sites become equivalent and the affinity for pyridoxamine‐P is smaller, meaning the occurence of an interaction between the substrate‐binding site and the coenzyme‐binding site.
The Subunit Structure of Lactate Dehydrogenase from Streptococcus cremoris US3
Authors: Dynon Maria K. , Jago G. Richard , Davidson Barrie E. , Gething Mary Jane H. , Davidson Barrie E.
Published: March 2005

Lactate dehydrogenase from Streptococcus cremoris US3, which has a molecular weight in solution of 140000, was found by dodecylsulphate‐gel electrophoresis to have a minimum molecular weight of 37000. It is, therefore, a tetramer.

Approximately 40 tryptic peptides were obtained by fingerprinting and in conjunction with the amino acid composition this indicates that the subunits are similar and probably identical.

There is one thiol group per subunit.

p‐Chloromercuribenzoate inhibits the enzyme.
The Purification and Properties of an NADPH‐Linked Aldehyde Reductase from Pig Brain
Authors: Turner Anthony J. , Tipton Keith F.
Published: March 2005

An NADPH‐linked aldehyde reductase has been purified some 600‐fold from the supernatant fraction of a pig brain homogenate.

Subcellular distribution studies show that the enzyme is almost exclusively located in the cytosol.

The enzyme has a molecular weight of 29000 and an isoelectric point of 5.8.

The enzyme reduces a wide range of aliphatic and aromatic aldehydes, including some biogenic aldehydes. Ketones are not reduced.

Specificity studies carried out on the enzyme suggest that substituted phenylglycolaldehydes are reduced far more readily than the corresponding substituted phenylacetaldehydes.

Ethanol, reserpine and several aldehyde dehydrogenase and liver alcohol dehydrogenase inhibitors do not affect the activity of brain NADPH‐linked aldehyde reductase. 0.1 mM sodium barbitone markedly inhibits the enzyme.
Benzene Metabolism of Moraxella Species
Authors: Högn Thomas , Jaenicke Lothar
Published: March 2005

A benzene‐utilizing strain of Moraxella B (genus/Acinetobacter) was isolated from Rhine water. Cell‐free extracts of this bacterium, which is highly specific for the oxidation of benzene, require for this process catalytic amounts of NADH. This is regenerated during the course of the reaction by the dehydrogenation of cis‐cyclohexadiene‐diol to pyrocatechol. The oxygenase system contains nonheme‐iron in the form of strongly bound Fe2+. It incorporates two atoms of oxygen simultaneously, as proven by isotope experiments; it is thus a dioxygenase. cis‐1,2‐Dihydroxy‐dihydrobenzene was isolated as the first identifiable stable intermediate. This is further oxidized to pyrocatechol which is then eventually completely metabolized via the 3‐oxoadipic acid pathway.
Replication of Bacteriophage M13
Authors: Staudenbauer Walter L. , Hofschneider Peter Hans
Published: March 2005

In Escherichia coli HfrH 165/70 (dnaB) infection with bacteriophage M 13 is abortive at the restrictive temperature of 41°C. However, if the progeny single‐strand synthesis has started at the permissive temperature (34°C), it will continue after a temperature shift by an asymmetric displacement synthesis as shown (a) by the flow of label from replicative form molecules to single strands and (b) by the almost exclusive labeling of the viral strand of the replicative form. Since a pulse label is found first in the membrane fraction and can be chased into the cytoplasm, M 13 single‐strand synthesis may occur at the cell membrane. Also at the elevated temperature, it is inhibited by rifampicin and chloramphenicol. Whereas rifampicin stops the synthesis of phage‐specific DNA immediately, chloramphenicol causes the conversion of newly synthesized single strands into double‐stranded replicative form molecules. This demonstrates that not only the synthesis of M 13 single strands but also their conversion into double strands is possible even in the absence of the dnaB function. Therefore for these types of DNA replication, contrary to the semiconservative replication of double‐stranded DNA, the product of the dnaB gene is not required.
Differences between the ATP/ADP Ratios in the Mitochondrial Matrix and in the Extramitochondrial Space
Authors: Heldt Hans W. , Klingenberg Martin , Milovancev Mirjam
Published: March 2005

The relationship between the ATP/ADP ratios inside and outside the mitochondria was studied using silicon‐layer centrifugation. As predicted from the properties of the adenine nucleotide translocation, the ATP/ADP ratio in the extramitochondrial space was found to be higher than in the mitochondria when in the state of respiratory control. Taking into account the content of inorganic phosphate, the phosphorylation potential in the mitochondria is calculated to be 2.3 kcal lower than in the medium.

This difference between the internal and external ATP/ADP ratios is abolished if uncoupler is added, When there is also no Mg2+ present in the medium, the internal ATP/ADP ratio may be even higher than the external ratio, provided that there was oligomycin added in order to inhibit ATP hydrolysis.

It is shown that the ADP is transported against a gradient from the extramitochondrial space into the mitochondria. The discussion of a model suggests that the physiological role of the energy‐requiring transport of ADP into the mitochondria is to maintain a constant level of the ATP in the cytosol.
On the Role of Alcohol Dehydrogenase in ω‐Oxidation of Fatty Acids
Authors: Björkhem Ingemar
Published: March 2005

Rat and horse liver alcohol dehydrogenase was found to oxidize ω2‐hydroxyfatty acids into the corresponding oxofatty acids but showed no significant activity towards ω1‐hydroxyfatty acids. However, ω1‐hydroxyfatty acids were oxidized into the corresponding dicarboxylic acids by a combination of horse or rat liver alcohol dehydrogenase with rat liver aldehyde dehydrogenase. The ratio between ω‐hydroxyfatty acid dehydrogenase activity and ethanol dehydrogenase activity increased about two‐fold through the steps used in the preparation of alcohol dehydrogenase from rat liver. Oxidation of 17‐dl‐hydroxystearic acid by rat liver alcohol dehydrogenase was inhibited by pyrazol, ethanol and 3β‐Lhydroxy‐5β‐cholanoic acid. Oxidation of ethanol and 3β‐hydroxy‐5β‐cholanoic acid by rat liver alcohol dehydrogenase was inhibited by ω‐hydroxy‐fatty acids. Long‐chain ω1‐ and ω2‐hydroxyfatty acids were oxidized by rat liver alcohol dehydrogenase much more efficiently than medium‐chain ω1‐ and ω2‐hydroxyfatty acids and the Km, values for oxidation of the long‐chain ω‐hydroxyfatty acids were 10–40 μM. The rate of oxidation of 17‐l‐hydroxystearic acid by rat and horse liver alcohol dehydrogenase was faster than that of oxidation of 17‐d‐hydroxystearic acid. Experiments with different monohydroxylated stearic acids as substrates for rat liver alcohol dehydrogenase showed that significant activity appeared when the distance between the hydroxyl group and the carboxyl group was more than 12 carbon units. The activity increased with increasing distance between the hydroxyl group and the carboxyl group. Lauryl alcohol but not stearyl alcohol was oxidized by rat liver alcohol dehydrogenase to a significant extent.
Different Pool Sizes of the Precursor Polypeptides of Cytochrome Oxidase from Neurospora crassa
Authors: Schwab Andreas J. , Sebald Walter , Weiss Hanns
Published: March 2005

Pulse‐labelling experiments with growing Neurospora crassa revealed that the polypeptides composing the protein moiety of a cytochrome oxidase preparation are derived from at least four independent pools of precursor polypeptides. The pool sizes range from 2% to 25% of the amount of the corresponding polypeptide present in cytochrome oxidase. The smallest pool is assigned to a polypeptide of mitochondrial origin. Serial pools were found for one of the polypeptides.
Reaction Mechanism of l‐Glutamate Dehydrogenase
Authors: di Franco Ann , Iwatsubo Motohiro
Published: March 2005

Presented here are the optical characteristics (absorbance, fluorescence, circular dichroism) of several enzyme · coenzyme complexes of l‐glutamate dehydrogenase, with and without the different substrates (or products). Three of the four complexes studied can be distinguished unambiguously by the following characteristics: the E · NADPH and E · NADPH ·l‐glutamate complexes have similar absorption and fluorescence properties, but markedly differ in their opposite ellipticities; the E · NADPH · 2‐oxoglutarate possesses characteristic absorption and elliptical properties, but its highly quenched fluorescence is its most distinctive feature.

A detailed study of the elementary kinetic parameters, k+ and k‐, was carried out with respect to each ligand. A comparison is made of the Kd values calculated from the k−/k+ ratio and from direct binding studies. As far as NADPH is concerned it is clear that its association rate constant is not affected, while its dissociation rate constant is very sensitive to the presence of the substrates. Thus, the stability of each complex is mainly governed by the dissociation rate. These dissociation rate constants are not very different from those obtained for the different substrates.
Fast‐Kinetic Studies of the Oxidative Deamination of Glutamate Catalyzed by Glutamate Dehydrogenase
Authors: D'albis Anne , Pantaloni Dominique
Published: March 2005

The release of protons, one of the products of the reaction, in the oxidative deamination of glutamate by glutamate dehydrogenase, has been followed in a stopped‐flow spectrophotometer by measuring the absorbance change of an indicator dye. The results obtained at pH 7.8 and 15 °C, in 0.05 M Na2SO4, with NADP+ as coenzyme, indicate that the liberation of the protons occurs after the electron transfer but before the release of NADPH, i.e. before the steady state; the results obtained in the same conditions, but in presence of the inhibitor GTP, indicate moreover that it occurs also before the liberation of 2‐oxoglutarate.

The oxidative deamination of glutamate has in addition been studied in the presence of equi‐molar amounts of the reduced coenzyme NADPH. The elimination of the presteady‐state step when all the enzyme is in the form of the enzyme · NADPH · glutamate complex supports the hypothesis that the rate‐limiting step in the overall mechanism is the dissociation of NADPH and the regeneration of the enzyme from this ternary complex.
Interaction of Concanavalin A with Rat Lymphocytes
Authors: Betel Ido , van den BERG Kor J.
Published: March 2005

Concanavalin A covalently bound to acrylic polymer particles (enzacryl AA), which cannot enter the cells, stimulated rat lymphocytes into DNA synthesis.

Evidence was obtained that concanavalin A binds to the lymphocyte membrane specifically through its sugar‐binding sites. No nonspecific adsorption was found.

The equilibrium constant found for the binding of concanavalin A to the cell was 12 μ−1 at 37°C and 3°−l at 0°C. No heterogeneity in the affinity of the binding sites was detected. The average maximum number of concanavalin A molecules (Mol. wt 108000) that could be bound was about 5 ×106 per cell.

Optimum stimulation of DNA synthesis was obtained when about 5%, of the available sites on the cells were occupied by concanavalin A.
The Theory of Alternative Substrates in Enzyme Kinetics and Its Application to Yeast Hexokinase
Authors: Ricard Jacques , Noat Georges , Got Claude , Borel Maguy
Published: March 2005

The theory of alternative substrates has been thoroughly investigated. The reciprocal rate equations, for ordered and random kinetic mechanisms, in the presence of an alternative substrate, have been developed. The conditions in which those equations give rise to either hyperbolic or linear reciprocal plots have been established. Degeneracy and constraint conditions of the various kinetic models have also been derived.

Definite conclusions concerning the relevant mechanism can be obtained only if the Dalziel coefficients pertaining to the substrate are different from those corresponding to the alternative substrate. It follows from this, that the method termed “isotope competition”, cannot be used to make a choice among various two‐substrate mechanisms, and appears to be unfounded.

The theory of alternative substrates has been applied to the study of yeast hexokinase. The results obtained are at variance with a random mechanism. They do agree, on the other hand, with a compulsory order of substrate binding on the enzyme, glucose being bound first.
Functional Properties of Native and Reconstituted Hemoglobins from Chironomus thummi thummi
Authors: Amiconi Gino , Antonini Eraldo , Brunori Maurizio , Formaneck Helmut , Huber Robert
Published: March 2005

A procedure for preparation of native globin from Chironomus hemoglobin has been developed and functional properties of native and reconstituted (proto‐, meso‐ and deutero‐) hemoglobins have been studied. Globin differs from the corresponding hemoglobin in a number of properties, but the differences are reversible and vanish on addition of protoheme. The oxygen affinity of protohemoglobin (p1/2 = 0.7 mm Hg at 20°C) is lower than that of meso‐ and deutero‐hemoglobins, respectively, the shape of oxygenation curve of all three hemoproteins being consistent with a simple mass‐law formulation. While protohemoglobin shows a small but definite Bohr effect, the oxygen affinity of the deuteroderivative is pH independent. Kinetic results show that binding of oxygen, carbon monoxide and ethylisocyanide to native and reconstituted Chironomus hemoglobins is a simple process, since the association process follows bimolecular behaviour and the dissociation reaction conforms to first‐order kinetics.
Regulation of d‐Triokinase and NAD‐Linked Glycerol‐Dehydrogenase Activities in Rat Liver
Authors: Veneziale Carlo M.
Published: March 2005

The specific activities expressed as [μmol × g wet wt−1× min−1 of d‐triokinase and NAD‐linked glycerol dehydrogenase, with d‐glyceraldehyde as substrate, were determined in the 27500 ×g supernatant fraction of livers from rats subjected to various dietary and hormonal programs. The specific activity of triokinase varied over a three‐fold to four‐fold range, being lowest in livers of diabetic rats fed a Purina Chow and highest in livers of intact rats fed 72% fructose diets containing casein; total triokinase activity per liver was five‐fold to six‐fold greater in the latter group than in the former group. The specific activity of triokinase also increased in livers of rats fed diets of 72% glucose or 72% dextrin plus 18% casein. Fructose feeding caused a decrease in specific activity of glycerol dehydrogenase. Glucagon had no effect on the specific activity of either enzyme in livers of fasted rats. Cortisone tended to increase the specific activity of triokinase as well as its total activity in livers of rats fed the Purina Chow. After only 48 h of a 72% fructose‐18% casein diet, liver weight was increased 50% relative to livers of rats fed the Purina Chow. Triokinase is subject to regulation in livers of adult male rats. The data indicate that the NAD‐linked glycerol dehydrogenase is also subject to regulation but to a lesser extent. If glucagon does stimulate the flux of carbon in the triokinase or the NAD‐linked glycerol dehydrogenase reactions, it is not as the result of increases in enzyme concentrations as determined by assayable activity measurements.
Metabolism of Isolated Kidney Tubules
Authors: Guder Walter G. , Wieland Otto H. , Stukowski Barbara
Published: March 2005

The effects of parathyroid hormone, adenosine 3′:5′‐monophosphate (Ado‐3′:5′‐P) and oleate on renal metabolism have been studied in isolated kidney‐cortex tubules from starved rats. Substrate uptake and glucose formation have been determined with l‐lactate, pyruvate, glutamate, and glycerol as substrates.

The results indicate that parathyroid hormone and Ado‐3′:5′‐P increase renal gluconeogenesis by the same mechanism, which however, differs from the effects brough about by free fatty acids:

Parathyroid hormone and Ado‐3′: 5′‐P increased glucose formation from l‐lactate, pyruvate and glutamate. The stimulatory effect of parathyroid hormone was always smaller compared with that of Ado‐3′:5′‐P. Glucose formation from l‐lactate was shown to be stimulated with hormone doses as low as 1–10 nM. Oleate had an increasing effect oil glucose formation from l‐lactate and glycerol, but no effect on glucose formation from 10 mM pyruvate and inhibited gluconeogenesis from glutamate.

The stimulatory action of parathyroid hormone and Ado‐3′:5′‐P was accompanied by an increase in substrate uptake, whereas oleate decreased substrate uptake.

The effects of parathyroid hormone and Ado‐3′:5′‐P on kidney‐tubule metabolism were not additive, when both substances were added together.

All effects of oleate were found to be unchanged in the presence of parathyroid hormone and Ado‐3′:5′‐P or both.

The results suggest two different mechanisms which may regulate renal gluconeogenesis additively.
Thermochemistry of Lysozyme‐Inhibitor Binding
Authors: Bjurulf Carin , Wadsö Ingemar
Published: March 2005

The binding of saccharide inhibitors to lysozyme has been studied using a microcalorimetric method. Measurements have been performed with N‐acetyl‐d‐glucosamine (pH 5 and 25 and 40°C), tri[N‐acetyl‐d‐glucosamine] (pH 2, 5 and 7.6 at 25°C and pH 5 at 40°C) and with tetra‐[N‐acetyl‐d‐glucosamine] (pH 2, 5 and 7.6 at 25°C). From the results of the calorimetric measurements values for ΔG0, ΔH0, ΔS0, and ΔCp0 were derived for the binding processes.

In addition to the saccharide‐binding reactions, some model experiments were performed.
Studies on Rat‐Liver Ribosomes, Ribosomal Subparticles and RNA Fixed with Formaldehyde
Authors: Dessev George N. , Grancharov Konstantin
Published: March 2005

Rat liver polysomes, single ribosomes, EDTA‐produced subparticles and free ribosomal RNA were treated with formaldehyde and their properties were studied. The following results were obtained.

RNA can be isolated from fixed polysomes or subparticles by exhaustive digestion with pronase. Such RNA shows a normal electrophoretic behaviour in agar gel.

In free RNA treated with formaldehyde the mobility of 18‐S and 28‐S RNA fractions is reduced; latent breaks are not revealed by heating; sensitivity towards ribonuclease is increased. No such effects are observed with RNA treated with formaldehyde while in the particles.

In fixed polysomes or single ribosomes the two subparticles appear to be covalently bound through their proteins. No intermolecular bonding occurs between 18‐S and 28‐S RNA either in fixed polysomes or in free RNA. The fixed ribosomes and subparticles can be fractionated by agar gel electrophoresis and give profiles similar to those of untreated particles. These results may prove useful in combining CsCl density gradient fractionation with gel electrophoretic methods.
Candida krusei Cytochrome c: a Correction to the Sequence
Authors: Lederer Florence
Published: March 2005

With the help of an automatic protein sequenator, we have verified a prediction made by us in a recent paper, namely that residue 16 in Candida krusei cytochrome c is a glutamine and not a glutamic acid. Neurospora crassa cytochrome c now remains the only mitochondrial cytochrome c which apparently does not have a glutamine at this position. In the course of this investigation, from two strains of Candida krusei we isolated two cytochromes which differ in amino acid composition. One of them seems to correspond to that described in the literature. Two of the differences have been located.
Rodent and Human Acid α‐Glucosidase
Authors: De Barsy Thierry , Jacquemin Paul , Devos Pierre , Hers Henri‐Géry
Published: March 2005

The lysosomal acid α‐glucosidase has been purified more than 10000‐fold from rat and mice liver and from human placenta. The kinetic properties of the human enzyme are similar to those previously described for the rodent enzyme, with the main exception of the absence of inhibition by an excess of maltose. Antibodies were obtained by injection of the pure rat liver, mouse liver or human placental α‐glucosidase to rabbits. The property of the enzyme of hydrolysing glycogen or to catalyse transglucosylation from maltose to glycogen could be nearly completely inhibited by the antibodies whereas the hydrolysis of small molecular substrates was much less inhibited. Within the same species, the same amount of enzyme was inhibited by a given amount of antibodies, whatever the tissue used as a source of enzyme. A partial cross‐reactivity was observed from species to species. The presence of an immunologically reacting protein in tissues from patients with type‐II‐glycogenosis could not be demonstrated either by double‐diffusion test or by antibodies consumption. Similar negative results were also obtained with normal human liver in which acid α‐glucosidase has been inactivated at alkaline pH.
Binding of Reduced Cofactor to Glutamate Dehydrogenase
Authors: Shafer Jules A. , Chiancone Emilia , Vittorelli Letizia M. , Spagnuolo Carla , Mackler Bruce , Antonini Eraldo
Published: March 2005

The binding of reduced cofactor (NADH) to glutamate dehydrogenase has been studied by gel filtration, equilibrium dialysis and fluorescence enhancement. The number of binding sites significantly exceeds 1 per polypeptide chain (1.3 per chain of molecular weight 56000), the saturation curve gives indications of slight positive interactions. The presence of GTP, glutamate and ADP, apart from increasing the affinity of NADH for glutamate dehydrogenase, appears to affect both the number of binding sites and the cooperativity of the binding.