bluuebunny/crossref_metadata_2025 huggingface dataset:
Magnetic Resonance Studies on Manganese‐Activated Phosphofructokinase
Authors: Jones Robert , Dwek Raymond A. , Walker Ian O.
Published: March 2005

Rabbit skeletal‐muscle phosphofructokinase exhibits similar kinetic behaviour and specific activity when manganese is used as the essential divalent cation in place of magnesium. At pH 7.5, the kinetics are Michaelis‐Menten with respect to fructose 6‐phosphate, ATP and manganous ion concentrations; the reaction, as a function of metal ion concentration, is inhibited competitively by excess free ATP and by calcium ions. Between pH 7.0 and 6.5, in the presence of high ATP concentrations, there is a sharp transition to sigmoid kinetics as indicated by an increase of the Hill interaction coefficient from 1 to 4.1. Preincubation of the enzyme with ligands has a marked effect on activity, particularly free divalent metals which cause dissociation and an almost total loss of activity.

Studies on the rates of water‐proton longitudinal relaxation at pH 7.5 and 35 MHz indicate that, in the absence of nucleotides, the phosphofructokinase‐manganese system shows low relaxation enhancements (ɛb∼ 2). On titrating with ATP at fixed enzyme and manganese ion concentrations, the enhancement rises sharply, then falls again at very high ATP levels. An analysis of enhancement data at fixed ATP concentrations as a function of enzyme and metal ion concentrations, using a combination of graphical and computational methods, indicates that the protomer of 90000 molecular weight has 2 Mn‐ATP sites with dissociation constant 240 μM and an enhancement for the ternary complex ɛt, of 18. Free ATP binds competitively with a dissociation constant of 3 mM. Line‐broadening studies on the H‐2 and H‐8 resonances of the adenine ring in the high resolution spectrum of ATP reveal that the proton‐manganese distances in Mn‐ATP are almost unchanged in forming the ternary complex with enzyme. The kinetic and magnetic resonance data indicate that Mn‐ATP is the true substrate, which probably binds to the enzyme via a nucleotide bridge.
Regulation of Gluconeogenesis in Acetobacter xylinum
Authors: Weinhouse Haim , Benziman Moshe
Published: March 2005

Gluconeogenesis in the form of cellulose synthesis from succinate and pyruvate has been studied in resting cells of Acetobacter xylinum. Substrate utilization and cellulose formation occurred at linear rates. At saturating levels of substrate the rate of cellulose formation from succinate was two to three times that from pyruvate. The cellulose formed accounted for 5% of the pyruvate utilized and for 33% of the succinate utilized. Acetate which alone was not gluconeogenic increased the rates of succinate and pyruvate conversion to cellulose several‐fold without significantly affecting their over‐all utilization. Acetate oxidation was strongly stimulated by both gluconeogenic substrates. A “crossing over” of carbon atoms from acetate to cellulose was observed in the presence of succinate but not in the presence of pyruvate. Succinate and pyruvate when added together yielded more cellulose than expected on the basis of mere additive effects, owing to an increase rate of cellulose formation from pyruvate. Possible mechanisms correlating these observations with the properties of the enzyme systems involved in the interconversion of the various substrates are discussed.
Effect of Sulfur Binding on Rhodanese Fluorescence
Authors: Finazzi Agrò Alessandro , Federici Giorgio , Giovagnoli Carlo , Cannella Carlo , Cavallini Doriano
Published: March 2005

Intrinsic fluorescences of the sulfur‐transferring protein rhodanese (or thiosulfate: cyanide sulfurtransferase) and of artificially persulfurated bovine serum albumin were studied. Both are attributable mainly to tryptophan side‐chains. Addition of cyanide or sulfite to these proteins causes a 20–25% increase in fluorescence.

Both proteins exhibit a low intensity absorption band at about 330 nm which disappears on addition of cyanide or sulfite. This absorption can be restored in rhodanese, after the treatment with cyanide, by excess thiosulfate; in the same time the fluorescence decreases to that of sulfur‐containing enzyme.

A change in solvent viscosity, obtained by addition of glycerol, reduces the quenching effect in both proteins. On the basis of these data a long‐range energy‐transfer mechanism between the donor (tryptophan) and an acceptor is proposed. The acceptor involved seems to be one or more persulfide groups (R‐SSH) in both proteins.

The presence of a persulfide group into rhodanese fits very well with the current knowledge of the mechanism of this enzyme.
On the Restriction of the Rotational Diffusion of Proteins in Polymer Networks
Authors: Laurent Torvard C. , ÖBrink Björn
Published: March 2005

The rotational relaxation time of dansyl‐labelled human serum albumin in solutions of dextran and polyethylene glycol of high concentrations (up to 0.3 g/ml) has been determined by fluorescence depolarization measurements. The increase in relaxation time due to the impeding effect of the polymer chains is moderate and the relaxation time is approximately doubled in 0.25 g/ml of polymer. The effect is independent of the molecular weight of the polymer; sucrose has approximately the same effect as dextran. It is concluded that, in contrast to the very restricted translational diffusion, the rotational diffusion of the albumin molecule is only moderately suppressed, even in very dense polymer networks.
Effect of Ionic Strength, pH, Amines and Divalent Cations on the Lytic Activity of T4 Lysozyme
Authors: Jensen Harald B. , Kleppe Kjell
Published: March 2005

The influence of ionic strength, pH, basic amines and divalent cations on the apparent lytic activity of T4 lysozyme on chloroform‐treated Escherichia coli B cells has been studied, and in some cases compared with the effect on hen egg‐white lysozyme.

The lytic activity of T4 lysozyme was found to be markedly dependent on ionic strength and pH. Maximum activity was observed at pH 7.3 at I= 0.07. Very low activity was observed above pH 8.5 whereas maximum activity of hen egg‐white lysozyme at I= 0.06 was obtained at pH 9.2.

Low concentrations of spermine, 40 μM, enhanced the lytic activity of T4 lysozyme 3 to 4‐fold. No activating effect was observed on hen egg‐white lysozyme.

Higher concentrations of spermine, above 100 μM inhibited lysis, presumably by stabilizing the protoplasts formed. The muramidase activity of T4 lysozyme was not inhibited, but rather stimulated also under these conditions.

Several other amines and Mg2+ and Ca2+ were also tested. They exhibited similar effects as spermine. In contrast to spermine, the inhibition observed at high concentrations of Mg2+ is presumed to be due to inhibition of the enzyme.
The Stereochemistry of Hydrogen Transfer from NADPH Catalysed by 3‐Hydroxy‐3‐methylglutaryl‐coenzyme A Reductase from Rat Liver
Authors: Beedle Alan S. , Munday Kenneth A. , Wilton David C.
Published: March 2005

3‐Hydroxy‐3‐methyl‐[3‐14C]glutaryl‐CoA was reduced to mevalonic acid in the presence of either [4A‐3H]‐ or [4B‐3H]NADPH and rat liver 3‐hydroxy‐3‐methylglutaryl‐CoA reductase. The resulting doubly labelled mevalonic acid incorporated tritium exclusively from the 4A (4R) position of NADPH. The distribution of tritium at C‐5 of the mevalonic acid was determined by a biological degradation. It was concluded that during the reduction of 3‐hydroxy‐3‐methylglutaryl‐CoA to mevalonic acid two hydrogen atoms from the 4A (4R) position of NADPH are transferred directly to mevalonic acid.
Influence of Thyroid Hormones on Enzyme Activities of Myelinating Rat Central‐Nervous Tissues
Authors: Wysocki Stanislaw J. , Segal Wolfe
Published: March 2005

The effects of triiodothyronine administration and of hypothyroidism on the rapidly developing enzymes UDP galactose: sphingosine galactosyltransferase and 2′:3′‐nucleotide 3′‐phosphohydrolase associated with central nervous system myelination were investigated. The activity of these enzymes in the spinal cords of young rats injected daily with triiodothyronine up to sacrifice on day 5 was significantly increased over control animals. In normal animals, circulating plasma thyroxine increased gradually from the second postnatal day to a maximum value at days 15–17. Rats, born of mothers treated with n‐propylthiouracil from the thirteenth day of pregnancy, did not exhibit the increase in plasma thyroxine. Determination of the specific activity of these enzymes in central nervous tissue of such hypothyroid rats at day 12 showed a significant reduction compared with normal animals. Intraperitoneal injection of the hypothyroid rats with triiodothyronine on day 8 resulted in a partial restoration of the activity of the enzymes in brain and of 2′:3′‐nucleotide 3′‐phosphohydrolase in spinal cord when assayed on day 12. Restoration of UDPgalactose: sphingosine galactosyltransferase activity did not occur in the spinal cord of such animals. However, when hypothyroid rats were injected with triiodothyronine on days 1, 4, and 7, the psychosine‐synthesising activity in their spinal cords on day 8 was restored to that of normal animals. This suggests that there is a critical period during the first 8 days of postnatal life when thyroid hormone levels must be adequate in order that spinal cord activity of this enzyme develops normally. It is concluded that the flux in circulating thyroid hormone is a factor in the normal development of these enzymes.
Studies on the Conformation of DNA‐Dependent RNA Polymerase in Solution by Small‐Angle X‐Ray Measurements
Authors: Pilz Ingrid , Kratky Otto , Rabussay Dietmar
Published: March 2005

The size and shape of DNA‐dependent RNA polymerase from Escherichia coli K 12 were studied by small‐angle X‐ray measurements. The equilibrium between the monomeric and dimeric form of the polymerase was investigated in dependence on the ionic strength of the solution.

Homodisperse solutions of the holo‐enzyme monomer and dimer and of the core enzyme monomer were investigated. For each particle the radius of gyration R, the molecular weight Mr and the volume V were determined; in detail the following values were obtained: RNA polymerase holo‐enzyme monomer (R= 6.2 nm, Mr= 4.9 × 105, V= 9.8 × 102 nm3), holo‐enzyme dimer (R= 9.5 nm, Mr= 9.95 × 105, V= 35 × 102 nm3), core enzyme (R= 6.0 nm, Mr= 3.8 × 105, V= 9.3 × 102 nm3).

The hole‐enzyme dimer is equivalent in scattering to a flat hollow cylinder of the following dimensions: length 10 nm, outer diameter 25 nm, inner diameter 5 nm. The shape of the holo‐enzyme monomer is not equivalent in scattering to any body which can be obtained by dividing this cylinder into two halves. The shape of the monomer is similar to that of the core enzyme; both are elongated bodies, which show a cross‐section factor.
Carboxyterminal Antigenic Determinants of Collagen from Calf Skin
Authors: Becker Udo , Timpl Rupert , Kühn Klaus
Published: March 2005

The prevalent antibody response in the rabbit to acid‐soluble calf collagen is specifically directed to sites on the C‐terminal cyanogen bromide peptides α1‐CB6a, α1‐CB61b and α2‐CB (3.5). Three different antigenic determinants each unique for one of these peptides could be defined serologically. After tryptic cleavage of α1‐CB6a or α1‐CB6b the full serologic activity was found on peptides composed of 39 and 28 amino acid residues, respectively. The center of the antigenic determinants could be located around a tyrosine residue in the nonhelical sequence of these peptides. Chymotrypsin which cleaved at this point completely abolished the serologic activity. Evidence is provided that α1‐CB6b is derived from α1‐CB6a by loss of 18 amino acid residues from the C‐terminal end. Therefore, the antigenic determinant on α1‐CB6b might be artificially created by tissue proteases. During this process a loss of activity for the antigenic determinant on α1‐CB6a was observed. Extraction of the collagen polypeptide chains with 8M urea prevented this degradation and revealed in comparison to α1‐CB6a an additional C‐terminal tyrosine residue.
Multiplicity and Origin of Isoenzymes of Lipoyl Dehydrogenase
Authors: Kenney William C. , Zakim David , Hogue Patricia K. , Singer Thomas P.
Published: March 2005

Reports in the literature on the number of isoenzymes of lipoyl dehydrogenase in mammalian heart mitochondria vary from 2 to 13 and one report claims that no isoenzymes occur in vivo. The present paper provides evidence that 6 main isoenzymes and 2 minor ones of lipoyl dehydrogenase occur in beef and pig heart, 3 of the main components being associated with 2‐oxoglutarate dehydrogenase, 3 with pyruvate dehydrogenase. Failure to detect these in some previous studies has been explained in terms of the experimental conditions used. Heart and liver mitochondria from several mammalian species present the same isoenzyme pattern. Evidence is presented that “free” (uncomplexed) lipoyl dehydrogenase detected in extracts of mitochondria by several investigators originates largely from the pyruvate dehydrogenase complex.
Octopine Dehydrogenase
Authors: Olomucki Anna , Huc Claude , Lefebure Francine , van Thoai Nguyen
Published: March 2005

The amino acid composition of octopine dehydrogenase from muscles of Pecten maximus was determined. The calculate dpartial specific volume of 0.74 allowed us to evaluate the molecular weight of the enzyme as 38000.

It is concluded that this enzyme consists of a single polypeptide chain on the basis of the molecular weight determined from equilibrium ultracentrifugation in 6 M guanidine hydrochloride and from electrophoresis on polyacrylamide gel containing sodium dodecylsulfate as well as on the basis of the number of peptides obtained by tryptic digestion. The presence of one essential –SH group per mole of enzyme and of one binding site for NADH are also consistent with the existence of a single active center.

No NH2‐terminal amino acid can be detected by reaction with dansyl chloride and with phenyl‐isothiocyanate.
Animal DNA‐Dependent RNA Polymerases
Authors: Kedinger Claude , Gissinger Francis , Gniazdowski Marek , Mandel Jean‐Louis , Chambon Pierre
Published: March 2005

This paper describes a large‐scale method for the solubilization and the separation of calf thymus A and B DNA‐dependent RNA polymerase activities. This method is reproducible, gives high yields and can handle up to several kg of tissue. The solubilization method involved homogenization of the whole tissue, sonication at high ionic strength, ammonium sulphate precipitation and protamine sulphate precipitation to remove nucleic acids. The separation of the two types of activities A (insensitive to α‐amanitin) and B (inhibited by this compound) was achieved by differential adsorption to DEAE‐cellulose using a batch procedure. Studies of the inhibitory effect of α‐amanitin on the various fraction during the solubilization and the separation stages suggest very strongly that the two types of activities pre‐existed in vivo and did not result from the solubilization procedure.
Animal DNA‐Dependent RNA Polymerases
Authors: Mellhac Madeleine , Tysper Zofia , Chambon Pierre
Published: March 2005

The inhibitory effect of various semi‐synthetic derivatives of rifamycin SV on calf thymus DNA‐dependent RNA polymerase AI and B activities was investigated. Some of these derivatives having highly hydrophobic side‐chains, like AF/013, completely inhibited both types of activities at a concentration of 20 to 40 μg/ml. Initiation, but not elongation, of RNA synthesis was inhibited by these derivatives. Studies on the mode of action of these inhibitors on B enzyme activity have shown that inhibition took place at two steps of the preinitiation process: both the primary binding of enzyme to DNA and a temperature‐and time‐dependent step were inhibited. After preincubation of B enzymes and calf thymus DNA at 37°C, RNA synthesis was almost completely resistant to AF/013, suggesting that the formation of the resistant complex was related to the opening of the two DNA strands over a short local region at the initiation site. Divalent cations, especially Mn2+, had a marked effect on the formation and the stability of the resistant complex. In the case of enzyme AI some AF/013‐resistant RNA synthesis was also observed. However, the same values were obtained whether the preincubation temperature was 37°C or 0°C, indicating that the preinitiation process on calf thymus DNA could be different for the two types of calf thymus RNA polymerase activities.
Esterase Activity of Chymotrypsin on Oxygen‐Substituted Tyrosine Substrates
Authors: Kundu Nakuleswar , Roy Sujata , Maenza Frederick
Published: March 2005

The replacement of the phenolic proton of tyrosine by alkyl, aryl or acyl groups completely abolishes the chymotryptic hydrolysis of tyrosine esters. Similarly, chymotrypsin fails to hydrolyze the tyrosyl bonds of dinitrophenyladrenocorticotropin.

Several model substrates have been synthesized and characterized. The action of chymotrypsin was followed potentiometrically in buffer containing some alcohol. The hydrolysis of the analogous compounds unsubstituted at the phenolic group was normal.

We conclude that inhibition is due, not to an inductive effect, but to a blocking effect which is markedly affected by replacement of the phenolic proton, even with a group as small as a methyl group.
Metabolism of Guanosine Tetraphosphate in Escherichia coli
Authors: Lund Elsebet , Kjeldgaard Niels Ole
Published: March 2005

In rel+ strains of Escherichia coli guanosine 2′(3′)‐diphosphate‐5′‐diphosphate (ppGpp) was found to accumulate (a) during amino acid starvation of an auxotrophic strain, (b) during heating to the non‐permissive temperature of a valSts strain, (c) during heating of a wild‐type strain to temperatures above 46°C and (d) during treatment with trimethoprim or hydroxylamine. A rapid decay of the accumulated ppGpp was observed after addition of chloramphenicol, tetracycline, fusidic acid, erythromycin, puromycin or colicin E3 to susceptible cultures. All of these antibiotics give a rapid and complete inhibition of protein synthesis. Streptomycin showed a delayed inhibitory action on protein synthesis and had little effect on the level of accumulated ppGpp. The addition of rifampicin also results in a delayed decay of ppGpp.

These results indicate that the synthesis of ppGpp is independent of RNA synthesis, but suggest that a coupling exists between ppGpp formation and an idling reaction of one of the initiation steps of protein synthesis.
Purification and Characterization of an Intracellular Dipeptidase from Mycobacterium phlei
Authors: Plancot Marie‐Thérèse , Han Kia‐Ki
Published: March 2005

A dipeptidase has been isolated from cell extracts of Mycobacterium phlei 689 in a highly purified form, by a five‐step procedure comprising streptomycin sulfate precipitation, hydroxyapatite adsorption, ammonium sulfate fractionation, gel‐filtration and preparative acrylamide gel electro‐phoresis. The final product exhibited only one band on acrylamide gel, one arc in immunodiffusion and one amino acid in end‐group determination; it was at least 1000‐times more active than the crude material. The enzyme thus purified specifically hydrolyzed typical dipeptide such as l‐leucyl‐glycine, and does require metal ions for its activity. The molecular weight of the enzyme was found to be 88000 by gel‐filtration and 45000 by gel‐electrophoresis. Effects of enzyme concentration and substrate concentration on the rate of catalysis have been respectively obtained for dipeptides alanyl‐glycine, leucyl‐glycine and d‐leucyl‐glycine as substrates. Different inhibitors were assayed toward enzyme. The effect of different metal ions on the enzyme were also considered. The enzyme possesses a leucine residue at the N‐terminal position. Amino acid composition reveals a high content of aspartic and glutamic acids, of leucine and alanine and a low content of cysteine, methionine, tryptophan and histidine residues.
Properties of the Sarcoplasmic ATPase Reconstituted by Oleate and Lysolecithin after Lipid Depletion
Authors: The R. , Hasselbach W.
Published: March 2005

The ATPase activity of the sarcoplasmic membranes and the phosphoryl transfer reaction from ATP to the membranes depend on the composition of their lipid phase.

Lipid depletion leads to a drastic (∼ 60%) reduction of phosphoprotein formation. Oleate raises and lysolecithin decreases phosphoprotein formation. In absence of calcium and/or magnesium neither the native nor the delipidated vesicular preparations are phosphorylated.

The ATPase activity of lipid‐depleted vesicular preparations reactivated by oleate or lysolecithin and that of the preparation containing the lipid hydrolysis products after phospholipase A treatment reaches half‐maximal activation at concentrations of ionized calcium between 0.2 and 0.3 μM. The maximal ATP‐splitting rates, however, differ considerably.

Half‐maximum activation by Mg · ATP for the oleate and lysolecithin‐reactivated ATPase is reached at 10 μM and 100 μM, respectively. The corresponding Hill coefficients are 0.5 and 0.6.

For the oleate‐reactivated ATPase an activation energy of about 35 kcal/mol has been calculated which is similar to that of the extra ATPase of native vesicles. The activation energy of the lysolecithin‐reactivated ATPase is about 8 kcal lower.

The ATPase activity of the reconstituted vesicular preparations shows an identical steep decline at pH values >7. An irreversible damage of the vesicular preparations at higher values could be excluded.

The properties of the reconstituted sarcoplasmic ATPase are discussed in relation to the sarcoplasmic calcium transport.
Isolation of a High‐Molecular‐Weight Hybrid between Ribosomal RNA and DNA
Authors: Spadari Silvio , Falaschi Arturo , Mazza Giorgio
Published: March 2005

The genes for rRNA of Bacillus subtilis have been purified as high molecular weight DNA: RNA hybrids with a DNA/RNA ratio of 1. Two different methods were used. (1) High molecular weight denatured DNA (Mr= 2.2 × 107) was hybridized with rRNA and the excess of single‐stranded DNA was digested with Neurospora crassa endonuclease and Escherichia coli exonuclease I. The hybrid was then purified on Sepharose 4B and two subsequent CsCl gradients. This is a marked improvement on the previously published procedure of Sgaramella, Spadari and Falaschi (1968) insofar as it gives high molecular weight products (8 × 105 for the DNA moiety of the hybrid). (2) A simplified non‐enzymic procedure that used relatively short single‐stranded DNA molecules (Mr= 4 × 106) and several subsequent CsCl gradients led to a hybrid with a DNA: RNA ratio of 1.1 and a molecular weight higher than the previous procedure (1.7 × 106 for the DNA moiety). The DNA of the isolated hybrid, after removal of the RNA moiety, rehybridizes completely with rRNA.

After hybridization of rRNA with DNA having a single‐stranded molecular weight of 2.2 × 107, the hybrid bands mainly at a density corresponding to that of denatured DNA; hybrid with a density corresponding to that of 1:1 DNA/RNA ratio can be obtained only if the DNA has been reduced by enzymic digestion or shearing to a single‐stranded molecular weight close to 2 × 106; this indicates that between each set of 16‐S + 23‐S rRNA genes, there must be regions of DNA non‐complementary to rRNA; these regions have a single‐stranded molecular weight that can be estimated as greater than 1.8 × 107.

When the isolated DNA is treated with N. crassa endonuclease, in conditions that cause 95% hydrolysis of denatured DNA and 5% digestion of native DNA, it is digested only up to 50%. This suggests the presence of self‐annealing sequences in the rRNA genes, in agreement with the secondary structure of rRNA.
An Attempt at the Identification of the Proteins Involved in the Incorporation of 5‐S RNA during 50‐S Ribosomal Subunit Assembly
Authors: Gray Peter N. , Garrett Roger A. , Stoffler Georg , Monier Roger
Published: March 2005

In an attempt to determine the RNA‐protein interactions involved in the biogenesis of the 50‐S ribosomal subunit we have developed a simple and specific system in vitro which reflects some of the intricacies of the Escherichia coli 50‐S subunit. By studying the interaction of isolated ribosomal proteins with 5‐S and 23‐S RNAs we have demonstrated the formation of a stable complex which contains 5‐S RNA and 23‐S RNA and which is dependent upon only two or three ribosomal proteins. The characteristics of this complex have been determined and the responsible proteins identified. The 5‐S · 23‐S RNA complex is dependent upon protein L18 in conjunction with either L6 or L25. Although it would be premature to conclude that these results in vitro are directly related to the assembly map in vivo of the 50‐S subunit, there is a strong correlation between our results and those obtained with various natural and artificial ribosomal subparticles.
An Induced Mitochondrial DNA Polymerase from Tetrahymena
Authors: Westergaard Ole , Lindberg Birte
Published: March 2005

The activity of mitochondrial DNA polymerase from Tetrahymena pyriformis can be induced up to 50‐fold by several kinds of DNA damage (irradiation, thymine starvation etc.). The induced mitochondrial enzyme has been purified more than 5900‐fold based on whole treated cells. The purification procedure involved the following steps: (a) isolation of mitochondria from cells with induced enzyme; (b) extraction of the polymerase; (c) chromatography on two columns of phosphocellulose at different pH. Homogeneity studies of the purified polymerase indicate that the enzyme is more than 50% pure.

The purified polymerase is able to use both native and heat‐denatured DNA as primer, although the optimal ion concentrations differ quite considerably with the primer. At low Mg2+ concentrations (5–12 mM) optimal conditions are observed for the reaction with heat‐denatured DNA, while the reaction with native DNA primer demonstrates optimum around 45 mM MgCl2. In the reaction with native DNA primer the high MgCl2 concentrations can be partially substituted by high KCl concentrations of same ionic strength. The reactions with the bihelical polymers poly[d(A‐T)] · poly[d(A‐T)] and poly[d(I‐C)] · poly[d(I‐C)] demonstrate optima similar to those found for the reaction with heat‐denatured DNA.

The purified DNA polymerase has a temperature optimum around 28°C and heat‐inactivation experiments demonstrate that the enzyme is very labile at the high Mg2+ concentrations required for the reaction with native DNA.
Revised Structure of Aspartokinase I‐Homoserine Dehydrogenase I of Escherichia coli K12
Authors: Falcoz‐Kelly Françoise , Janin Joël , Saari John C. , Véron Michel , Truffa‐Bachi Paolo , Cohen Georges N.
Published: March 2005

After revision, the molecular weight of the subunit of Escherichia coli K12 aspartokinase I‐homoserine dehydrogenase I is 86000 ± 4000 instead of 60000 as previously published. The enzyme is a tetramer, not an hexamer. The electrophoretic homogeneity of the subunits, the number of unique sequences around cysteinyl and tryptophanyl residues, the determination of the N‐terminal and C‐terminal sequences all point to the probable identity of the four subunits.

After revision of the molecular absorption coefficient, a new set of binding data for various ligands of the enzyme is given.
Tetrahydrobiopterin, a Cofactor in Mitochondrial Electron Transfer
Authors: Rembold Heinz , Buff Klaus
Published: March 2005

Submitochondrial particles, obtained from sonified rat liver and beef heart mitochondria, exhibit a strong increase of oxygen consumption in the presence of tetrahydropterin. As with to intact mitochondria, ultraviolet difference spectra indicate a concomitant reduction of cytochromes c and a/a3. A soluble electron transfer system, which consists of the essential components tetrahydropterin, cytochrome c, and cytochrome oxidase, is able to reduce molecular oxygen by NADH or NADPH. Submitochondrial particles and mitochondria are activated by cytochrome c in the same way, but less effectively. Radioactivity of 14C‐labelled tetrahydrobiopterin is incorporated into mitochondria in the range of 1/4 to 2/3 of equilibrium distribution. The possible physiological function of tetrahydropterins in the control of cellular reducing power is discussed.
Phylogeny of the Neurohypophysial Hormones
Authors: Acher Roger , Chauvet Jacqueline , Chauvet Marie‐Thérèse
Published: March 2005

Two oxytocin‐like hormones have been isolated from a cartilaginous fish, the spiny dogfish Squalus acanthias, belonging to the order Pleurotremata. They have been characterized by amino acid analysis and partial sequence determination. The chemical data suggest that the oxytocin‐like hormones are [Val8]‐oxytocin (Valitocin) and [Asn4]‐oxytocin (Aspartocin). The biological properties of natural products are compatible with those previously published for the synthetic analogues of oxytocin. On the other hand arginine‐vasotocin, present in very small amount, has been pharmacologically recognized.

In contrast to the other classes of vertebrates, the cartilaginous fishes show a particular neurophyophysial hormone heterogeneity since the three main groups, rays, sharks and chimaeras seem to have different oxytocin‐like hormones. If four species of rays have the same active principles, only one species of shark has so far been investigated and the distribution of the two new hormones remains to be studied. It is not known whether valitocin and aspartocin are present in a single gland of Squalus acanthias or each one in a particular strain.

Cartilaginous fishes have arginine‐vasotocin as other nonmammalian vertebrates but the amount in the pituitary gland is comparatively much lower; the quantity per mg of pituitary powder is roughly one thousand times weaker in marine cartilaginous fishes than in bony fishes. This decrease might be explained by the existence of an uraemic regulation of osmotic pressure in the former instead of the current ionic regulation found in the latter.
Complexes of Histone F1 with DNA in 0.15 M NaCl
Authors: ŠPonar Jaroslav , ŠOrmová Zora
Published: March 2005

Aggregated complexes of histone F1 with DNA of different composition and origin were prepared by gradual dialysis from a high salt concentration to 0.15 M NaCl. Analysis of the DNA composition both in the supernatant and in the precipitate showed that in equilibrium complexes in this medium, histone F1 selectively prefers sequences of native DNA rich in adenine and thymine. The mechanism of the selective binding is discussed.
Utilization of Methionine‐Accepting tRNA Species from Escherichia coli, Ascites‐Tumor Cells, and Yeast in Homologous and Heterologous Cell‐Free Systems
Authors: Drews Jürgen , Grasmuk Hans , Weil Rudolf
Published: March 2005

The methionine‐accepting tRNA species from Escherichia coli, ascites tumor cells, and yeast were tested for their capacity to function in peptide‐chain elongation in homologous and heterologous cell‐free systems from these organisms comprising polysomes and elongation factors. The following results were obtained.

The utilization of Met‐tRNAfMet in chain elongation is not restricted to mammalian systems but can also be observed in a polysome system from E. coli with initiator tRNA from ascites cells and homologous Met‐tRNAfMet.

In polysome systems from E. coli and ascites tumor cells the magnesium concentrations optimal for the utilization of the homologous methionine‐accepting tRNAs in chain elongation were found to be higher than the Mg2+ ion concentrations at which the discrimination between homologous Met‐tRNAMet and Met‐tRNAfMet is maximal.

Of the three systems studied, the polysome system from yeast is characterized by the most stringent exclusion of any initiator tRNA from functioning in peptide‐chain‐elongation.

Initiator‐tRNA from yeast, while recognized by initiation factors from E. coli, is not utilized for chain elongation in any of the cell‐free polysome systems tested.

Additional results pertaining to the T factor‐induced attachment of all six Met‐tRNA species to ribosomes from E. coli, ascites cells and yeast were in complete agreement with these findings.

Finally, the influence of a formylation or acetylation of the α‐amino group on the recognition of Met‐tRNA species from E. coli and ascites tumor cells by factors T1, Tu,s, and a crude initiation factor preparation from E. coli was studied. The presence of a free α‐amino group could be shown to be an important prerequisite for the interaction of aminoacyl‐tRNA and factors T1 or Tu,s. On the other hand, the acetylation of the α‐amino group of Met‐tRNAMet from E. coli appeared to introduce a critical structural element into this tRNA which rendered it suitable for partial recognition by bacterial initiation factors.
Studies on Alkaline Phosphatase
Authors: Byers David A. , Fernley H. Neville , Walker Peter G.
Published: March 2005

The reaction of l‐phenylalanine, a stereospecific and uncompetitive inhibitor, with human placental alkaline phosphatase in the free and phosphoryl forms was investigated, using a rapid mixing and quenching technique. It is established that, at pH 8—9, l‐phenylalanine reacts instantaneously with phosphoryl phosphatase preventing dephosphorylation at concentrations similar to those causing inhibition in the steady state. It is further established that at pH 9 l‐phenylalanine produces an increase in the steady state concentration of phosphoryl phosphatase, amounting to over 80% of the total enzyme with 5 mM l‐phenylalanine. At high inhibitor concentration the same steady‐state activity was observed with a variety of phosphate ester substrates that different widely in affinity and reactivity. All these results are consistent with a mechanism whereby l‐phenylalanine prevents dephosphorylation while other schemes giving rise to uncompetitive inhibition are excluded. It is suggested that only the basic form of l‐phenylalanine is inhibitory and possible mechanisms are discussed.
Studies on Alkaline Phosphatase
Authors: Byers David A. , Fernley H. Neville , Walker Peter G.
Published: March 2005

The three common homozygous variants of human placental alkaline phosphatase, types F1, S1 and I1 were partly purified and the concentration of active sites determined using a stopped‐flow procedure. A comparison of the variant steady‐state activities indicated that any differences in catalytic‐centre activity are marginal, both at alkaline pH (with 4‐nitrophenyl phosphate) where phosphorylation is rate limiting and at acid pH (with 4‐methylumbelliferyl phosphate) where dephosphorylation is rate limiting.

Though the I1 variant is significantly more heat labile, it is suggested that differences in activity on starch gel and in placental tissue are due to a lower rate of synthesis of the I1 subunit, approximately one half that of the F1 and S1 subunits.

It is proposed that where kinetic identity between closely‐related phosphatases is observed, especially with regard to inhibition by l‐phenylalanine, then the catalytic activities are probably identical.
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.
Conservative Amino‐Acid Replacement in the Tyrosine Region of the Lysine‐Rich Histones
Authors: Bustin Michael
Published: March 2005

A comparison has been made of the sequence of the tyrosine‐containing tryptic peptide derived from the purified, unfractionated, lysine‐rich histone complement of the calf thymus, rat thymus and calf liver. Two tyrosine‐containing peptides, one of which is a fragment of the other, have been obtained from each tissue. The major tyrosine‐containing peptide was isolated by a single electrophoretic step in about a 50%, yield. The results indicate that the sequence of amino acids around the single tyrosine present in the lysine‐rich molecule is constant from tissue to tissue. However, this sequence varies among the molecular species comprising the lysine‐rich histone complement of a single tissue. Within a tissue, a conservative amino acid replacement in the residue penultimate to tyrosine (from the amino terminus) has been detected. The sequence of the tyrosine‐containing region was found to be Lys‐Ala‐Leu‐Ala‐AlaGly‐Gly‐Tyr‐Asp‐Val‐Glu‐Lys.
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.
Biological Activity of Cross‐Linked Escherichia coli tRNAfMet
Authors: Berthelot Francis , Gros François , Favre Alain
Published: March 2005

A covalent bond between 4‐thiouridine in position 8 and cytidine in position 13 can be quantitatively introduced in Escherichia coli tRNAfMet by irradiation at 335 nm. The cross‐linked tRNAfMet can be acylated and formylated like the unmodified molecule though the rate of acylation is lower. It is bound normally to 70‐S ribosomes by crude initiation factors in the presence of pAUG or T4 mRNA, and can transfer N‐formyl‐methionine to puromycin. Thus, the initiation process does not require any separation of the 4‐thiouridine and cytidine residues.
Properties of Qβ‐Replicase Protein Synthesized in a Cell‐Free System
Authors: Jockusch Harald , Hindennach Ingrid
Published: March 2005

The largest polypeptide obtained as a product of Qβ RNA‐stimulated cell‐free protein synthesis is the replicase (R) protein. The R polypeptide synthesized in vitro has a chain length of approximately 550 residues and is identical in size to the β chain of purified replicase. From its electrophoretic behavior at pH 8.5, a net charge of ‐27 and a cysteine content of > 15 residues can be deduced. A portion of R polypeptide synthesized in vitro binds to added Qβ RNA and this binding is more resistant to increased ionic strength than the binding of the positively charged coat protein to Qβ RNA. Another portion of the R polypeptide binds to some constituent of the Escherichia coli extract to form a 20‐S complex.
Dissociation and Reassociation of Globin‐Synthesizing Polyribosomes from Immature Avian Red Cells
Authors: Gander Eugen S. , Luppis Benedetta , Stewart Andrew , Scherrer Klaus
Published: March 2005

Duck immature red‐cell polyribosomes were dissociated by different concentrations of EDTA At 0.02 μmol EDTA260 unit of ribosomes the dissociation was complete and reversible as shown by the ability of the reassociated ribosomes to synthesize globins. The behaviour of the mRNA during dissociation and reassociation was studied. Possible mechanisms of the reassociation are discussed.