bluuebunny/crossref_metadata_2025_split huggingface dataset:
Spectrometric Investigation of Thaumatin I and II, Two Sweet‐Tasting Proteins from Thaumatococcus daniellii Benth
Authors: Korver Onno , van Gorkom Maarten , van der Wel Henrik 
Published: March 2005

The sweet‐tasting proteins, thaumatin I and II, isolated from the fruits of Thaumatococcus daniellii Benth, both lose their sweetness on heating. Circular dichroism and proton magnetic resonance measurements indicate that the proteins undergo reversible conformational changes as the temperature is increased. Then at a certain temperature, which varies with pH, irreversible heat denaturation occurs. At pH 5.0, the native conformation is most stable and the conformational change is reversible up to 75 °C even on prolonged heating.We concluded from the spectroscopic data that the proteins are flexible molecules and that tyrosine residues and at least one disulphide chromophore are involved in the conformational change. The results justify the assumption that the irreversible heat‐induced conformational transition is responsible for the loss of sweetness of the proteins.
A Comparative Study of Pig and Sheep‐Brain Glutamine Synthetases: Tryptic Peptides and Thiol Groups
Authors: Rajagopal Rao Desiraju , Beyreuther Konrad , Jaenicke Lothar 
Published: March 2005

A comparative study of the tryptic peptides of glutamine synthetase from pig and sheep brain has been made. Individual peptide maps of S‐[14C]carboxamidomethylated enzymes as well as mixed peptide maps of S‐[14C]carboxymethyl and S‐[3H]carboxymethyl enzymes reveal close similarities of the enzyme. The S‐carboxyamidomethylated tryptic peptides have been separated by column chromatography. The amino acid composition of several of these peptides as well as their N‐terminal groups have been determined. Some of the tryptic peptides from both sources have very similar composition and properties. The major peptides obtained by cyanogen bromide cleavage of both the enzymes have identical N‐terminal groups.The various thiol groups of both the pig and sheep brain glutamine synthetase have similar reactivity towards iodoacetic acid and iodoacetamide. The [14C]alkylated peptides from both sources have very similar properties. The pig brain enzyme is more sensitive to inhibition by N‐ethylmaleimide than by iodoacetamide. The inhibition can be prevented by ATP and Mg2+. Some thiol group or groups are located close to the active centre and are involved in the formation of active conformation rather than binding of ATP.
Activation of Human Plasminogen by an Insoluble Derivative of Urokinase
Authors: Wiman Björn , WallÉN Per 
Published: March 2005

Activation of plasminogen was performed by perfusing a plasminogen solution through a small column loaded with insolubilized urokinase. The effluent was immediately collected in an excess of plasmin inhibitor (p‐nitrophenyl‐p‐guanidino‐benzoate). This provides a practically instantaneous inhibition of the plasmin formed and autocatalytic degradation is thus reduced to a minimum. Different degrees of activation are obtained by changing the perfusion rate.Dodecylsulfate‐polyacrylamide electrophoresis, amino acid and NH2‐terminal amino acid analyses show that the activation of human plasminogen involves cleavage of at least two peptide bonds, the first event being the release of a peptide of 64 amino acid residues from the NH2‐terminal end. An intermediate inactive product with a molecular weight of about 86000 and methionine as NH2‐terminal amino acid is formed. Cleavage at the second site results in formation of a molecule with two chains connected by disulfide bridges. One chain which has a molecular weight of about 63000 is very sensitive to proteolytic degradation in its NH2‐terminal part. However, there is evidence that methionine is the true NH2‐terminal amino acid of this fragment. The other chain has a molecular weight of about 25000 and has valine as NH2‐terminal amino acid.
Mitochondrial Translation Products before and after Integration into the Mitochondrial Membrane in Neurospora crassa
Authors: Michel Rainer , Neupert Walter 
Published: March 2005

Nascent translation products on mitochondrial ribosomes were selectively labeled in vivo in the presence of cycloheximide with radioactive leucine. They were isolated together with the ribosomes.
The labeled polypeptides show a high tendency to aggregate and can only be kept in solution in the presence of detergents such as dodecylsulfate. Also, mitochondrial ribosomes carrying nascent peptide chains easily form aggregates.
The polypeptides adhering to mitochondrial monomeric ribosomes differ from those adhering to polymeric ribosomes. Gel electrophoresis in the presence if dodecylsulfate shows for the peptidyl transfer RNA products at the monomer, an apparent molecular weight of 27000. After removing the transfer RNA, an apparent molecular weight of less than 10000 is registered. The peptides adhering to mitochondrial polymeric ribosomes display a broad range of apparent molecular weights. In contrast, translation products associated with cytoplasmic monomeric and polymeric ribosomes all show quite disperse molecular weights.
Using gel‐chromatographic analysis no difference in the elution characteristics between translation products associated with mitochondrial monomeric and polymeric ribosomes was found. In both cases apparent molecular weights of about 11000 were obtained.
A kinetic study of the appearance of mitochondrial translation products in the mitochondrial membrane was carried out. A conversion process of products with lower apparent molecular weights to those with higher apparent molecular weights is observed. This suggests that mitochondrial ribosomes form polypeptides which are modified during or after integration into the membrane.
The hypothesis is discussed that mitochondria possess their own system of transcription and translation, because the hydrophobic nature of the translation products makes it necessary that they are formed inside the inner mitochondrial membrane, into which they are integrated.
The Reaction of Hexokinase with Equilibrated d‐Glucose
Authors: Wurster Bernd , Hess Benno 
Published: March 2005

In the hexokinase reaction with equilibrated d‐glucose (36.2%, α‐d‐glucopyranose and 63.8%β‐d‐glucopyranose) at saturating concentrations of ATP‐Mg, the ratio of phosphorylation of α‐d‐glucose and β‐d‐glucose was investigated.It is shown that, knowing the Michaelis constants Km,(α), Km,(β), and the maximum velocities V(α), V(β), of hexokinase with α‐d‐glucose and β‐d‐glucose respectively, one can calculate the ratio of phosphorylation of α‐d‐glucose and β‐d‐glucose starting from the equilibrium mixture of d‐glucose.In addition, the ratio of generation of α‐and β‐d‐glucopyranose‐6‐phosphate in the hexokinase reaction was determined from steady‐state analysis of the hexokinase/glucose‐6‐phosphate dehydrogenase system with equilibrated d‐glucose. The experimentally obtained and the calculated values were in agreement.
On the Regulation of the Activity of Immobilized Enzymes
Authors: Gestrelius Stina , Mattiasson Bo , Mosbach Klaus 
Published: March 2005

Glucose oxidase, hexokinase, trypsin and urease were entrapped, either separately or together, within polyacrylamide particles, using a bead polymerization technique. The pH optima, for the immobilized enzymes in 5 mM buffer, were displaced compared to those of the enzymes free in solution.Thus the pH optimum for trypsin activity towards benzoyl‐l‐arginine ethyl ester shifted 1.3 pH unit (to pH 9.6) and the pH optimum for glucose oxidase towards glucose shifted 0.3 pH units to the alkaline side (to pH 6.9). Reversely, urease activity towards urea leading to consumption of protons shifted its optimum 0.4 unit to the acidic side (to pH 5.8).The effect on glucose oxidase activity of pH changes caused by activity of coentrapped trypsin or urease was studied. It was found that glucose oxidase activity in the alkaline region was stimulated during simultaneous trypsin activity, whereas at pH values below that of the optimum for glucose oxidase inhibition occurred. A reversed effect was measured during urease activity. At pH 8.6, for example, glucose oxidase activity was increased by a factor of three to 75% of its activity found at pH optimum due to trypsin activity. On the other hand, at pH 6.0, i.e. below the optimum, it increased by a factor of two to about 80% due to urease activity. Both simultaneous trypsin or urease activity gave distorted “two‐peak” pH‐activity profiles of glucose oxidase.The effect of protons produced by trypsin on the simultaneous activities of glucose oxidase and hexokinase, both competing for the same substrate, glucose, was studied with a gel containing all three enzymes. It was found that the amount of glucose converted per minute by each enzyme reaction was effected. In one case, at pH 8.5 in the absence of trypsin activity, about one seventh of the glucose consumption was due to hexokinase activity. On addition of benzoyl‐l‐arginine ethyl ester, hexokinase activity decreased almost to zero, while glucose oxidase activity was stimulated, resulting in practically all conversion being due to the latter.
Isolation and Characterization of Cytoplasmic l‐Glycerol‐3‐Phosphate Dehydrogenase from Rabbit‐Renal‐Adipose Tissue and Its Comparison with the Skeletal‐Muscle Enzyme
Authors: Warkentin Donald L. , Fondy Thomas P. 
Published: March 2005

Cytoplasmic NAD‐linked l‐glycerol‐3‐phosphate dehydrogenase was isolated from rabbit renal adipose tissue in a preparation that was apparently homogeneous by preparative isoelectric focusing and polyacrylamide‐disc‐gel electrophoresis. The adipose tissue and skeletal muscle enzymes were indistinguishable by disc‐gel electrophoresis, immunological comparisons, apparent Km, values for substrates and coenzymes, isoelectric point in preparative isoelectric‐focusing runs, and apparent molecular weight and Stokes radius on polyacrylamide‐gel‐filtration columns. The ultraviolet spectrum of the adipose tissue enzyme was identical to that of the muscle enzyme including the broad maximum at 275 nm indicative of the non‐protein component characteristic of the muscle enzyme. Crude homogenates of rabbit adipose tissue and muscle electro‐focused in polyacrylamide gels and stained for glycerol‐3‐P dehydrogenase activity showed four major zones present in slightly different proportions in the two homogenates, but fully superimposable in runs on mixed tissue homogenates.Comparison of amino acid composition of the adipose tissue enzyme with analyses of the muscle done in five different laboratories showed that the adipose tissue enzyme cannot be distinguished from the muscle enzyme by this criterion. Similar comparison of the adipose tissue and muscle enzymes with the liver enzyme suggests that the liver enzyme is not identical to the muscle‐adipose form of the enzyme. Possible similarity in physiological role for the muscle‐adipose form of glycerol‐3‐P dehydrogenase in these two tissues and a rationale for their absence in rapidly growing cancer cells is suggested.
Enzymatic Degradation of O‐Antigenic Lipopolysaccharides by Coliphage Ω8
Authors: Reske Konrad , Wallenfels Barbara , Jann Klaus 
Published: March 2005

Incubation of the lipopolysaccharide from Escherichia coli O8: K27–: H– with coliphage Ω8 resulted in enzymatic degradation of the polysaccharide moiety. Investigation of the water‐soluble products showed that the polysaccharide was split into the trisaccharide repeating unit (α‐mannosyl‐1,2‐α‐mannosyl‐1,2‐mannose), its dimer, trimer and tetramer. As in the polysaccharide the trisaccharides are α‐1,3‐linked, the action of coliphage Ω8 may be described as that of an endo‐α‐1,3‐mannosidase.Coliphage Ω8 also splits the lipopolysaccharides of E. coli O93 and Klebsiella O5 in the same fashion. These lipopolysaccharides are also receptor of coliphage Ω8; coliphage Ω8 is adsorbed on the respective bacteria and the lipopolysaccharides inhibit the infection of E. coli O8 with coliphage Ω8. In addition, all lipopolysaccharides susceptible to the enzyme action of coliphages Ω8 cross‐react serologically with that of E. coliΩ8. Two polysaccharides from E. coli strains O9 and O100 which do not cross react with O8 material are not receptors of coliphage Ω8 and are not degraded by it.
Studies on the Mechanism of the Oxygen‐Induced Synthesis of Cytochrome Oxidase in Yeast
Authors: Schmitt‐Verhulst Anne‐Marie , Bex Françoise , Sels André A. 
Published: March 2005

At concentrations where benzimidazole inhibited partially total protein synthesis in yeast, the drug also inhibited protein synthesis mediated by cytoplasmic ribosomes and by isolated mitochondria in vitro.
Except for cytochrome oxidase, the effects of benzimidazole on the anabolism of hemoproteins were also partial and unspecific; the induction by oxygen of the particulate cytochromes b and c1, the synthesis of which is determined by the obligate co‐operation of both protein‐synthesizing systems, was not affected more than the induction of catalase, an extramitochondrial hemo‐protein, which is synthesized exclusively in cytoplasm. The drastic inhibition of cytochrome oxidase was the consequence of a specific blocking of cytochrome (a, a3) production.
The delayed adaptation to oxygen, following the exclusion of benzimidazole, was characterized by its exceptionally high rate and correlated with the simultaneous increase of hemo‐protein (a, a3) content and the development of cytochrome oxidase activity; the latter activity ruled respiration rate. The process was largely unsensitive to the concerted actions of cycloheximide and chloramphenicol; under identical conditions, the synthesis of other hemoproteins was immediately stopped.
The reconstitution of cytochrome oxidase in vivo at the expense of the double set of cytoplasmic and mitochondrial adaptation products, which were simultaneously accumulated, was established by reference to the specific effects of cycloheximide and chloramphenicol. As the prevailing effect of benzimidazole was operationally to switch off the assembly of cytochrome (a, a3), it is inferred that the drug will afford new experimental approaches in further studies of cytochrome oxidase anabolism.
The Mechanism of Action of Methionyl‐tRNA Synthetase from Escherichia coli
Authors: Blanquet Sylvain , Iwatsubo Motohiro , Waller Jean‐Pierre 
Published: March 2005

Titration of native methionyl‐tRNA synthetase from Escherichia coli with tRNAfMet and tRNAmMet leads to 37% and 38% quenching of the enzyme fluorescence respectively. The quenching curves for the two tRNAs are not additive. Analysis of the binding data indicates the presence of a single site for tRNA per mole enzyme, with a three‐fold higher affinity for tRNAmMet. The tRNA binding parameters do not vary significantly in the presence of a saturating concentration of methionine. On the other hand, titration of the enzyme · methioninyl‐adenylate complex (two moles of the ligand bound/mole enzyme) with tRNAfMet and tRNAmMet, leads to an extensive decrease of fluorescence, from an initial level of 134% to 40% and 35%, respectively, indicative of a major structural or conformational change of the enzyme. Analysis of the quenching curves reveals two distinct binding processes, corresponding to occupation of two tRNA sites on the enzyme.Requirements for optimal interaction between enzyme and tRNA were investigated. Magnesium ions are essential for complex formation. Maximal interaction is attained at 1 mM and 4 mM MgCl2, respectively, for tRNAmMet and tRNAfMet. However, close to 40 mM MgCl2 are required to reach equivalence of the two tRNA sites which are available in the presence of methioninyl adenylate. Both spermine and spermidine are able to simulate the role of magnesium in the binding of tRNAMet to the free enzyme, but not in the occupation of the second tRNA site on the enzyme · methioninyl‐adenylate complex.Addition of KCl results in a decrease of the fluorescence quenching produced by tRNA and in a lowering of the affinity for tRNA, both in the presence of MgCl2 and spermidine. Essentially similar quenching levels and affinities are obtained when titration experiments are performed in imidazole‐HCl or Tris‐HCl buffer in the pH range of 6.3 to 8.7. In contrast, in potassium phosphate buffer which inhibits the enzyme‐tRNA interaction, raising the pH leads to a progressive decrease of the fluorescence quenching caused by tRNA, with concomitant lowering of its affinity for the enzyme.For comparative purposes the interaction of tRNAMet with trypsin‐modified methionyl‐tRNA synthetase was investigated. Analysis of the binding data indicates the presence of a single tRNA site per mole enzyme, even in the presence of saturating amounts of methioninyl adenylate (one mole of this ligand bound per mole enzyme). Furthermore, the magnesium ion concentration required to reach maximal quenching is the same (i.e. 4 mM) in the presence or absence of methionyl adenylate.
Microbial Assimilation of Methanol
Authors: Sahm Hermann , Wagner Fritz 
Published: March 2005

An NAD‐dependent alcohol dehydrogenase has been isolated from the yeast Candida boidinii grown on methanol. A study of the properties of the enzyme has shown that it is very similar to the alcohol: NAD oxidoreductase from baker's yeast. The alcohol dehydrogenase from Candida boidinii does not catalyze the oxidation of methanol and is constitutively formed.
A mutant, 48, has been isolated which is unable to grow on methanol as sole carbon and energy source. This strain lacks the methanol‐oxidizing enzyme but not NAD‐dependent alcohol dehydrogenase.
Cells grown on glucose or ethanol do not contain the methanol‐oxidizing enzyme. During adaptation from glucose to methanol, enzyme activity appears before growth on methanol begins.
The methanol‐oxidizing enzyme has been partially purified. It catalyzes the oxidation of methanol to formaldehyde and hydrogen peroxide, and is independent of the addition of a hydrogen acceptor.

The prosthetic group of this enzyme is FAD. The molecular weight was calculated to be 600000, one subunit has a molecular weight of 74000.The optima of pH and temperature for enzyme activity are 7.5–9.5 and 30°C, respectively. It is unstable in acidic pH.The enzyme is not specific for methanol. It also oxidizes lower primary alcohols. The Km value for methanol is 2.0 mM and that for ethanol 4.5 mM.
An Insoluble Receptor for Oestrogens in the “Residual” Nuclear Proteins of Chick‐Liver
Authors: Lebeau Marie‐Claire , Massol Nelly , Baulieu Etienne‐Emile 
Published: March 2005

When chick liver nuclei are incubated at 37°C with [3H]oestradiol in buffer or in liver cytosol, the majority of the incorporated radioactivity is not extractable by high or low salt solutions or by the usual detergents. Total extraction is obtained only by using polar organic solvents, such as methanol. This was also observed when liver minces were incubated with the radioactive hormone and the nuclei were prepared subsequently.The hormone recovered in this insoluble fraction of the nuclei is bound to a protein component, as determined by treatment with various hydrolytic enzymes. This protein binds oestrogens specifically (oestradiol with a Kd∼ 1 nM at 37°C) and is found in the liver but not in the lung nor the kidney. There is a larger number of oestradiol binding sites in the “residual” nuclear fraction of oestradiol‐treated chicks or laying hens than in immature or male chickens.
Relationships between Intracellular Contents of Nucleotides and 5‐Phosphoribosyl 1‐Pyrophosphate in Escherichia coli
Authors: Bagnara Aldo S. , Finch Lloyd R. 
Published: March 2005

The intracellular contents of nucleotides and 5‐phosphoribosyl 1‐pyrophosphate (P‐Rib‐PP) in cells of Escherichia coli were quantitatively determined under various conditions of growth. When the growth rate was limited by the carbon source available in the growth medium the intracellular content of each of ATP, CTP, GTP, UTP, ADP and GDP was proportional to the growth rate. The intracellular content of P‐Rib‐PP showed a similar, though stronger dependence, suggesting that at the slower growth rates, the availability of P‐Rib‐PP could become limiting for nucleotide biosynthesis.Several treatments which are known to specifically stimulate the rate of net RNA synthesis in E. coli had a common effect in causing a transient depletion of intracellular ribonucleoside triphosphates. For each treatment, the depletion of triphosphates coincided with transiently elevated levels of P‐Rib‐PP, despite increases in the ADP:ATP ratio, an effect which might be expected to inhibit P‐Rib‐PP synthetase activity in E. coli (Atkinson and Fall [15], Klungsøyr et al. [16]). Explanations of this apparent anomaly might be made on the basis of the properties of P‐Rib‐PP synthetase from Salmonella typhimurium, (Switzer, [22], Switzer and Sogin, [21]). One explanation is that the enzyme is regulated by total concentrations of nucleotides so that decreases in nucleoside triphosphate concentrations result in stimulation of the reaction. Another is that the decrease in nucleoside triphosphate concentrations releases from chelation a pulse of free Mg2+ which stimulates the synthetase. And thirdly, the concentration of the substrate, ribose 5‐phosphate, might be initially high enough to exert its inhibitory effect on the enzyme, so that stimulation of the reaction can occur if the substrate concentration falls.
Les stérols et leurs précurseurs chez Astasia longa Pringsheim
Authors: Rohmer Michel , Brandt Roger D. 
Published: March 2005

Sterols and sterol precursors of Astasia longa were characterised by thin‐layer chromatography, gas chromatography and mass spectrometry. Sterols were found in liposoluble fractions in the form of free alcohols, esters or glycosides, in hydrosoluble fractions and in the culture medium in the form of free alcohols and esters. Triterpenes and 4α‐methyl sterols were encountered only in the form of free alcohols in both liposoluble and hydrosoluble fractions of the cell.The following compounds have been found in cell cultures: cycloartenol, 24‐methylene‐cycloartanol, cycloeucalenol, obtusifoliol, 4α‐methyl‐Δ8‐ergostenol, Δ7‐cholestenol, cholesterol, Δ7‐ergostenol, Δ5‐ergostenol, isofucosterol, Δ7‐chondrillastenol, clionasterol and poriferasterol.In one culture, some new compounds were accumulated for unknown reasons: 4α,14α‐di‐methyl‐(24S)‐Δ8,25(26)‐stigmastadienol, 14α‐methyl‐(24S)‐Δ8,25(26)‐stigmastadienol, 24‐methylene pollinastanol and 24‐methylpollinastanol.The sterol composition of Astasia longa is consistent with the hypothesis that this alga is a natural mutant of Euglena which has lost all potential for photosynthesis. The presence of cycloartenol and its metabolites shows that in this nonphotosynthesizing organism sterol biosynthesis follows a plant‐type pathway. It is also suggested that several biosynthetic pathways coexist and are expressed differently depending upon the physiological condition of the alga.
The Actinidin‐Catalysed Hydrolysis of N‐α‐Benzyloxycarbonyl‐l‐lysine p‐Nitrophenyl Ester
Authors: Boland Michael J. , Hardman Michael J. 
Published: March 2005

The hydrolysis of N‐α‐benzyloxycarbonyl‐lysine p‐nitrophenyl ester catalysed by the thiol protease, actinidin, from Actinidia chinensis has been studied under steady state and non‐steady state ([E]0 > [S]0) conditions.
The Michaelis constant Km is dependent on ionising groups of pKa 3.75 and 8.1 and lowest at neutral pH. The catalytic centre activity, kcat, is almost pH independent below pH 7.0 but increases greatly at high pH with a pKa of 8.1.
A biphasic reaction was observed under conditions of [E]0 > [S]0 indicating that the simple acylation‐deacylation mechanism does not apply and at least one additional step is necessary. The rapid phase of reaction involves production of p‐nitrophenol bound to an acylated enzyme; p‐nitrophenol is released in the slow phase.
From the amplitude and rate constant of the rapid phase, and the steady state parameters, the rate and equilibrium constants of some of the steps of the reaction were determined.
The biphasic behaviour under conditions of excess enzyme was found from pH 5.0 to 7.9 but the reaction was monophasic at pH 4.0.