bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kinetic Properties of Phenylalanyl‐tRNA and Seryl‐tRNA Synthetases for Normal Substrates and Fluorescent Analogs
Authors: Hertz Harry S. , Zachau Hans G. 
Published: March 2005

The kinetics of phenylalanyl‐tRNA and seryl‐tRNA formation were investigated with tRNAs and aminoacyl‐tRNA synthetases from yeast.Phenylalanyl‐tRNA synthetase yielded linear Lineweaver‐Burk plots with tRNAphe, phenylalanine, and 1,N6‐ethenoadenosine triphosphate (ɛATP) as variable substrates. According to equilibrium dialysis in the absence or presence of phenylalaninyl adenosine 5′‐phosphate, phenylalanyl‐tRNA synthetase possesses one binding site for phenylalanine. For ATP as variable substrate, the deviation from linearity in the Lineweaver‐Burk plot, observed by other investigators, was confirmed. The slope of the curve indicates the presence of more than two ATP binding sites.Seryl‐tRNA synthetase yielded a linear Lineweaver‐Burk plot only with ɛATP as variable substrate. The Lineweaver‐Burk plots for serine and tRNASerwere non‐linear; the interpretation we favor involves positive cooperativity between amino acid binding sites and between tRNA binding sites. Hill plots of the kinetic data showed that the enzyme possesses at least two binding sites for each of these substrates. The kinetic data for ATP could be interpreted as showing more than two binding sites with negative and positive cooperativity in binding of successive ATP molecules.The aminoalkyl adenylates, phenylalaninyl adenosine 5′‐phosphate and serinyl adenosine 5′‐phosphate, competitively inhibited the aminoacylation reaction with respect to amino acid.ɛATP functions in place of ATP in phenylalanyl‐tRNA and seryl‐tRNA formation although with rather different kinetic properties. Modified tRNApheand tRNASer, in which the 3′‐terminal adenosine was replaced by ethenoadenosine, were prepared by a C‐C‐A transferase‐catalyzed reaction of ɛATP. These modified tRNAs show kinetic properties very similar to those of the unmodified tRNAs and can therefore be used, in place of the unmodified tRNAs, as fluorescent probes in synthetase‐tRNA interaction studies.The kinetic behavior of phenylalanyl‐tRNA synthetase appears to be much simpler than that of seryl‐tRNA synthetase, despite the fact that the former enzyme is twice as big and contains twice as many subunits as the latter one. The comparative simplicity of the one enzyme relative to the other correlates with previous results on interactions with substrates, which were obtained by fluorescence measurements and nuclease protection studies.
Acetyl‐CoA‐Dependent Cleavage of Citrate on Inactivated Citrate Lyase
Authors: Buckel Wolfgang , Ziegert Kathrin , Eggerer Hermann 
Published: March 2005

Citrate lyase, an acetyl enzyme which catalyzes the cleavage of citrate in two consecutive steps, the acyl exchange and acyl lyase reactions, on deacetylation yields inactivated enzyme. On treatment with iodoacetate, this is irreversibly converted to another inactivated lyase.
Both inactivated lyase species are catalytically active in the presence of certain acyl‐CoA derivatives such as acetyl‐CoA or citryl‐CoA. 3S‐Citryl‐CoA is cleaved stereospecifically to acetyl‐CoA and oxaloacetate in this catalysis, and citrate, in the presence of acetyl‐CoA, is cleaved to acetate and oxaloacetate.
The acetyl group of acetyl‐CoA however is liberated as acetate and the pro‐S acetyl group of citrate is transformed to acetyl‐CoA during the acetyl‐CoA‐dependent cleavage of citrate. This exchange reaction was demonstrated by isotopic experiments.
Kinetic analysis of the reaction indicated the formation of an intermediate. This conclusion was substantiated by performing the acetyl‐CoA‐dependent cleavage of citrate in the presence of EDTA. The complexing agent inhibits the lyase activity of the enzyme and therefore leads to the accumulation of the intermediate, 3S‐citryl‐CoA.
The formation of 3S‐citryl‐CoA from acetyl‐CoA and citrate in the presence of EDTA demonstrates the acyl exchange activity, the cleavage of this intermediate demonstrates the lyase activity of inactivated citrate lyase.
Comparison of these results with the mechanism of action of native citrate lyase led to the conclusion that the acetylated acyl carrier groups (enzyme‐S‐acetyl in citrate lyase; CoA‐S‐acetyl on inactivated citrate lyase) are alike in both systems. This was supported by the demonstration of the presence of phosphopantetheine in the lyase.
From these and other results, citrate lyase was characterized as a multienzyme complex. It was concluded that phosphopantetheine most likely represents the acyl‐carrying group in this complex.
The Acyl‐Carrier Protein of Citrate Lyase
Authors: Dimroth Peter , Dittmar Wolfgang , Walther Gundel , Eggerer Hermann 
Published: March 2005

When [14C]acetyl citrate lyase was dissociated in sodium dodecylsulfate and the products were separated by gel electrophoresis, all of the radioactive label appeared in a small protein. This had a molecular weight of about 10000 and contained phosphopantetheine. It was concluded, therefore, that this protein component represents the acyl carrier protein of citrate lyase.
[14C]Acetylated acyl carrier protein was purified by conventional methods to homogeneity as judged from the results of gel electrophoretic analysis.
The well‐known dissociation of citrate lyase into subunits and their recombination were studied in detail for optimal conditions. It was found that inactivated, deacetylated citrate lyase could also undergo the dissociation‐association process.
Recombination of this inactivated and dissociated lyase species in presence of the purified [14C]acetyl acyl carrier protein produced enzymatically active citrate lyase. The biological activity of the lyase acyl carrier protein thus demonstrated was confirmed by correlating enzymic activity and radioactive label of the reconstituted complex.
An acyl carrier protein present in crude extracts of Klebsiella aerogenes, but not the deacetylated, purified lyase acyl carrier protein, was active in the malonyl‐CoA‐CO2 exchange reaction catalyzed by an Escherichia coli fatty acid synthetase preparation lacking acyl carrier protein. It was concluded, therefore, that two different acyl carrier proteins are present in K. aerogenes, one for fatty acid synthetase, the other for citrate lyase.
Based on experimental results a model is proposed for the construction of citrate lyase. It was assumed that four moles of acyl carrier protein are present in one mole of the native complex which is additionally composed of 16 protein subunits of identical or nearly identical size. The basic functional unit appears to be a pentamer containing one acyl carrier protein and four of these protein subunits. Hence the native complex would consist of a tetramer of the functional basic pentamers.
Some of the properties of the lyase acyl carrier protein are described. A remarkable feature is the presence of two sulfhydryl groups, one contributed by a cysteamine, the other by a cysteine residue. Both groups were shown to be acetylated in the isolated lyase acyl carrier protein by labelling experiments performed with iodo[14C]acetate. Whereas these results amply demonstrate the thioester linkage of the acetyl group to acyl carrier protein, the question whether both of the thioesters are enzymically active has not yet been clarified.
The Effect of Stress Induced by Ether Anaesthesia on Cholesterol Content and Cholesteryl‐Esterase Activity in Rat‐Adrenal Cortex
Authors: Trzeciak Wieslaw H. , Boyd George S. 
Published: March 2005

The cholesterol content and the activities of cholesteryl esterase and cyclic‐AMP‐dependent protein kinase have been studied in the adrenal glands of rats. These studies have been performed on the adrenal glands of rats injected with a protein‐synthesis inhibitor, cycloheximide, and in the adrenal glands of rats exposed to ether anaesthesia to increase the blood concentration of adrenocorticotrophic hormone.The analysis of the adrenal steroids revealed that acute stress induced by ether anaesthesia stimulated cholesterol ester depletion in adrenal lipid droplets. Cycloheximide injection into rats did not significantly alter the concentrations of cholesterol and cholesterol esters in the lipid droplets. Injection of cycloheximide followed by ether anaesthesia resulted in an increase in free cholesterol with a concomitant decrease in cholesterol ester concentration within the lipid droplets.Cholesteryl esterase and protein kinase activities in rat adrenal 105000 × g supernatant fractions were significantly higher in animals subjected to stress by ether anaesthesia. Injection of cycloheximide did not prevent the stress‐induced enhancement of the activities of either enzyme.Cholesteryl esterase activity was significantly enhanced by the addition of cyclic AMP, ATP and theophylline in the presence of cyclic‐AMP‐dependent protein kinase. The combination of all three activators proved to be more effective than any one or combinations of any two factors. The degree to which the activators stimulated cholesteryl esterase was higher in untreated animals than in animals subjected to ether anaesthesia.It is postulated that the enhancement of cholesteryl esterase activity induced by ether anaesthesia is due to the effect of adrenocorticotropic hormone on the adrenal gland, and that in vitro activation of cholesteryl esterase by cyclic AMP and ATP involves a protein‐kinase‐dependent phosphorylation reaction.Subcellular distribution of cholesteryl esterase activity in rat adrenal gland and some properties of the enzyme have been studied.
Studies on Rat‐Liver Microsomal Cholesterol 7α‐Hydroxylase
Authors: Boyd George S. , Grimwade Alexander M. , Lawson Margaret E. 
Published: March 2005

Cholesterol 7α‐hydroxylase or cholesterol, reduced NADP: oxygen oxidoreductase (7α‐hydroxylating), the initial and possibly rate‐limiting step in bile acid formation is a mixed function oxidase of rat liver microsomes. This enzyme requires NADPH and oxygen. The enzyme system is induced by feeding the bile salt sequestering agent, cholestyramine, this compound effectively breaks the enterohepatic circulation of bile salts and cause about a five‐fold increase in the activity of the enzyme within four days.The cholesterol 7α‐hydroxylase enzyme has a Km for oxygen of approximately 20 μM. It is inhibited by carbon monoxide and this inhibition is released by irradiation with light of 450 nm. The enzyme is not inhibited by cyanide but is inhibited by cytochrome c and p‐chloromercuribenzoate. The enzyme therefore appears to be a typical liver microsomal mixed function oxidase, involving NADPH, cytochrome c reductase and cytochrome P450.The cholesterol 7α‐hydroxylase has an activation energy of 22 kcal/mol and is inhibited by the product of the reaction, namely, 7α‐hydroxycholesterol and also inhibited by bile acids and bile salts.
Immunochemistry of Lobster Arginine Kinase
Authors: Benyamin Yves , Robin Yvonne , van Thoai Nguyen 
Published: March 2005

The effect of the modification of essential amino‐acid residues of lobster‐muscle arginine kinase on the antigenic reactivity towards rabbit antiserum to native enzyme has been investigated.It was found that the carbethoxylation of the essential histidine residue and the 1‐dimethyl‐aminonaphthalene‐5‐sulphonylation (dansylation) of the essential lysine residue have no effect on the immunochemical properties of the enzyme. The same result was observed with the reversible S‐sulfenylsulfonation of the SH groups, despite the fact that a great part of the helical structure was lost.Nitration of the tyrosine residue was carried out on S‐sulfenylsulfonated enzyme. The modified protein has lost a major part of its helical structure and 20% of its antigenic reactivity. The latter was fully recovered upon reduction of the 3‐nitrotyrosyl to 3‐aminotyrosyl residue, in spite of the persistence of the whole conformational change. In contrast, unmasking of the SH groups of the nitrated enzyme restored neither the antigenic activity nor the helical structure of the enzyme.It is suggested that the essential tyrosine residue of lobster‐muscle arginine kinase, which is known to be exposed at the protein surface, is part of an antigenic determinant of the enyzme. The partial loss of the antigenic reactivity upon nitration is strictly related to the nature of the chemical modification at the tyrosine residue, which is likely to be connected to the change in the pKa value of the phenolic hydroxyl.
Studies with Isolated Surviving Rat Hearts
Authors: Davis E. Jack , Bremer Jon 
Published: March 2005

The effects of various substrates on the concentrations of free amino acids, citrate cycle intermediates, acyl‐carnitines and on ammonia production by glucose‐perfused rat hearts were studied in order to assess the mode of regulation of the level of citrate cycle intermediates in this tissue.1. Pyruvate caused a ten‐fold increase in the level of citrate cycle intermediates which was accompanied by an increase in alanine and a decrease in glutamate.2. Acetate or acetoacetate effected a three‐fold increase in cycle intermediates and accumulation of acetyl‐carnitine. Accompanying these changes were decreases in the levels of alanine, aspartate and pyruvate, and a marked increase in the lactate/pyruvate ratio. The concentrations of other free amino acids and ammonia production were not significantly affected.3. l‐Adrenalin caused an elevation of cycle intermediates and a decrease in glutamate and more than doubled the rate of ammonia production.4. Perfusion with 2‐oxo‐3‐methylvalerate, 2‐oxoisovalerate or 2‐oxoisocaproate promoted increases in the level of citrate cycle intermediates, a marked drop in the concentration of glutamate, accumulation of branched‐chain acyl‐carnitines and the appearance of isoleucine, valine and leucine, respectively.5. Long‐term perfusion without substrate led to release of essential amino acids and appearance of propionyl and branched‐chain acyl‐carnitines.6. It is concluded that citrate cycle intermediates are produced by rat heart in at least three manners: (a) conditions which perturb the concentration of pyruvate cause shifts in the levels of alanine and cycle intermediates in the same direction via transamination reaction; (b) in addition to (a), acetate and acetoacetate are capable of eliciting an increase in cycle intermediates without increasing alanine or the rate of ammonia production and (c) l‐adrenalin promotes increases both in the level cycle intermediates and in ammonia release. An overall pathway for the production of citrate cycle intermediates and pyruvate through the degradation of protein amino acids is proposed.
The Function of myo‐Inositol in the Biosynthesis of Raffinose
Authors: Lehle Ludwig , Tanner Widmar 
Published: March 2005

1. An enzyme from Vicia faba seeds is described which transfers the galactosyl moiety of galactinol to sucrose giving rise to raffinose and myo‐inositol.2. The enzyme was purified about 400‐fold through 6 steps. A molecular weight of 80000 has been determined by gel‐filtration and of 100000 by glycerol density gradient centrifugation.3. The enzyme galactinol:sucrose 6‐galactosyl transferase is different from α‐galactosidase; these two activities as well as the stachyose‐synthesizing enzyme separate during purification.4. The transferase showed a high acceptor specificity. Out of 10 acceptors tested a transfer only to sucrose took place. This transfer was 5 times faster than the hydrolysis of galctinol. Galactinol, p‐nitrophenyl‐α‐d‐galactopyranoside and raffinose, but not UDP‐galactose, could act as donors.5. The enzyme catalyzes an exchange reaction between raffinose and [14C]sucrose. This partial reaction is less sensitive towards heat inactivation and SH‐poisons than the total reaction.6. The pH‐optimum of the reaction was found to be pH 7.0, the temperature optimum 42 °C. Heat inactivation could be prevented to some extent by galactinol and raffinose. In the presence of 0.4 mM sucrose the Km‐value for galactinol was 7 mM and for raffinose 10 mM. For sucrose a Km‐value of 1 mM in the synthesis reaction has been determined.7. The transferase activity is high enough to explain the synthesis rate in vivo of all the raffinose‐type sugars present in the seeds.8. The physiological meaning of the results as well as the metabolic function of myo‐inositol is discussed.
Crystalline Kidney‐Bean Leucoagglutinin
Authors: Räsänen Vesa , Weber Theodor H. , Gräsbeck Ralph 
Published: March 2005

1. Kidney‐bean (Phaseolus vulgaris) leucoagglutinin was purified on a preparative scale by fractional precipitation with ethanol followed by ion‐exchange chromatography on DEAE‐cellulose and SP‐Sephadex and exclusion chromatography on Sephadex G‐150. The final purification step consisted of crystallization in aqueous solution.2. The crystallized leucoagglutinin was homogeneous by analytical ultracentrifugation, disc electrophoresis, isoelectric focusing and immuno‐diffusion.3. Leucoagglutination was demonstrable at 1–3 μg/ml and optimum lymphocyte stimulation occurred at 1–5 μg/ml. No erythroagglutinating activity could be detected.4. The leucoagglutinin lacks sulphur‐containing amino acids completely, but contains the other common amino acids, aspartic acid and serine being predominant. It contains glucosamine and mannose as the only carbohydrates. In addition it contains considerable amounts of calcium and manganese, which are essential for the lymphocyte‐stimulating and leucoagglutinating activities.5. The leucoagglutinin has the following physico‐chemical properties: Mr= 126000, s020,W= 6.87 S, f/f0= 1.45, partial specific volume = 0.728 ml/g, pI= 5.1, A1%280 nm/cm= 11.4. The leucoagglutinin is composed of 4 identical subunits, Mr= 31000.
On the Substrate‐Binding Sites of the Active Centre of Mitochondrial Monoamine Oxidase
Authors: Severina Irene S. 
Published: March 2005

Mitochondrial monoamine oxidase from a pig liver was rendered soluble by two‐fold extraction of mitochondrial phospholipids with methylethyl ketone according to Holluger and Oreland, and purified about 400 times.The influence of different compounds on the deamination of 2‐phenylethylamine its 4′‐chloro, 4′‐amino, 4′‐hydroxy derivatives and serotonin was studied.The interaction of monoamine oxidase with 1‐anilino‐8‐naphthalene sulphonate results in 15‐fold increase of the dye fluorescence accompanied by the shift of fluorescence maximum from 520 nm to 475 nm. It has been shown that 1‐anilino‐8‐naphthalene sulphonate inhibits deamination of tyramine, 2‐phenylethylamine and serotonin to the same degree.Alcohols, 1‐naphthol, 2‐naphthol and 8‐hydroxyquinoline inhibit deamination of tyramine and 2‐(4′‐aminophenyl) ethylamine in a much more pronounced fashion than that in the case of 2‐phenylethylamine and its 4′‐chloro derivative.Substitution of the benzene ring of 2‐phenylethylamine by pyridine or picoline causes the loss of the ability of corresponding amines to be affected by monoamine oxidase. 4‐Ethylpyridine and its iodomethylated and chlorobenzylated derivatives inhibit deamination of the unsubstituted 2‐phenylethylamine, its 4′‐chloro, 4′‐amino and 4′‐hydroxy derivatives and serotonin in different degree.Introduction of an electron‐accepting group into the 4‐ethylpyridine molecule significantly increased the inhibitory effect of the corresponding iodomethylate and chlorobenzylate on deamination of tyramine. At the same time the inhibition of oxidative deamination of serotonin is decreased.The data obtained confirm the conception that in the active centre of mitochondrial monoamine oxidase there are at least two substrate‐binding sites: a hydrophobic area and a definite polar site, which participate to a variable degree in binding of different amines.
Kinetics of the Middle and Terminal Phases of Degradation of DNA by Spleen Acid DNAase
Authors: Soave Carlo , Thiery Jean‐Paul , Ehrlich Stanislav D. , Bernardi Giorgio 
Published: March 2005

The kinetics of the middle and terminal phases of calf thymus DNA degradation by spleen acid DNAase has been investigated in detail with the main purpose of providing a background for a study of the specificity of the enzyme and its use in investigations of nucleotide sequences in DNA.During the middle phase the ultraviolet absorption, the acid solubility and the reciprocal average degree of polymerization of the hydrolysate, ¯n−1, increase linearly with digestion time, while the elution profile of the DEAE‐cellulose‐urea chromatogram is progressively shifted to the left. Empirical relationships between ¯n−1 and hyperchromic shift have been established. Extrapolation of ¯n−1 to zero digestion time indicates that bond splitting proceeds at a faster, yet decreasing rate during the early part of digestion.The beginning of the terminal phase is characterized by a sudden slow down of the reaction rate; this phenomenon has been shown to be essentially due to the melting of double‐stranded DNA fragments; the single‐stranded fragments so originated are a poor substrate for the enzyme, in spite of the fact that they still contain a large number of sequences which can be split. During the terminal phase, the ultraviolet absorption, the acid solubility and ¯n−1 increase at a progressively slower rate until an end‐point is practically reached. Investigations on the liberation of mono‐ and dinucleotides, the base composition and termini of isostichs and the effects of digestion temperature and of dephosphorylation on the isostich patterns are also reported.
Specificity of Spleen Acid DNAase
Authors: Thiery Jean‐Paul , Ehrlich Stanislav D. , Devillers‐Thiery Anne , Bernardi Giorgio 
Published: March 2005

The kinetics of liberation of 3′‐phosphate terminal and of 5′‐hydroxy terminal and penultimate nucleotides (termini) by spleen acid DNAase was investigated using as substrates calf thymus DNA, three bacterial DNAs of different base composition, and poly‐[d(A‐T] · d(A‐T)].The relative amounts of the nucleotides deoxyguanosine, deoxyadenosine, thymidine and deoxycytidine in the termini released by the enzyme from all DNAs were found to be constant in the terminal phase of digestion. In contrast, some variations were seen during the middle phase, particularly in the amount of 3′‐phosphate terminal deoxyguanosine, which decreased with digestion time, specially in the calf thymus DNA digest. This phenomenon appears to be due to an initial accumulation of breaks in some nucleotide sequences which are preferentially split at the beginning of digestion, possibly by a diplotomic mechanism, with predominant release of 3′‐phosphate deoxyguanosine. In contrast, no change in the composition of 3′‐phosphate terminals released from poly[d(A‐T) · d(A‐T)] took place in the 40–15 average size range; deoxyadenosine and thymidine formed 80% and 20%, respectively, of these terminals.The composition of the 3′‐phosphate terminal nucleotides was very little or not at all affected by changes in the ionic environment of the incubation mixture, whereas it was significantly changed in degradations of native DNA digested at neutrality and low ionic strength, or of heat‐denatured DNA.Linear relationships were found to hold, at all degradation stages investigated here, between the relative amounts of termini liberated from different bacterial DNAs and their (dG + dC) contents. In contrast, the termini obtained from calf thymus DNA were released in relative amounts which did not fit the relationships established for bacterial DNAs.