bluuebunny/crossref_metadata_2025 huggingface dataset:
Photosystem‐II Electron Transport and Phosphorylation with Dibromothymoquinone as the Electron Acceptor
Authors: Gould J. Michael , Izawa Seikichi
Published: March 2005

Dibromothymoquinone has two effects on isolated chloroplasts. At very low concentrations it inhibits the reduction of conventional hydrophilic electron acceptors, probably by acting as a plastoquinone antagonist. At higher concentrations it acts as an electron acceptor, intercepting electrons either before or at the site of its inhibitory activity. Reduced dibromothymoquinone can be readily reoxidized by excess ferricyanide in the reaction mixture or by molecular oxygen. The transfer of electrons to this substance from water is coupled to phosphorylation. The pH optima for this reduction and associated phosphorylation are both at 7.3, considerably lower than the pH optimum of 8.4 observed with the normal Hill reaction. The ratio of molecules of ATP formed to pairs of electrons transported is relatively constant (0.3 –0.4) between pH 6 and 9. The rate of reduction is independent of the presence or absence of ADP and phosphate or uncouplers.

The reduction of dibromothymoquinone resembles the reduction of other lipid‐soluble oxi‐dants such as oxidized p‐phenylenediamines and 2,5‐dimethylquinone in several respects. Both reactions are insensitive to the plastocyanin inhibitors KCN and polylysine, but are sensitive to 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea. They support phosphorylation with a similar efficieney. However, dibromothymoquinone reduction differs from the reduction of other lipophilic oxidants in that its sensitivity to dichlorophenyl‐dimethylurea decreases with increasing light intensities. This implies that it may be reduced via a pool (plastoquinone?) which is a common electron acceptor for independent photosystem II units whereas oxidized p‐phenylenediamines and 2,5‐dimethylquinone are reduced directly by these independent units.
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.
Interaction of Mixed‐Function Oxidation with Biosynthetic Processes
Authors: Thurman Ronald G. , Scholz Roland
Published: March 2005

Lipogenesis from glucose was studied in perfused livers from normal and phenobarbital‐pretreated rats by measurement of the incorporation of tritium from tritiated water and 14C from from [U‐14C]glucose into the lipid fraction. In the presence of aminopyrine, a substrate for NADPH utilizing mixed‐function oxidations, rates of tritium incorporation decreased to 82 and 57% of control in livers from normal and phenobarbital‐pretreated animals, respectively. Aminophenylpyrazolone, a demethylated product of aminopyrine metabolism, had no effect implicating that inhibition of lipogenesis by aminopyrine is due to the process of mixed‐function oxidation. It is concluded that both mixed‐function oxidation and fatty‐acid synthesis compete for extramitochondrial NADPH. These data indicate that NADPH supply may, under the extreme conditions of enhanced mixed‐function oxidation, be the rate‐limiting step for lipogenesis in liver.
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.
Initiation Complexes of Escherichia coli RNA Polymerase and T7 DNA
Authors: McConnell David J. , Bonner James
Published: March 2005

Complexes between Escherichia coli RNA polymerase and T7 DNA have been studied by three techniques measuring their stability either to 0.4 M (NH4)2SO4 or to rifampicin in 0.15 M KCl. In each case a nucleoside triphosphate was required for stabilisation, the order of efficiency in general being GTP > ATP > UTP ∼ 0. This did not correspond to the order with which the nucleotides initiate RNA synthesis (ATP > GTP ≫ CTP = UTP) contradicting the hypothesis of Anthony et al., which suggested that complexes formed in the presence of the 5′‐terminal nucleotide in the absence of polymerisation are stable to 0.4 M (NH4)2SO4. The complexes formed in the presence of [3H]GTP were shown to have tightly bound oligonucleotides. The degree of stabilisation produced by GTP could be reduced markedly by purification while in the case of ATP purification completely removed the ability to stabilise. These results suggest that the interpretation of previous experiments on the initiation of RNA synthesis may need to be revised.
Effects of Alcohols on β‐Galactosidase‐Catalyzed Hydrolysis of n‐Alkyl‐β‐d‐galactopyranosides
Authors: van der Groen Guido , Wouters‐Leysen Josée , Yde Marc , De Bruyne Clemens K.
Published: March 2005

The effect of an homologous series of n‐alkyl β‐d‐galactopyranosides on the enzymic activity of β‐d‐galactosidase from Escherichia coli ML 309 was investigated. The affinity of the enzyme for these substrates increases with the length of the apolar chain, indicating that the aglycon is bound through hydrophobic forces. Addition of alcohols as nucleophilic competitors indicates that the reaction of aryl and alkyl β‐d‐galactosides proceeds via a common galactosyl·enzyme intermediate, whose formation is rate‐limiting. The reactivity of the alcohols cannot be explained merely in terms of their relative nucleophilicity. It is necessary to assume that the alcohols interact with the enzyme to form adsorptive complexes, and that the conformation of the enzyme changes during the formation of the enzyme·galactosyl complex. Alcohols also function as competitive inhibitors by binding to the free enzyme.
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.
The Covalent Structure of Collagen
Authors: Fietzek Peter P. , Rexrodt Friedrich W. , Hopper Kelvin E. , Kühn Klaus
Published: March 2005

Using automated stepwise Edman degradation of suitable overlapping peptides, the complete amino acid sequence of the 268 residue peptide α1‐CB7 from calf skin collagen was determined. The preparation and ordering of the chymotryptic, tryptic and thermolytic peptides is described in the preceding paper. As shown previously for other peptides from the helical region of collagen, glycine appears in every third position and proline is present in high amount. Hydroxylation of proline to 4‐hydroxyproline and lysine to hydroxylysine only occurred in the Y position of the tripeptide unit Gly‐X‐Y. Non‐random distribution of other amino acids between the X and Y positions was also observed.
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.
Some Properties of NADPH‐Cytochrome c Reductase Reconstitutively Active in Fatty‐Acid ω‐Hydroxylation
Authors: Ichihara Kosuke , Kusunose Emi , Kusunose Masamichi
Published: March 2005

A NADPH‐cytochrome c reductase (called Fraction II) has been solubilized with Triton X‐100 from porcine liver microsomes and purified.

1. Fraction II can reconstitute with a cytochrome P‐450 fraction (called Fraction I) solubilized with Triton X‐100 from porcine kidney cortex microsomes to exhibit laurate ω‐hydroxylation activity.

2. When the purified Fraction II is subjected to disc electrophoresis on polyacrylamide gel in the presence of 0.1% Triton X‐100, it shows a single band. In contrast, on electrophoresis in the absence of Triton X‐100, a polydisperse state is obtained. Fraction II is estimated to have a molecular weight of about 400000 by gel‐filtration with detergent, and it does not contain iron.

3. Either gel filtration on a Sephadex G‐100 column or treatment with trypsin at 4 °C results in a conversion of Fraction II to a form, which migrates at a much faster rate on disc gel electrophoresis than dose Fraction II. This fast‐moving form (called Fraction IIf) is indistinguishable from either steapsin or trypsin‐solubilized NADPH‐cytochrome c reductase, judging from several criteria including electrophoretic and sedimentation behaviors, and molecular size.

4. Fraction IIf is reconstitutively inactive in the hydroxylation, unless supplemented with spinach ferredoxin‐NADP reductase. Incubation of Fraction IIf with trypsin at 30 °C results in a remarkable enhancement in the reconstitution activity in the presence of ferredoxin‐NADP reductase. The results in this paper along with an earlier report [11] suggest that Fraction II may contain two different electron carrier components, possibly participating in the electron transfer from NADPH to cytochrome P‐450.
Primary Structure of Peptides Released during Activation of Human Plasminogen by Urokinase
Authors: Wiman Björn
Published: March 2005

Activation of human plasminogen by urokinase is a two‐stage process. Initially peptide material is released from the NH2‐terminal part of the proenzyme during formation of an inactive intermediate compound, from which plasmin is formed in a second step by proteolytic cleavage of an internal peptide bond. The released peptide material is shown to consist of two peptides, API and APII, containing 63 and 5 amino acid residues, respectively. The amino acid sequences of these peptides, and of two cyanogen bromide fragments situated in the NH2‐terminal part of the plasminogen molecule (CNBr II and CNBr III), have been determined. From these results it is shown that API and APII, in intact plasminogen, occupy the positions 1–63 and 64–68 respectively. The residue in position 69 is shown to be methionine, which also is the NH2‐terminal amino acid found in the intermediate compound formed during activation.
Isolation and Characterization of Chloroform‐Soluble Proteins from Rat‐Liver Mitochondria and Other Fractions
Authors: Hadváry Paul , Kadenbach Bernhard
Published: March 2005

Chloroform‐soluble proteins have been isolated free from phosphatides from rat liver mitochondria, microsomes and cytosol by chromatography on Sephadex LH‐20 columns.

The amino acid composition of the chloroform‐soluble and insoluble proteins has been estimated. The chloroform‐soluble proteins from mitochondria and cytosol have a higher content of hydrophobic and a lower content of acidic amino acids compared to the corresponding chloroform‐insoluble proteins. No marked differences were found between chloroform‐soluble and insoluble proteins from microsomes. In contrast to the acidic chloroform‐insoluble proteins, all chloroform‐soluble proteins are basic.

From the amino acid composition the amount of chloroform‐soluble proteins has been estimated. Mitochondrial membranes contain 1.6%, mitochondrial matrix proteins less than 0.025%, microsomes 3.0% and the cytosol 0.15% from total proteins.

The molecular weight distribution of chloroform‐soluble proteins synthesized in mitochondria has been measured by gelelectrophoresis. The molecular weights range from about 7000 to 30000.

The turnover of chloroform‐soluble proteins from mitochondria and cytosol is higher than from the corresponding chloroform‐insoluble proteins. A part of mitochondrial chloroform‐soluble proteins (about 30%) is synthesized within the mitochondria.
Yeast 3‐Phosphoglycerate Kinase: Evidence for a Glutamyl Residue in the Phosphoryl Transfer
Authors: Brevet Annie , Roustan Claude , Desvages Gisèle , Pradel Louise‐Anne , van Thoai Nguyen
Published: March 2005

The possible involvement of two residues (histidylor carboxylic acid) in the reaction catalyzed by yeast 3‐phosphoglycerate kinase, has been investigated. The direct implication of a histidyl residue seems to be improbable. The implication of a carboxyl residue is first supported by the stoichiometric inactivation of 3‐phosphoglycerate kinase by 1‐cyclohexyl‐3‐(2‐morpholinoethyl)‐carbodiimide and glycine ethyl ester. The ability of the modified enzyme to bind substrates (ADP, ATP, 3‐phosphoglycerate or ATP‐Mg + 3‐phosphoglycerate) while the partial isotopic exchange between ATP and ADP is inhibited, points out the role of a carboxylic residue in the transphosphorylation process. Furthermore the reaction catalyzed by 3‐phosphoglycerate kinase occurs via a transitory phosphoryl‐enzyme complex. The enzyme can be phosphorylated by ATP‐Mg and the phosphoryl‐enzyme is isolated. This phosphoenzyme can transfer its phosphoryl group to ADP. It reacts with hydroxylamine and the hydroxamate formed is submitted to Lossen rearrangement. The identification of the resulted 2,4‐diaminobutyric acid evidences that a glutamyl residue is essential to the catalytic activity of yeast 3‐phosphoglycerate kinase.
The Amino‐Acid Sequence of the αA2 Chain of Bovine α‐Crystallin
Authors: van der Ouderaa Frans J. , de Jong Wilfried W. , Bloemendal Hans
Published: March 2005

The αA2 chain of bovine α‐crystallin was fragmented by means of cyanogen bromide treatment and by tryptic, chymotryptic and thermolytic digestions. Twenty tryptic peptides were obtained from the S‐aminoethylated αA2 chain, accounting together for the complete sequence. The direct Edman degradation and the dansyl‐Edman technique were used to determine the sequences of the tryptic peptides. The order of the tryptic peptides was deduced from overlapping peptides obtained by cyanogen bromide treatment, tryptic digestion of the maleylated chain and chymotryptic and thermolytic digestion of the S‐aminoethylated chain. The sequence of the αA2 chain comprises 173 residues and corresponds to a molecular weight of 19 832.
Luteinizing Hormone
Authors: Maghuin‐Rogister Guy , Hennen Georges
Published: March 2005

The sequences of the 119 amino acids of bovine and porcine luteinizing hormone β‐subunits were determined, 17 amino acid replacements being observed between these species. Variability in the primary structure was observed for porcine β‐subunit, in that position 10 is occupied by both arginyl and glutamyl residues. No free amino terminal residue was detected for both porcine and bovine β‐polypeptide chains and both exhibited carboxy‐terminal heterogeneity. The homology of the β‐subunits of luteinizing hormone from various species and the β‐subunit of the bovine thyroid‐stimulating hormone is discussed as the subunit of both hormones can combine with a similar α‐subunit.
Ethoxyformylation of Helix pomatia Haemocyanin
Authors: Engelborghs Yves , Lontie René
Published: March 2005

Ethoxyformylation of Helix pomatia haemocyanin was studied to investigate the role of histidine residues in the binding of copper and in the dissociation properties of the protein. At neutral pH ethoxyformylation gives rise to a difference spectrum with a maximum at 240 nm, characteristic of ethoxyformylimidazole.

Initial‐rate studies at 250 nm reveal the reaction to be bimolecular. The rate constant depends on pH and follows the titration of the histidine residues. The complete time course of the reaction is complicated by hydrolysis of the reagent and of the reaction products.

Ethoxyformylation of α‐haemocyanin at pH 6.0 causes the protein to dissociate into halves, at pH 6.5 and higher the dissociation proceeds to tenths. β‐Haemocyanin does not dissociate at pH 6.0, at higher pH values it dissociates completely into tenths. An induction period is seen in the dissociation reaction, indicating that a certain number of groups have to be modified before dissociation starts. This number is lowered by raising the pH.

The hydrolysis of the ethoxyformylimidazole groups can be enhanced with NH2OH · HCl. During hydrolysis the halves reassociate quantitatively into wholes, the tenths reassociate into large aggregates.

Modification at pH 7.0 and above destroys the copper binding site at high reagent concentration. Ethoxyformylation of the apoprotein prevents reconstitution. In both cases a linear relationship was found between the copper band and the copper content. Parallel to the liberation of copper, tyrosine residues become accessible, as shown by the negative difference spectrum at 278 nm upon ethoxyformylation or by spectrophotometric tyrosine titrations.
Two Forms of the DNA Ligase of Human Cells
Authors: Pedrali Noy Guido C. F. , Spadari Silvio , Ciarrocchi Giovanni , Pedrini Antonia M. , Falaschi Arturo
Published: March 2005

We have further characterized the polynucleotide ligase purified from cultures of the human heteroploid line EUE [Spadari, Ciarrocchi and Falaschi, Eur. J. Biochem. 22, 75 (1971)]. The 350‐fold purified enzyme gives positive response with four different assays; the rates with the different substrates are different, but the variations are identical to those observed with the purified ligase induced by T4‐phage. The enzyme can reconstitute the transforming activity of Bacillus subtilis DNA inactivated by pancreatic DNAase. The human cell enzyme is unable to use a hybrid substrate where an interrupted polydeoxynucleotide is annealed to a polyribonucleotide, whereas in the same conditions the T4 enzyme gives appreciable activity. The partial dependence of the purified enzyme on proteins present in a boiled crude extract, reported in the previous paper, seems due to an aspecific protective effect of the soluble proteins of cell extracts. The enzyme does not show any appreciable sequence specificity in the phosphodiester bond it can form.

The purified enzyme can be fractionated into two molecular forms, one having a molecular weight of 190000, the other 95000; fresh extracts of EUE cells contain almost exclusively the high molecular‐weight form; ageing of the extract or purification lead to the appearance of the second peak, without variations in the total activity. This could correspond to the conversion of a dimer structure into a monomer. The “dimer” has a pH optimum close to 8.1, whereas the “monomer” has its optimum at 7.5; this explains the bimodal pH curve previously described in the purified enzyme.
Studies on the Metabolism of Steroids in the Foetus
Authors: Plasse Jean‐Claude , Lisboa Belisário Penna
Published: March 2005

Biosynthesis and site of formation of epitestosterone in the human foetus was investigated by incubation of androstenedione with subcellular fractions of mid‐gestational foetal liver. Testosterone and epitestosterone were purified by thin‐layer chromatography and resolved by gas‐liquid chromatography.

Epitestosterone (and testosterone) were identified by gas chromatography‐mass spectrometry as silyl ether and methoximesilyl ether derivatives. The ratio testosterone: epitestosterone found in the incubations was about 20:1.

Both 17β‐ and 17α‐hydroxysteroid oxidoreductase activities were found in the 105000 X g microsome fraction as well as in the cytosol fraction.

It may be concluded that the human foetal liver is one of the sources of production of epitestosterone, and that androstenedione is one of its precursors. The possibility that androstenedione and epitestosterone are precursors of maternal urinary 17α‐hydroxyphenolic steroids during pregnancy is discussed.
The Association of Ribosomal Subunits of Escherichia coli
Authors: van Diggelen Otto P. , Bosch Leendert
Published: March 2005

Association between ribosomal subunits of Escherichia coli can lead to the formation of two classes of 30‐S · 50‐S couples. The first class sedimenting between 50 S and 70 S, arises when native subunits associate, the second sedimenting at 70 S upon coupling of so‐called derived subunits. From cross experiments it is concluded that it is the 50‐S subunit which determines the nature of the association product, whereas the 30‐S subunit determines whether an association product can be formed. Native and derived 50‐S subunits are indistinguishable by sucrose gradient centrifugation and do not differ qualitatively in their ability to form polypeptides under the directions of bacteriophage RNA. Native 30‐S subunits lose a factor during isolation, probably IF‐3. They are therefore able to associate at 0° and 37 °C. On the other hand derived 30‐S subunits are unable to associate at 0 °C but they do so at 37 °C in the presence of 10 mM Mg2+.

A number of trivial factors have been excluded to explain the difference between the two types of 50‐S subunits. The preparation method is not responsible for the difference. Nor do the derived subunits contain peptidyl‐tRNA, known to influence subunit association. The content of tRNA and aminoacyl‐tRNA has been determined quantitatively by polyacrylamide‐gel electrophoresis. None of the subunits nor the 70‐S ribosomes from which the derived subunits are prepared contain a tRNA species.
The Association of Ribosomal Subunits of Escherichia coli
Authors: van Diggelen Otto P. , Oostrom Henk , Bosch Leendert
Published: March 2005

The two types of 30‐S · 50‐S couples described in the preceding paper, differ markedly in their sensitivity to hydrostatic pressure evoked during centrifugation in sucrose gradients. The sedimentation coefficient of about 60 S recorded with couples formed from native subunits is only apparent, the real s value being 70 S. The latter couples can be stabilized against pressure‐induced dissociation by bound tRNA. On the other hand this binding renders couples formed from derived subunits more sensitive to this dissociation.

As the factor determining this intriguing difference in pressure sensitivity lies in the 50‐S subunit, a comparative analysis of a number of ribosomal constituents has been performed for native and derived 50‐S particles. No qualitative differences in the proteins L1–L34 have been detected. Quantitative differences cannot yet be excluded. It is considered unlikely that differences in rRNAs or low‐molecular‐weight components are responsible for the distinct association behaviour. Partial reconstitution of ribosomal γ‐cores and split proteins detached by CsCl reveals that the determinant factor is located in the core particles. This suggests that native and derived 50‐S subunits do not differ in conformation only. The possibility may be envisaged therefore, that the two types of 50‐S differ in a ribosomal protein.
Quaternary Structure of Polynucleotide Phosphorylase from Escherichia coli
Authors: Portier Claude , Thang Minh Nguy , Grunberg‐Manago Marianne , van Rapenbusch Roland
Published: March 2005

Purification of polynucleotide phosphorylase from Escherichia coli by a modified technique yeilds a protein that appears to be homogeneous when analyzed by disc‐gel electrophoresis. Two species of polypeptide chains are obtained by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate or by filtration on Sephadex G‐200 column in the presence of guanidinium chloride; their molecular weights are 92000 and 48000.

Renaturation experiments show that reassociation of the purified heavy chains yields an active enzyme endowed of both polymerization and phosphorolysis activities; accordingly, the active site(s) must reside on the heavy component.

Reassociated renatured 92000‐Mr chains have a behavior very similar to that of the native enzyme on sucrose gradient and in disc gel electrophoresis, strongly suggesting a structure of the α2 type for polynucleotide phosphorylase. The origin and possible role of the 48000‐Mr component is discussed.
Studies on the Specificity of an Acid Deoxyribonuclease from Helix aspersa (Müll.)
Authors: Thiery Jean‐Paul , Bernardi Giorgio , Laval Jacques , Paoletti Claude , Ehrlich Stanislav D.
Published: March 2005

The kinetics of degradation of calf thymus DNA by the DNAase from Helix aspersa has been investigated in its middle and terminal phase by determining both hyperchromic shift and average chain length of the digest. The results obtained are practically identical with those previously reported for acid DNAase from hog spleen. In contrast, the distribution of the isostichs in snail DNAase digests is significantly different from those found for spleen acid DNAase digests of similar sizes.

The 3′‐phosphate and 5′‐hydroxy terminal nucleotides were found not to vary in their compositions in the average size range 34 to 10 and 70 to 12, respectively. In the 3′‐phosphate terminal position, deoxyadenosine is by far predominant (78%) and deoxycytidine almost absent (less than 1%); deoxyguanosine and thymidine form 6% and 16%, respectively, of the terminals. In the 5′‐hydroxy terminal position deoxyguanosine is predominant (45%), followed by deoxycytidine (31%), thymidine (14%) and deoxyadenosine (10%); in the 5′‐hydroxy penultimate position thymidine forms 38% of the nucleotides, deoxyguanosine 24%, deoxyadenosine 21% and deoxycytidine 17%.
Studies on Tyrosine Environments of Chicken Ovomucoid
Authors: Krysteva Milka A. , Mancheva Ivanka N. , Dobrev Ivan D.
Published: March 2005

The particular environments of tyrosine side chains were suggested to be responsible for the non‐denaturation acidic difference spectra of ovomucoid as well as of abnormal ionization of its phenolic groups. The micro environment of the most exposed tyrosine in ovomucoid was studied. The tyrosine was first labelled by means of tetranitromethane. Nitrotyrosine peptide was isolated. The study of the nine steps of nitrotyrosine peptide degradation results in the following amino acid sequence: Ala‐Gly‐Ala‐Lys‐Thr‐Gln‐NO2Tyr‐Gly‐Gly. The analysis of neutral sugars as well as glycosamine gave negative results. The presence of sialic acid however was detected.

On the basis of evidence obtained, the existence of a glycosidic bond formed from the hydroxyl group of tyrosine and the glycosidic hydroxyl group of sialic acid was suggested. The spectral behaviour of the tyrosine studied was discussed in the light of above assumption.