bluuebunny/crossref_metadata_2025 huggingface dataset:
Dual Control of Phenyladanine Ammonia‐Lyase Production and Removal by Its Product Cinnamic Acid
Authors: SHIELDS Susan E. , WINGATE Vincent P. , LAMB Christopher J.
Published: March 2005

l‐Phenylalanine ammonia‐lyase (EC 4.3.1.9, which catalyses the first reaction in the biosynthesis of plant phenylpropanoid products, undergoes a transient increase in activity in excised sections of pea (Pisum sativum) epicotyl tissue. Exogenous supplies of pathway intermediates inhibit the initial development of phenylalanine ammonia‐lyase activity and, if added at the time of high enzyme levels, cause a rapid decay in enzyme activity. The inhibitory effect of exogenous cinnamic acid, the immediate product of the enzyme, is expressed very rapidly (lag < 1 h) with the initial rate of decay of enzyme activity and the final steady‐state level of enzyme activity being dependent on the concentration of cinnamic acid.

The response has been studied by density labelling in vivo with 2H from 2H2O with analysis of the equilibrium distribution of enzyme activity in high‐resolution KBr density gradients. Cinnamic acid, at a concentration of 1 mM completely inhibits further incorporation of label and also markedly stimulates the removal of pre‐existing, unlabelled enzyme. Inhibition of de novo production and stimulation of removal occur at concentrations of cinnarnic acid as low as 10 μM.

The initial increase in enzyme activity reflects an increase in the rate of de novo enzyme production against a low background rate of enz:yme removal. The subsequent decay in enzyme activity is a result of a decline in production of the enzyme concomitant with a marked increase in the rate of removal of enzyme activity. In the presence of α‐ aminooxy‐β‐phenylpropionic acid, which specifically inhibits phenylalanine ammonia‐lyase and hence endogenous generation of cinnamic cid, high rates of enzyme production and low rates of enzyme removal are maintained leading to high levels of zyme activity in the later stages of enzyme induction. These data indicate the operation of dual feedback modulation of phenylalanine ammonia‐lyase in vivo following endogenous production of cinnamic acid. Such dual control by a product over both production and removal of an enzyme may be of general importance in slowly growing plant cells for the rapid removal of biosynthetic enzymes no longer required by the cell.
Transbilayer Organization of the Chlorophyll‐Proteins of Spinach Thylakoids
Authors: ANDERSSON Bertil , ANDERSON Jan M. , RYRIE Ivan J.
Published: March 2005

To investigate the transverse bilayer organization of the chlorophyll‐proteins of the three intrinsic chlorophyll‐ protein complexes, the effects of proteolytic enzymes, and an antibody against the light‐harvesting complex were compared using right‐side‐out and inside‐out thylakoid vesicles. The vesicles were isolated by aqueous polymer phase partitioning following the fragmentation of spinach thylakoids by passage through a Yeda press. Both vesicle types were agglutinated by an antiserum specific for the light‐harvesting complex, although proteo‐ lytic degradation of the complex occurred only in right‐side‐out vesicles. In addition, there are different anti‐ genic sites for the light‐harvesting complex on the inner and outer thylakoid surfaces. Polypeptides of the chlorophyll‐a‐protein complex of photosystem I1 were degraded by proteases at both membrane surfaces. We conclude that both these chlorophyll‐protein complexes are membrane spanning and transversely asymmetric, but that the light‐harvesting complex polypeptides accessible at the inner thylakoid surface are more resistant to proteolytic attack. In contrast, the main chlorophyll‐containing polypeptide (Mr= 64500) of photosystem I complex was resistant to proteolytic attack at both the outer and inner thylakoid surfaces.
Heterogeneous Distribution of ATP Citrate Lyase in Rat‐Liver Parenchyma
Authors: KATZ Norbert R. , FISCHER Wilhelm , ICK Margit
Published: March 2005

A radiochemical microtest was established for the determination of ATP citrate lyase in tissue samples of 0.2–1.0 μg dry weight. The specificity of this test system was guaranteed by its coenzyme A dependence as well as by inhibition of the activity measured in presence of a specific antibody.

Using this test system ATP citrate lyase activity was determined in microdissected periportal and perivenous liver tissue of fed, fasted and refed animals. The perivenous activity was 1.8‐fold and 2.4‐fold higher than the periportal one in fed male and female rats respectively.

The perivenous to periportal gradient was decreased during starvation‐dependent reduction of the ATP citrate lyase activity. On the other hand it was not only restored but enhanced up to 2.8 after refeeding‐dependent enhancement of the enzyme activity.

The predominance of the ATP citrate lyase activity in the perivenous, mainly glycolytic zone supports the hypothesis of the coordinate zonation of the carbohydrate and the lipid metabolism in the liver parenchyma.
NAD‐Linked l(+)‐Lactate Dehydrogenase from the Strict Aerobe Alcaligenes eutrophus
Authors: STEINBÜCHEL Alexander , SCHLEGEL Hans G.
Published: March 2005

An NAD‐linked l(+)‐lactate dehydrogenase (EC 1.1.1.27) was isolated from the strictly respiratory bacterium Alcaligenes eutrophus and purified about 700‐fold to homogeneity. Derepression of enzyme formation required prolonged incubation of the cell suspension under conditions of restricted oxygen supply. The purification procedure included precipitation by cetyltrimethylammonium bromide, and subsequent chromatography on MatrexTM green A gel, DEAE‐Sephacel and Cibacron blue F3G‐A Sepharose. Affinity chromatography on Matrex green A resulted in an about 140‐fold purification. The overall enzyme recovery was 41.7% of the initial activity, and the specific activity (pyruvate reduction) of the final preparation was 825 units · mg protein−1. The enzyme is a dimer consisting of two equal subunits of a relative molecular mass of 37000 each as determined at pH 7.0 in potassium phosphate buffer. It is specific for the l(+)‐stereoisomer of lactate. It is able to reduce, besides pyruvate, a variety of 2‐oxocarbonic acids and 3‐fluoropyruvate. The optimum temperature is 42–45°C, and the activation energy amounts to EA= 19.2 kJ · mol−1 for both directions. The enzyme is highly sensitive to p‐chloromercuribenzoate and heavy metal ions. It does not belong to the type of lactate dehydrogenase dependent on fructose 1,6‐bisphosphate for activity. Among various compounds tested for affecting enzyme activity only NAD, phosphoenolpyruvate, 3‐fluoropyruvate, oxalate, oxamate and oxaloacetate exhibited significant effects, the latter inhibiting the enzyme almost completely at 1 μM concentration.
Rotational Motion of Actin Monomer at Low and High Salt Concentration
Authors: BELÁGYI József , GRÓF Pál
Published: March 2005

Treatment of F‐actin pellet with 1 M MgCl2 results in a high concentration of monomer actin in solution [Biró, E. N. A. and Venyaminov, S. Yu. (1979) Acta Biochim. Biophys. Acad. Sci. Hung. 14, 31–42]. The physico‐chemical properties of this actin solution were studied by the spin‐labelling method and were compared with the normal actin solutions. The electron paramagnetic resonance (EPR) spectra produced the following results.

The rotational correlation time for monomer actin at low ionic strenght, which was calculated by the extrapolation procedure, increased from 16 ns to 26 ns in the presence of 1 M MgCl2.

The polymerization of actin is accompanied by a change in the polarity of the environment of the label (increasing hydrophobicity).

When heavy meromyosin (HMM) was added to monomer actin at high ionic strengh, the EPR data provided evidence that HMM induced the polymerization of the MgCl2‐depolymerized actin.
Evidence for a Restriction/Modification‐Like System in Anacystis nidulans Infected by Cyanophage AS‐1
Authors: SZEKERES Miklós , SZMIDT Alfred E. , TÖRÖK István
Published: March 2005

Anacystis nidulans infected by AS‐1 cyanophage contains an endnuclease (AS‐1 endonuclease) which splits host DNA but not AS‐1 phage DNA [Szekeres, M. (1981) Virology, III, 1–10]. AS‐1 phage DNA proved to be resistant not only to AS‐1 endonuclease but also to a number of restriction endonucleases the recognition sites of which contain a central dG‐dC dinucleotide. Since an unmodified 5′dG‐dC dinucleotide was shown to be present at the sites at which DNA is cleaved by AS‐1 endonuclease, the results suggest that the sites attacked preferentially by the AS‐1 endonuclease are specifically protected on the AS‐1 DNA molecule. The modification of AS‐1 DNA was shown to occur specifically in infected Anacystis because AS‐1 DNA fragments which are normally resistant to AS‐1 endonclease became susceptible to this enzyme if inserted into pBR322 plasmid and cloned in Escherichia coli. AS‐1 DNA was shown to contain about 5% of a modified nucleotide which was not 5‐methyldeoxycytidylic acid. Results presented and our earlier data suggest that in Anacystis infected by AS‐1 phage, a restriction/modification‐like system operates which is able to eliminate ‘unwanted’ (host) DNA selectively.
Seven Mammalian Aminoacyl‐tRNA Synthetases Associated within the Same Complex are Functionally Independent
Authors: MIRANDE Marc , CIRAKOGLU Beyazit , WALLER Jean‐Pierre
Published: March 2005

A heterotypic multienzyme complex from sheep liver containing seven aminoacyl‐tRNA synthetases specific for isoleucine, leucine, methionine, glutamine, glutamic acid, lysine and arginine was subjected to kinetic analyses to examine the possibility that association of these enzymes may impart kinetic properties which differ from those of their unassociated counterparts. The evidence obtained by two different approaches leads to the conclusion that the associated enzymes are functionally independent. Firstly, the kinetic constants of the methionyl‐tRNA and lysyl‐tRNA synthetase components of the complex do not differ significantly from those of their unassociated counterparts obtained after controlled proteolysis of the complex. Secondly, the methionyl‐tRNA synthetase component of the complex displays identical kinetic constants, whether assayed in the presence of [14C]methionine, ATP and highly enriched tRNAMet alone, or in the additional presence of the substrates required for unlabeled aminoacyl‐tRNA formation by each of the other six enzymes. Similarly, the initial rates of [14C]aminoacyl‐tRNA formation catalyzed by any of the six other enzymes was unaffected by the concomitant functioning of the other aminoacyl‐tRNA synthetase. The sedimentation behaviour of the aminoacyl‐tRNA synthetase components of the complex under conditions prevailing in the tRNA aminoacylation assay indicates that they remain associated under these conditions. The implications of these findings on the structural organization of the enzymes within the complex are discussed.
Patterns of Amino Acids near Signal‐Sequence Cleavage Sites
Authors: VON HEIJNE Gunnar
Published: March 2005

According to the signal hypothesis, a signal sequence, once having initiated export of a growing protein chain across the rough endoplasmic reticulum, is cleaved from the mature protein at a specific site. It has long been known that some part of the cleavage specificity resides in the last residue of the signal sequence, which invariably is one with a small, uncharged side‐chain, but no further specific patterns of amino acids near the point of cleavage have been discovered so far. In this paper, some such patterns, based on a sample of 78 eukaryotic signal sequences, are presented and discussed, and a first attempt at formulating rules for the prediction of cleavage sites is made.
Semisynthesis of Phospholipase A2
Authors: van SCHARRENBURG Gustaaf J. M. , PUIJK Wouter C. , de HAAS Gerard H. , SLOTBOOM Arend J.
Published: March 2005

The N‐terminal α‐helical region of phospholipase A2 is an important part of the enzyme for catalytic activity and lipid binding. Porcine pancreatic phospholipase A2 has Arg‐Ser at positions 6 and 7, whereas the bovine enzyme has Asn‐Gly. To pursue further the effects of these variable residues on differences in enzymatic properties, we prepared and studied the following semisynthetic analogs of →‐amidinated phospholipase A2 (AMPA): porcine [Ala7]AMPA, [Gly7]AMPA, [Asn6]AMPA, [Asn6‐Gly7]AMPA and bovine [Ser7]AMPA and [Arg6‐Ser7]AMPA. As we had previously found for the Asn6→ Arg bovine substitution, an Asn6‐Gly7→ Arg5‐Ser7 bovine substitution similarly improves the catalytic activity, the affinity for neutral lipid‐water interfaces and the capacity to penetrate lecithin monolayers, while just changing Gly7→ Ser produces almost no effect on these properties. Ser7→ Ala and Ser7→ Gly substitutions in porcine AMPA did not affect penetration or lipid binding, although they did diminish catalytic activity (which is true of all substitutions made in the porcine enzyme). Arg6→ Asn substitution in porcine AMPA decreases penetration of lecithin monolayers, but not as much as it was improved by the Asn6→ Arg substitution in bovine AMPA. In contrast to the dramatic increase in affinity for lipid‐water interfaces of Asn6→ Arg substitution in bovine AMPA, no decrease in affinity was found for Arg6→ Asn substitution in procine AMPA. This difference is most likely due to the fact that the porcine enzyme has positively charged Lys and His in place of the Lys10, Glu17 pair that lie very close to residue 6 in the bovine structure. It can thus be conclude that (with the exception of Gly7→ Ser in bovine AMPA) all the substitutions tried at positions 6 and 7 in bovine and porcine AMPAs have definite effects on the catalytic activity.
The Reactivity of Arginine Residues Interacting with Glucose 1‐Phosphate in Glycogen Phosphorylase
Authors: VANDENBUNDER Bernard , BUC Henri
Published: March 2005

Modification of pyridoxal‐reconstituted phosphorylase b with two arginine‐directed reagents, butanedione and [14C]phenylglyoxal, has been investigated and compared with the results obtained on the active and inactive conformations of the native enzyme; the reactivity of the various arginine residues has been directly described using autoradiography of chymotryptic maps derived from [14C]phenylglyoxal‐labelled phosphorylase.

In the native enzyme this method demonstrates that the same arginine residue (568) is reactive on both activated phosphorylase a and b, non‐reactive on inactive forms of phosphorylase and protected by glucose 1‐phosphate. Another residue is reactive, but its reactivity does not drastically depend upon phosphorylase conformation; it interacts with glucose 1‐phosphate.

In the pyridoxal‐reconstituted phosphorylase, the residue Arg‐568 is reactive. This reactivity does not correlate in a simple manner with the ionisation state of the coenzyme, since it is high when this group is either absent or in a dianionic form, and low when it is monoanionic. The reactivity of Arg‐568 rather correlates with the quaternary structure of the enzyme. The protection offered by glucose 1‐phosphate, pyrophosphate and phosphite on this pyridoxal‐reconstituted phosphorylase also provides information about the relative disposition of the substrate, the coenzyme and this particular arginine residue.
Ca2+ and Mg2+‐Dependent Complex Formation of Tropomyosin with Phosphotroponin (P1TI2C) or Dephosphotroponin (TI2C)
Authors: JAHNKE Ulrike , HEILMEYER Ludwig M. G.
Published: March 2005

The reduced viscosity of troponin and dephosphotroponin is independent of the protein concentration in both states, either metal‐free or with troponin C saturated with Ca2+ or Mg2+; that of tropomyosin increases linearly as function of the protein concentration, indicating aggregation. Addition of troponin to tropomyosin increases the reduced viscosity over the expected value being maximal at a 1:1 molar ratio of both proteins.

The reduced viscosity of a 1:1 molar mixture of phosphotroponin‐Mg4 or dephosphotroponin‐Mg3 increases in two phases as function of the total protein concentration, indicating the formation of two kinds of troponin‐tropomyosin complexes. In the first phase, troponin and tropomyosin form a non‐aggregating 1:1 complex, which is characterized by a value of 0.45 dl/g for the intrinsic viscosity and a sedimentation coefficient of 3.6 S. Employing these two values a molecular weight of 150 000 can be calculated, which is in the range of the sum of molecular weights for troponin and tropomyosin (156 000).

In the second phase the troponin‐tropomyosin complex aggregates further, a process described by: n (troponin‐tropomyosin) → (troponin‐tropomyosin)n. This further aggregation occurs upon saturation of the Ca2+‐specific sites in troponin C. A model is discussed which explains the shortening of 1.5 nm per tropomyosin molecule upon the shift of tropomyosin from the periphery into the groove of the actin filament by tropomyosin aggregation.
Metabolism of N8‐Monoacetylspermidine in Rat Hepatoma Cells
Authors: MAMONT Pierre S. , JODER‐OHLENBUSCH Anne‐Marie , SIAT Marlyse
Published: March 2005

Recent evidence has indicated a role for the acetyl derivatives of polyamines, particularly N8‐monoacetylspermidine, as activators of l‐ornithine decarboxylase in rat hepatoma tissue culture (HTC) cells. This is in contrast with the well‐described negative regulatory control of ornithine decarboxylase exerted by their non‐acetylated counterparts. Because of the possibility of a rapid extracellular and intracellular catabolism of the acetyl derivatives of polyamines, the metabolism of N8‐monoacetylspermidine and its effect on HTC cell ornithine decarboxylase have been investigated, under conditions which eliminate its extracellular catabolism.

Differing from previous reports, we demonstrate that N8‐monoacetylspermidine does not elevate ornithine decarboxylase activity when added at low concentrations to the culture medium of HTC cells. Higher concentrations decrease ornithine decarboxylase activity in a dose‐dependent manner. This effect cannot be unambiguously attributed to the effect of the acetyl derivative itself, because of the presence in situ of a very active N8‐monoacetylspermidine deacetylase, which generates spermidine intracellularly.
The Amino‐Acid Sequence of a Purothionin from Triticum monococcum, a Diploid Wheat
Authors: JONES Berne L. , MAK Alan S.
Published: March 2005

Purothionins were extracted and purified from the diploid wheat Triticum monococcum. Two proteins were obtained, one of which was present in only very small amounts. The major purothionin of T. monococcum was sequenced and it had an amino acid sequence identical with that of the β‐purothionin of Triticum aestivum (hexaploid bread wheat). It is known that T. monococcum contains the wheat A genome, so the structural gene coding for the β‐purothionin must comprise a part of the A genome. There have been no observable (as amino acid replacements) changes in the DNA comprising either the β‐purothionin gene of T. aestivum or the purothionin gene of T. monococcum, since T. monococcum (or its wild equivalent, Triticum boeoticum) hybridized with the diploid wheat B genome progenitor and started the evolution from diploid to allohexaploid wheat. All of the investigated characteristics of the purothionin‐like protein isolated in small amounts suggested that it was essentially identical in amino acid sequence with the T. monococcum purothionin. It may be a dimerized form of β‐purothionin.
Bovine Lens γ‐Glutamylcysteine Synthetase
Authors: GANDER John E. , SETHNA Shirley S. , RATHBUN William B.
Published: March 2005

Steady‐state kinetic analysis shows that glutathione binds reversibly to both Mg · enzyme and Mg · enzyme ·l‐glutamate forms of γ‐glutamylcysteine synthetase to form inactive complexes. The Ki values for binding to these two species of enzyme are 4 mM and 0.4 mM, respectively; those for S‐methyl glutathione are 16 mM and 0.5 mM, respectively. These data suggest that glutathione is an important feedback inhibitor and contributes to the regulation of glutathione synthesis by modulating the synthesis rate of the precursor dipeptide. Adenosine 5′‐diphosphate (5′ADP) is also an inhibitor and competes with both ATP and l‐β‐chloroalanine for Mg · enzyme ·l‐glutamate and Mg · enzyme ·l‐glutamylphosphate, respectively. Under physiological conditions in the lens, 5′ADP competes effectively with l‐cysteine for Mg · enzyme ·l‐glutamylphosphate, owing to the low concentration of l‐cysteine, and less effectively with ATP for Mg · enzyme ·l‐glutamate, because of a high concentration of ATP.
The Nucleotide Sequence of cDNA Complementary to the C1 Component of Rat Prostatic Binding Protein
Authors: DELAEY Bernard , DIRCKX Luc , PEETERS Ben , VOLCKAERT Guido , MOUS Jan , HEYNS Walter , ROMBAUTS Wilfried
Published: March 2005

The mRNA for component C1 of rat prostatic binding protein has been cloned and characterized. A partially purified mRNA fraction for this complex protein was reverse‐transcribed into double‐stranded cDNA and cloned into the PstI site of plasmid pBR 322. The 426‐base‐pair insert of the recombinant plasmid pC1A75 was completely sequenced. The coding region corresponds precisely to the 88 amino acid residues of C1 and in addition contains the information of a signal peptide of 23 residues. The 5′non‐coding region counts only 19 nucleotides and is incomplete but the 3′‐terminal non‐coding part of 60 nucleotides extends into the poly(A) tail. Sequence analysis of other C1‐positive clones indicates the presence of sequence rearrangements which must have occurred during the cloning procedure. Possible mechanisms for the generation of these cloning artefacts are discussed.
Purification and Some Properties of Bacteriophage T4 Particle‐Associated Lysozyme
Authors: SZEWCZYK Boguslaw , SKÓRKO Romuald
Published: March 2005

Bacteriophage T4 particle‐associated lysozyme, purified to electrophoretic homogeneity, was found to be a protein with a relative molecular mass of 15000. The lysozyme was purified from the particles of bacteriophage T4 e mutant and from the lysates of the 5tsl e T4 mutant, in which the enzyme is in soluble form. In the purification procedure advantage was taken of the affinity of the enzyme for GlcNAc‐MurNac‐LAla‐DGlu‐msA2pm‐DAl a (C6 muropeptide), one of the product of the digestion of Escherichia coli murein with lysozyme. The test for the quick estimation of bacteriolytic activity of the enzyme, using E. coli B freeze‐dried cells, is described. The pH optimum of the particle‐associated lysozyme was equal to about 6.0, ionic strength optimum to 0.05–0.1 M, and optimum Triton X‐100 concentration to 1%, when this substrate was used. Some of the aspects of the possible biological significance of the particle‐associated lysozyme in bacteriophage T4 infection are discussed.
Kinetic Analysis of the Protonation of a Surface Group of a Macromolecule
Authors: GUTMAN Menachem , NACHLIEL Ester , GERSHON Eli , GINIGER Rina
Published: March 2005

The dynamics of proton transfer between a surface‐attached acidic moiety and the bulk of the solution was measured using the laser‐induced proton pulse technique. [Gutman, M., Huppert, D. and Pines, E. (1981) J. Am. Chem. Soc. 103, 3709–3713]. The model system for this study consists of pH indicators (either neutral red or bromcresol green) adsorbed on Brij 58 micelles, as defined targets for protonation and a non‐adsorbed proton emitter (2‐naphthol‐3,6‐disulfonate) for generation of protons in bulk. The reaction was measured with 50‐ns time resolution over a time period of about 200 μs. The results were analyzed by a numerical solution of the coupled nonlinear differential equation corresponding with the reaction system. [Gutman, M., et al. (1983) J. Am. Chem. Soc. 105, 2210–2216].

Quantitative analysis reveals two independent reactions which govern the observed dynamics: (a) a diffusion‐controlled reaction between the proton and the surface targets; (b) translocation of the protonated target between the hydration layer of the interface and a more hydrophobic one.

The contribution of the translocation reaction to the dynamics of surface protonation is more pronounced for compounds like carboxylates or phenolates which increase their hydrophobicity upon protonation. Amines and azoaromatic structures are more hydrophilic in their protonated states, the dynamics of their protonation is less affected by post‐protonation distribution within the microenvironments of the interface.

The interrelation between the partial rate constants and the macroscopic time constants and equilibrium parameters is analyzed.
The Role of Magnesium in Binding of the Nucleotide Polyphosphate Chain to the Active Site of Myosin Subfragment‐1
Authors: SCHAUB Marcus C. , WATTERSON John G. , LOTH Klaus , FOLETTA Daniela
Published: March 2005

The binding of adenosine 5′‐[β,γ‐imido]triphosphate, pyrophosphate and triphosphate to the active site of myosin subfragment‐1 was assessed in the presence and absence of Mg2+ by direct and indirect methods. In addition, the affinity and stoichiometry of Mg2+in the ternary complexes formed by protein, Mg2+ and each of these phosphate compounds have been determined. As direct methods, equilibrium dialysis, sedimentation and quantitative affinity chromatography were used in conjunction with the indirect method of monitoring reactivity changes of the critical thiol‐1 and thiol‐2 groups, which occur upon binding of the ligands at the active site. There was good agreement between the results yielded by the different methods. All three phosphate compounds alone bind just one molecule per isolated myosin head portion with similar affinities lying in the range 1−4×103 M−1. Again only one molecule/head portion binds when they exist in the form complexed with Mg2+, but now show much higher affinities of between 106−107 M−1. In all cases Mg2+ was found to be associated in the ternary complexes with the very high affinity of 108−109 M−1. It is postulated that this ion plays a prominent role in fixing the phosphate chain in the myosin active site. In contrast, Mg2+ scarcely affects the affinity of ADP and shows only a low affinity around 4 × 104 M−1 in the ternary complex [Watterson, J. G., Foletta, D., Kunz, P. A., and Schaub, M. C. (1983) Eur. J. Biochem. 131, 89–96]. As pyrophosphate displays binding parameters similar to the triphosphate compounds and widely different from ADP, it is argued that it may bind in the β,γ‐phosphate positions at the active site.
500‐MHz 1H‐NMR Studies of Ribosomal Proteins Isolated from 70‐S Ribosomes of Escherichia coli
Authors: van de VEN Fransk J. M. , de BRUIN Simon H. , HILBERS Cornelis W.
Published: March 2005

A method for the large‐scale isolation of ribosomal proteins is described avoiding pre‐separation of 30‐S and 50‐S subunits. Five proteins isolated in this way were studied with high‐resolution 1H NMR at 500 MHz. These are S21, L18, L25, L30 and L33. The results show that L18, L25 and L30 exhibit tertiary structure in solution and indications for secondary structure in S21 are found. Protein L33 appears to be a random coil. Several resonances in the 1H NMR spectra are assigned to particular protons of amino acid residues, e.g. the aromatic ring protons of tyrosines and histidines, and ɛ‐protons of lysines.
Factors Affecting the Cold Inactivation of an Acetyl‐Coenzyme‐A Hydrolase Purified from the Supernatant Fraction of Rat Liver
Authors: ISOHASHI Fumihide , NAKANISHI Yoko , SAKAMOTO Yukiya
Published: March 2005

An extramitochondrial acetyl‐coenzyme‐A hydrolase from rat liver is shown to be a cold‐labile oligomeric enzyme that undergoes a reversible conformational transition between a dimeric and a tetrameric form in the presence of adenosine 5′‐triphosphate or adenosine 5′‐diphosphate at 25–37°C, and between a dimeric and a monomeric form at low temperature. The enzymatically active dimer is fairly stable at 25–37°C, but much less stable at low temperature, dissociating into monomer with no activity. At 37°C and low concentrations of enzyme protein (≤ 14 μg/ml), the activity decreased rapidly and only 10% of the initial activity remaining after 60 min. Addition of bovine serum albumin or immunoglobulin G to the medium completely prevented inactivation of the dimeric enzyme at low concentration at 37°C, but had little effect on cold inactivation of the enzyme. Cold inactivation of the dimeric enzyme was partially prevented by the presence of various CoA derivatives. The order of potency was acetyl‐CoA (substrate) ≥ butyryl‐CoA > octanoyl‐CoA > CoA (product) > acetoacetyl‐CoA. Another enzyme product, acetate, had little effect on cold inactivation. Polyols, such as sucrose, glycerol, and ethylene glycol, and high concentrations of NaCl, KCl, pyrophosphate and phosphate also greatly prevented cold inactivation. Cold inactivation was scarcely affected by pH within the pH range at which the enzyme was stable at 37°C.
A Type I DNA Topoisomerase from Trypanosoma cruzi
Authors: RIOU Guy F. , GABILLOT Michéle , DOUC‐RASY Setha , KAYSER Alain , BARROIS Michel
Published: March 2005

A type I DNA topoisomerase has been isolated from the nuclei of the flagellate Trypanosoma cruzi, using poly(ethylene glycol) fractionation and chromatography on hydroxyapatite and on phosphocellulose. The relaxation activity was ATP‐independent, enhanced by Mg2+ and spermidine. The enzyme removed supercoils from negative and positive superhelical DNAs. Topoisomerase activity was associated with a polypeptide of Mr about 65000 as shown by glycerol gradient centrifugation and by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels.
Identification and Characterization of an UDP‐Gal: N‐Acetyllactosaminide α‐1,3‐d‐Galactosyltransferase in Calf Thymus
Authors: VAN DEN EIJNDEN Dirk H. , BLANKEN Willem M. , WINTERWERP Herrie , SCHIPHORST Wietske E. C. M.
Published: March 2005

Calf thymus was found to contain a high activity of a galactosyltransferase, which transfers galactose from UDP‐galactose to asialo‐α1‐acid glycoprotein and N‐acetyllactosamine. By means of competition and acceptor‐specificity studies the enzyme could be distinguished from an N‐acetylglucosaminide β‐1,4‐galactosyltransferase and an N‐acetylgalactosamine‐protein β‐1,3‐galactosyltransferase, which in addition occur in calf thymus, as well as from the blood‐group‐B‐associated α‐galactosyltransferase. Identification of the products revealed that the enzyme accomplishes an α1 → 3 linkage resulting in a terminal Gal(α1 → 4)GlcNAc sequence. The enzyme is membrane‐bound and is activated by Triton X‐100. It shows optimal activity over a broad pH range (5.5–7.0) and has a pronounced requirement for Mn2+ ions (Km= 6.1 mM) for its action. It is suggested that the α‐1,3‐galactosyltransferase functions in the biosynthesis of calf thymocyte cell‐surface glycoconjugates including glycoproteins.
Towards Purification of the Scrapie Agent
Authors: DIRINGER Heino , HILMERT Horst , SIMON Dietrich , WERNER Edgar , EHLERS Bernhard
Published: March 2005

A method for the partial purification of scrapie infectivity from hamster brain is described. About a 100–1000‐fold, 20‐fold, and 200‐fold enrichment in scrapie infectivity with respect to protein, RNA, and DNA content has been achieved using differential centrifugation, enzyme and detergent treatment. The inbred CLAC strain of hamsters used in our experiments contained about 10 times less infectivity in brain than has been found in randomly bred animals or other inbred strains.
A novel protein programmed by the mRNA conserved in dry wheat embryos
Authors: HANLEY‐BOWDOIN Linda , LANE Byron G.
Published: March 2005

If bulk mRNA from dry wheat embryos (wheat germ) is used to direct cell‐free incorporation of [35S]cysteine into proteins, a striking proportion of the total radioactivity is channeled into a single protein. During early postimbibition development, when protein synthesis is directed by the mRNA conserved in dry embryos, incorporation of cysteine is preponderantly (20–25%) directed into synthesis of this one protein: the ‘early’ cysteine‐labeled protein (Ec). When conserved mRNA from the dry embryos has been fully degraded, as when cellular or cell‐free protein synthesis is directed by the mRNA in germinated embryos, synthesis of Ec is not detected. Reliable detection of Ec requires prior alkylation of wheat embryo proteins, and it was especially interesting to find that when wheat embryo proteins are alkylated by iodo[14C]acetamide, two proteins co‐dominate the distribution of radioalkylated products in dodecylsulphate/polyacrylamide gels: Ec and wheat germ agglutinin. Using co‐electrophoresis with the isotopically labeled protein to detect a dye‐staining counterpart, Ec has been purified by combined cation‐exchange and gel‐filtration chromatography of alkylated wheat germ proteins. The purified protein can be recovered in milligram quantity (5–10 mg/100 g wheat germ) and compositional analysis shows that it is unusually rich in cysteine (approx. 15%) and glycine (approx. 17%), as is wheat germ agglutinin.
Molecular properties of 5‐aminolevulinic acid dehydratase from Spinacia oleracea
Authors: LIEDGENS Walter , LÜTZ Cornelius , SCHNEIDER Hansjörg A. W.
Published: March 2005

5‐Aminolevulinic acid dehydratase from spinach (Spinacia oleracea), highly purified by immunoprecipitation, was characterized by inhibitor studies, amino acid composition, the mode of substrate binding and electron photomicrography. The results show that the conversion of 5‐aminolevulinate to porphobilinogen requires an active arginine residue and the formation of a Schiff base between the enzyme and 5‐aminolevulinate. The formation of a Schiff base is well known for bacterial and animal dehydratases. Spinach dehydratase, however, is distinguished by its insensitivity to iodoacetamide, a low content of cysteine residues and a high proportion of acidic amino acids. In addition, electron photomicrographs of spinach dehydratase molecules do not resemble the corresponding images of beef liver dehydratase. The finding that an arginine residue is essential for substrate conversion corroborates the suggestion that the right orientation of the substrate in the active center is dependent on a positive charge.
A soluble metalloendopeptidase from rat brain
Authors: ORLOWSKI Marian , MICHAUD Charlene , CHU Thomas G.
Published: March 2005

A metalloendopeptidase, optimally active at a neutral pH, was purified from the soluble fraction of brain homogenates. The enzyme (molecular weight about 67000) is strongly inhibited by metal chelators such as EDTA and o‐phenanthroline. An EDTA‐treated enzyme can be reactivated by several divalent metal ions including Zn2+, Co2+, and Mn2+. The specificity and kinetic parameters of the enzyme were studied with a series of model synthetic substrates. The enzyme preferentially cleaves peptide bonds in which the carbonyl group is contributed by an aromatic amino acid residue in the P1 position. The lowest Km values and the highest Kcat/Km ratios were obtained with substrates having aromatic residues in the P′3 and P1 position or in the P′3 and both the P1 and P2 positions. Lower Kcat/Km ratios were obtained with substrates having arginine residues in position P1, and even lower values with those substrates having a glycine or aspartyl residue in this position. Introduction of a D‐amino acid residue in either position P1 or P′1 renders the substrate totally resistant to hydrolysis. The specificity studies suggest that the active site of the metalloendopeptidase can accomodate at least five amino acid residues, with two of those residues binding on the N‐terminal side and three binding on the C‐terminal side of the hydrolyzed bond. Several biologically active peptides are cleaved by the enzyme at sites consistent with the specificity deduced from studies with model synthetic substrates.
Histones from Chinese hamster ovary cells
Authors: JOSEPH Gérard , CAIZERGUES‐FERRER Michèle , AMALRIC François
Published: March 2005

A quantitative analysis of histone variants and of their modified forms in exponentially growing Chinese hamster ovary cells is described. A two‐step analytical procedure has been developed. Total nuclear proteins were first fractionated on a sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis. After electroelution from the corresponding bands cut out of the gel, each individual histone was analysed on an acetic acid/urea/Triton X‐100 slab gel. All the variants and most of the acetylated and phosphorylated molecules were thus resolved. In absence of butyrate, we have detected four, three and two different levels of acetylation respectively for H4, H3, H2A and H2B. Scanning of the stained gel has allowed the determination of the relative amount of each forms. H3 and H4 present a higher turnover of the acetate groups than H2A and H2B. For the four histones, acetate label appeared preferentially in the multi‐acetylated forms then gradually in the less acetylated forms. The data suggest that the deacetylation‐acetylation process of a given histone molecule involves all the acetate groups of the molecule (three for a triacetylated histone, one for a monoacetylated histone).
Change in the reactivity of the active‐site serine OH of butyrylcholinesterase caused by a new reversible inhibitor
Authors: GAÁL József , BARTHA Ferenc , BATKE József
Published: March 2005

The 2‐chloro‐12‐(2‐piperidinoethyl)‐dibenzo[d,g] (1,3,6)‐dioxazocine · HCI (EGYT‐2347), a new specific inhibitor of butyrylcholinesterase inhibits reversibly and non‐competitively the enzymatic hydrolysis of butyrylthiocholine iodide (Ki= 0.15 μM, at 37°C in 0.1 M Tris/HCI, pH 7.5).

The theoretical progress curve of product accumulation has been developed for the case when a non‐competitive reversible inhibitor (EGYT‐2347) and an active‐site‐directed irreversible inhibitor (diisopropylfluorophosphate) act simultaneously. By the aid of this approach it was concluded that the butyrylcholinesterase–EGYT‐2347 binary complex does not react with diisopropylfluorophosphate either because of a structural change caused by binding or by the direct steric hindrance of EGYT‐2347.
Tissue‐specific formation of transcription‐initiation complexes at the 5′ end of the mouse β major globin gene
Authors: TRIADOU Patrick , LELONG Jean‐Claude , GROS François , CREPIN Michel
Published: March 2005

Accurate initiation of transcription in vitro requires, in addition to RNA polymerase II, factors present in soluble extracts of cultured cells. We have developed transcription system in vitro, which permits us to visualize the transcription‐initiation complexes on a mouse β globin restricted fragment from a recombinant β globin bacteriophage DNA. Using the λ fragments as internal controls this system has allowed us to assess the specificity of transcription with RNA polymerase II. Comparing extracts from cells and tissues expressing the globin genes (Friend cells, spleen and blood from phenylhydrazine‐induced anemic mice) with those which do not (thymus, liver, PCC3 cells), we observed that specific initiation of transcription on the β globin gene occurs only with soluble extracts from erythroid tissues. This tissue‐specific transcription is partially sensitive to α‐amanitin and occurs at the 5′ end of the globin gene.
Chromatin structure in barley nuclei
Authors: MITHIEUX Gilles , ROUX Bernard
Published: March 2005

In order to study the chromatin structure of a higher plant we used a high‐yield method, which allows one to obtain up to 109 nuclei/kg fresh barley leaves. Significant amounts of low‐ionic‐strength‐soluble chromatin can be extracted from these nuclei. Physicochemical properties were examined and discussed. Electric birefringence allowed us to observe the same transition in electro‐optical properties as has been observed for animal chromatin, and suggested the existence of a symetrical structure occurring for approximately six nucleosomes. Circular dichroism showed that barley oligonucleosomes exhibit a higher molar ellipticity at 282 nm than total soluble chromatin and than their animal counterparts.
Glutamyl‐tRNA synthetases from wheat
Authors: RATINAUD Marie Hélène , Thomes Jean Claude , JULIEN Raymond
Published: March 2005

Three dimeric glutamyl‐tRNA synthetases (GluRS) were isolated from extracts of quiescent wheat germ and wheat chloroplasts.

The chloroplast enzyme (Mr= 110000), called GluRS C, exhibits a prokaryotic (Escherichia coli) tRNA specificity.

Two enzymes were found in the quiescent germ and were separated on phosphocellulose P11: one called GluRS P, probably the mitochondrial enzyme, has the same tRNA specificity as GluRS C; the other, called GluRS E, has eukaryotic (wheat germ) tRNA specificity. Both enzymes exhibit a molecular weight close to 160000.

Each of these enzymes co‐eluate on hydroxyapatite and phosphocellulose chromatographies with an unstable active monomer whose molecular weight is approximately half that of the corresponding dimer. Two assumptions are discussed about these monomers.
Nucleotide sequence of the lipoamide dehydrogenase gene of Escherichia coli K12
Authors: STEPHENS Paul E. , LEWIS Hilary M. , DARLISON Mark G. , GUEST John R.
Published: March 2005

The nucleotide sequence of a 1980‐base‐pair segment of DNA, containing the lpd gene encoding the lipoamide dehydrogenase component (E3) of the pyruvate dehydrogenase complex of Escherichia coli K 12, has been determined by the dideoxy chain‐termination method. The lpd structural gene comprises 1419 base pairs (473 codons, excluding the initiating AUG codon). It is preceded by a good promoter and an excellent ribosome binding site and it ends with a typical rho‐independent terminator sequence. The results confirm that the lpd gene is an independent gene linked to, but not part of, the ace operon that encodes the E1 and E2 components of the pyruvate dehydrogenase complex. The location and transcriptional polarity of the lpd gene relative to the restriction map of the corresponding region of DNA, are completely consistent with previous genetic and post‐infection labelling studies. The composition, Mr (50554 or 51274 if the FAD cofactor is included), aminoterminal sequence and carboxy‐terminal sequence predicted from the nucleotide sequence are in excellent agreement with previous studies on the purified enzyme. The enzyme also exhibits a remarkable degree of sequence homology with peptides of the pig heart enzyme and with other pyridine nucleotide disulphide oxidoreductases whose sequences have been defined: human erythrocyte glutathione reductase and plasmid‐encoded mercuric reductase.
The effect of quercetin on the phosphorylatio activity of the Rous sarcoma virus transforming gene product in vitro and in vivo
Authors: GRAZIANI Yosef , ERIKSON Eleanor , ERIKSON Raymond L.
Published: March 2005

The phosphotransferase activity of the Rous sarcoma virus src gene product, pp60src, was inhibited both in vitro and in vivo by the bioflavonoid quercetin. The Ki for the inhibitory effect was in the range of 6–11μM under conditions in vitro. The inhibitory effect of quercetin was competitive towards the nucleotides ATP and GTP as substrates for pp60src and was non‐competitive towards α‐casein as the protein substrate of this kinase activity. In contrast, studies in vitro of the phosphotransferase activity of the catalytic subunit of the cAMP‐dependent protein kinase showed that this flavonoid did not inhibit the phosphorylation of physiological substrates of this enzyme. In cultured cells the half‐maximal inhibition of tyrosine phosphorylation of pp60src as well as the phosphorylation of the Mr= 34 000 protein, a physiological substrate of pp60src, was in the range 0.06–0.08 mM.
Purification, subunit structure and immunological comparison of fructose‐bisphosphate aldolases from spinach and corn leaves
Authors: KRÜGER Ingo , SCHNARRENBERGER Claus
Published: March 2005

The cytosol and chloroplast fructose‐bisphosphate aldolases from spinach leaves were separated by ion‐exchange chromatography on DEAE‐cellulose, and were purified by subsequent affinity chromatography on phosphocellulose to apparent homogeneity as judged from polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The two aldolases had specific activities of 7.2 and 7.8 units mg protein−1. Molecular weight determinations by electrophoresis in sodium dodecyl sulfate gels and by sedimentation velocity centrifugation in sucrose gradients showed that the aldolases contained four subunits of Mr 38000 and 35000, respectively. Antibodies against the cytosol and chloroplast aldolase from spinach leaves were raised in a guinea pig and in a rabbit, respectively. In the Ouchterlony double‐diffusion test, the two aldolases did not cross‐react. A small degree of cross‐reaction was observed by a test in which immune complexes were adsorbed to a solid‐phase support (Staphylococcus aureus Cowan 1 cells) and nonbound enzyme activity was determined after centrifugation. These results imply major structural differences between the two spinach leaf aldolases.

Only one major aldolase could be resolved on DEAE‐cellulose from corn leaves. The aldolase was purified and had a specific activity of 6.4 units · mg protein−1. The corn leaf aldolase cross‐reacted with the antiserum raised against the chloroplast enzyme from spinach leaves, but not with the other antiserum. Thus, the corn leaf aldolase could be identified as a chloroplast enzyme. Since aldolase activity is mostly restricted to the bundle sheath cells of corn leaf, it was concluded that it is compartmentalized in the chloroplasts of these cells but not in chloroplasts of the mesophyll cells.
On the mechanism by which glucocorticoids cause the activation of glycogen synthase in mouse and rat livers
Authors: LALOUX Monique , STALMANS Willy , HERS Henri‐Géry
Published: March 2005

The administration of glucocorticoids to mice caused within 3 h an inactivation of glycogen phosphorylase and activation of glycogen synthase in their livers. In a Sephadex filtrate of liver extract, as well as in a purified glycogen fraction obtained from treated mice, but not in the same preparations obtained from control mice, glycogen synthase was activated without previous inactivation of phosphorylase. The initial rate of synthase activation in a Sephadex filtrate was proportional to the rate of glycogen synthesis in vivo in the same animal. When the glycogen fraction was isolated in the presence of soluble starch, it could be separated from phosphorylase, phosphorylase phosphatase and synthase phosphatase. When added to a control Sephadex filtrate, this purified glycogen fraction obtained from prednisolone‐treated mice relieved synthase phosphatase from inhibition by phosphorylase a, indicating that it contained a transferable ‘deinhibiting factor’. This deinhibiting factor appears to be a protein and was further purified by alkyl‐Sepharose or DEAE‐cellulose chromatography. Another modification introduced by treatment with prednisolone was that phosphorylase phosphatase was 1.5–2‐fold more active than in the liver of control mice. This property however did not correlate with the rate of glycogen synthesis in vivo. Administration of actinomycin D prevented the expression of the glucocorticoid effects on the rate of glycogen synthesis in vivo and on the protein phosphatases in vitro. The deinhibition of synthase phosphatase was also observed in isolated rat hepatocytes incubated in the presence of glucocorticoids, but in these preparations synthase was not activated.
Association of rabbit muscle glycolytic enzymes with filamentous actin
Authors: WESTRIN Hjalmar , BACKMAN Lars
Published: March 2005

The association between purified glycolytic enzymes and filamentous actin from rabbit muscle has been studied by counter‐current distribution. The co‐distribution of a glycolytic enzyme and filamentous actin leads to a significant change in the counter‐current distribution profile of the enzyme whereas that of actin is unaffected. The changes in the distribution profiles clearly demonstrated that all glycolytic enzymes studied, though to different extents, bind to filamentous actin.

The aqueous two‐phase system used for the studies contained dextran, poly(ethyleneglycol) and 150 millimolal potassium phosphate buffer, pH 7.0. Since the ionic strength of the two‐phase system is determined mainly by the buffer, the glycolytic enzymes are evidently able to associate with filamentous actin, at least in the presence of neutral polymers, at ionic strengths comparable to or higher than those assumed to prevail in vivo.