bluuebunny/crossref_metadata_2025 huggingface dataset:
Measurement of Testosterone and Dehydroepiandrosterone 16α‐Hydroxylase Activities by a Tritium Exchange Method
Authors: KREMERS Pierre , De GRAEVE Jean , AZHIR Ari , GIELEN Jacques E.
Published: March 2005

A new isotopic method, based upon the stereospecific replacement of a proton (3H) by a hydroxyl group has been developed for the measurement of rat liver testosterone and dehydroepiandrosterone 16α‐hydroxylase activity. Specifically 16‐tritiated substrates were prepared by microbiological (Cylindrocarpon radicicola) transformation of the [16‐3H]progesterone and [16‐3H]pregnenolone. The incubation medium consists of a phosphate buffer (pH 7; 150 mM), NADPH (0.1 mM), nicotinamide (10 mM) and magnesium chloride (4 mM). Tween 80 (1 mg/ml) is used to solubilize saturating concentrations of [16‐3H]testosterone (50 μM) or [16‐3H]dehydroepiandrosterone (100 μM). The enzymatically relased tritium is recovered in the incubation medium as tritiated water which is distilled under reduced pressure and counted by liquid scintillation. The method is easy to perform, very sensitive (50 pmol of 16α‐hydroxylated metabolites) and is independent of any further metabolism of the 16α‐hydroxylated products.
A Hydrogen‐Deuterium Exchange Study of the Amide Protons of Polymyxin B by Nuclear‐Magnetic‐Resonance Spectroscopy
Authors: PERKINS Stephen J. , RADDA George K. , RICHARDS Rex E.
Published: March 2005

Proton magnetic resonance spectra at 270 MHz of polymyxin B, a cationic oligopeptide antibiotic, show the influence of the inorganic counteranion present in solution.

Hydrogen‐deuterium exchange rates for the amide protons are of two types, depending on whether the anion is monovalent or polyvalent. Polyvalent anions catalyse the acid‐catalysed reaction more than the monovalent anions.

The structure in solution was monitored using the proton signals of the amides, the phenylalanine aromatic protons, and the leucine methyl and γ‐CH protons in several polymyxin salts. The temperature coefficients of the chemical shifts of the N‐H protons are used to identify two β turns in the cyclic ring of polymyxin B. The variation in chemical shift of the N‐H protons, the aromatic protons and the leucine protons are correlated with anionic size and electronegativity.
Do Mitochondria Produce Oxygen Radicals in vivo?
Authors: NOHL Hans , HEGNER Dietmar
Published: March 2005

Heart mitochondria from rats at 3 months and 23 months of age were investigated for their capacity to generate oxygen radicals and hydrogen peroxide.

Highest values for O2+‐formation were obtained with submitochondrial particles freed from superoxide dismutase after the addition of succinate and antimycin A to start the reaction. Under these conditions superoxide‐radical formation with mitochondria from old rats (2.54 nmol × min−1× mg−1) exceeded formation rates in the young controls (1.9 nmol × min−1× mg−1) by 25%.

A constant fraction of 20% of the radicals produced escaped quenching by intramitochondrial superoxide dismutase. This fraction was independent of the rate of radical formation; its magnitude was deduced from generation rates of hydrogen peroxide in the presence and absence of exogenous superoxide dismutase.

Free oxygen radicals could be obtained with intact rat‐heart mitochondria as well following the addition of ATP, a system which is closer to physiological states. Formation rates of oxygen radicals observed under these conditions were similar to those seen in the particulate fractions which escaped quenching by mitochondrial superoxide dismutase.

Peroxidative degradation of membrane lipids was found to parallel steady‐state concentrations of free oxygen radicals.

The results are discusssed in terms of a radical‐generating mechanism which functions in vivo at the level of the mitochondrial electron‐transferring system.
The Use of Netropsin with CsCl Gradients for the Analysis of DNA and Its Application to Restriction Nuclease Fragments of Ribosomal DNA from Physarum polycephalum
Authors: MATTHEWS Harry R. , JOHNSON Edward M. , STEER Wendy M. , BRADBURY E. Morton , ALLFREY Vincent G.
Published: March 2005

Netropsin binds to DNA in caesium chloride density gradients and reduces the density of the DNA. The DNA is saturated at a netropsin/DNA weight ratio of about 6 and the change in density, Δϱ, at saturation is given by Δϱ= ‐109 (dA + dT content)1.87 mg/ml for the six DNAs tested covering dA + dT contents from 0.28 to 0.69. At lower netropsin/DNA ratios the observed density shifts are consistent with a two‐site model for netropsin binding to DNA. Netropsin approximately doubles the resolution of Physarum polycephalum nucleolar satellite DNA from main‐band DNA. The fragments of P. polycephalum nucleolar satellite DNA obtained with the restriction endonuclease HindIII do not separate on CsCl gradients, even in the presence of netropsin, which shows that the transcribed and non‐transcribed sequences in this DNA have similar nucleotide compositions.
Evidence for the Involvement of a Glucose‐6‐Phosphate Carrier in Microsomal Glucose‐6‐Phosphatase Activity
Authors: NILSSON Olle S. , ARION William J. , DEPIERRE Joseph W. , DALLNER Gustav , ERNSTER Lars
Published: March 2005

Protease and diazobenzene sulfonate have been used to probe the transverse topology of the microsomal glucose‐6‐phosphatase system.

Treatment of intact microsomes with these reagents under the conditions used here did not affect the permeability of the membrane to mannose 6‐phosphate, nucleoside diphosphatase, or dextran of 70 000 molecular weight. Nor did these treatments inactivate the hydrolytic site of glucose‐6‐phosphatase, a finding in agreement with earlier conclusions that this site is on the inside of the membrane.

On the other hand, treatment of intact microsomes with diazobenzene sulfonate or proteases does inactivate (or increase the apparent Km of) some other component which is rate‐limiting for glucose‐6‐phosphatase activity in intact but not in disrupted microsomes. The simplest explanation for this phenomenon is that there is a protein carrier in the microsomal membrane which transports glucose 6‐phosphate from the medium to the lumen, where it is hydrolyzed, and that diazobenzene sulfonate and proteases attack this carrier.

The lack of effect of these reagents on microsomal inorganic pyrophosphatase activity suggests that the glucose‐6‐phosphate carrier cannot transport pyrophosphate.

Finally, it was found that treatment of microsomes with ammonia breaks down their permeability barrier but also removes significant amounts of microsomal phospholipid and inactivates a number of microsomal enzymes. We do not recommend it as a general approach to altering microsomal permeability.
Nature of the Collagenous Protein in a Tumor Basement Membrane
Authors: TIMPL Rupert , MARTIN George R. , BRUCKNER Peter , WICK Georg , WIEDEMANN Hanna
Published: March 2005

The EHS sarcoma (named after its discoverer, Engelbreth‐Holm, and its primary characterizer, R. Swarm) contains an extracellular matrix of basement membrane. The collagenous component of the membrane was extracted with dilute acetic acid from tumors grown in lathyritic mice and separated from other proteins by chromatographic methods. This collagen has a composition resembling that reported for other basement membrane collagens. The polypeptide chains of the tumor collagen are linked by disulfide bonds and migrate after reduction intermediate between the α chains und β components of type‐I collagen. The circular dichroism spectra obtained from it resemble those obtained from other collagens.

The intact protein contained about 280 residues of glycine per 1000 residues. After reduction of disulfide bonds in the basement membrane collagen, treatment with either pepsin or trypsin gave rise to resistant fragments containing one‐third glycine. These fragments formed segment‐long‐spacing crystallites 210 nm long in the case of those treated with pepsin and 350 nm long in the case of those treated with trypsin. The band pattern differs from that found with other collagens.

Antibodies prepared against the tumor collagen were found to localize to the tumor matrix and to known basement membranes occurring in mouse tissues. The data suggest that these basement membranes may contain a collagenous protein similar to the tumor protein.
Synthesis of an Oligonucleotide Inhibitor of Protein Synthesis in Rabbit Reticulocyte Lysates Analogous to that Formed in Extracts from Interferon‐Treated Cells
Authors: HOVANESSIAN Ara G. , KERR Ian M.
Published: March 2005

A heat‐stable, low‐molecular‐weight inhibitor of protein synthesis is formed on incubation of haemin‐supplemented rabbit reticulocyte lysates with ATP and double‐stranded RNA (dsRNA). It inhibits the translation of both added encephalomyocarditis virus RNA (EMC RNA) and endogeneous messenger RNA in reticulocyte lysates and mouse L‐cell extracts.

The enzyme responsible for the synthesis of the inhibitor binds to dsRNA and can be purified on a column of poly(I) · poly(C) bound to an inert support. The highly purified enzyme in its stable column‐bound state can be conveniently employed to synthesise the inhibitor and to label it with [3H]ATP, or [α‐32P]ATP or [γ‐32P]ATP as substrate. The radioactive inhibitor synthesised in this way with material from rabbit reticulocyte lysates shows the same spectrum of resistance and sensitivity to alkali and a variety of enzymes as corresponding material similarly synthesised with extracts from interferon‐treated mouse L‐cells. The inhibitors from the two systems have comparable absorbance spectra, are chromatographically and electrophoretically indistinguishable and are apparently identical in specific activity in the inhibition of protein synthesis in the cell‐free system.

The inhibitor is also formed on inhibition of protein synthesis by dsRNA in reticulocyte lysates. On comparison of the spectrum of polypeptide products synthesised in response to EMC RNA in the reticulocyte lysate, the effects of the inhibitor or dsRNA were similar: a distinctly different effect was obtained with the haemin‐controlled repressor, a known inhibitor of initiation. The significance of these results with respect to the mechanism of action of the inhibitor and its role in the inhibition observed in response to dsRNA is discussed.
Yeast DNA Polymerases: Antigenic Relationship, Use of RNA Primer and Associated Exonuclease Activity
Authors: WINTERSBERGER Erhard
Published: March 2005

Highly purified preparations of DNA polymerases A and B from yeast were compared with respect to antigenic relationship, ability to use ribonucleotide primers and associated nuclease activity. The following results were obtained.

Antiserum directed against DNA polymerase A inhibits this enzyme but does not interfere with the activity of DNA polymerase B or of mitochondrial DNA polymerase, nor does it precipitate the latter two enzymes.

DNA polymerase A is capable of using oligo(ribouridylic acid) as a primer for the polymerization of dTMP. This reaction is not catalysed by polymerase B to any significant extent.

Whereas DNA polymerase A is devoid of nuclease activity, DNA polymerase B catalyses an exonucleolytic release of mononucleotide units from the 3′ end of polynucleotides. The results of several experiments suggest that this nuclease activity is associated with the DNA polymerase B molecule.
An Analysis of the Bovine Genome by Density‐Gradient Centrifugation
Authors: MACAYA Gabriel , CORTADAS Jordi , BERNARDI Giorgio
Published: March 2005

The dG+dC‐rich fractions obtained by density gradient centrifugation of bovine DNA in Cs2SO4/BAMD [J. Cortadas, G. Macaya & G. Bernardi (1977) Eur. J. Biochem. 76, 13–19] were centrifuged in Cs2SO4/Ag+ density gradients. These experiments led to the preparation of the DNA components which had been detected (by analytical centrifugation in CsCl) in the Cs2SO4/BAMD fractions, and also of DNA components which had identical behaviors in Cs2SO4/BAMD gradients and identical buoyant densities in CsCl. A total of eight satellite components and 11 minor components, accounting for 23% and 4% of the bovine genome, respectively, were thus isolated and characterized in their relative amounts and buoyant densities. The implications of these results on the interpretation of renaturation kinetic data on the bovine genome are discussed.
β‐Lapachone, an Inhibitor of Oncornavirus Reverse Transcriptase and Eukaryotic DNA Polymerase‐α
Authors: SCHUERCH Alfred R. , WEHRLI Walter
Published: March 2005

β‐Lapachone is a naturally occurring compound that can be isolated from a number of tropical trees. It is shown to be a potent inhibitor of reverse transcriptase activity from both avian myeloblastosis virus and Rauscher murine leukaemia virus. In addition, it affects eukaryotic DNA‐dependent DNA polymerase‐α activity; 50% inhibition is reached in 60‐min incubation time by about 8 μM β‐lapachone. Enzyme activity is inhibited irrespective of the purity of the enzyme used or of the amount or type of template/primer or substrate present. The inhibitory effect of the drug is only observed in the presence of dithiothreitol. The primary site of action of β‐lapachone appears to be the enzyme protein, as is also borne out by the specificity of its action. Eukaryotic DNA‐dependent DNA polymerase‐β, prokaryotic DNA‐dependent DNA polymerase I, several other nucleic acid polymerases and some completely unrelated enzymes are not affected. Reverse transcriptase and DNA‐dependent DNA polymerase‐α may be in someway related in possessing similarly exposed ‘– SH structures’ in their active sites. β‐Lapachone thus affords a novel means of studying such interrelationships and of further characterizing enzymes.
The Linear Electric Field Effect in Stellacyanin, Azurin and in Some Simple Model Compounds
Authors: PEISACH Jack , MIMS William B.
Published: March 2005

All mononuclear Cu(II) sites in frozen solution are non‐centrosymmetric and, unless physically constrained, will have a tetrahedral distortion away from the usual square planar structure often presented for Cu(II) complexes. Blue copper sites such as are found in azurin and stellacyanin have a greater distortion towards a tetrahedral geometry than do simple Cu(II) complexes. The distortion is comparable to that which is observed for Cu(II)‐o‐phenanthroline dichloride, a known tetrahedral complex. Blue copper sites posses as axis of asymmetry directed away from g¶ which could arise from a metal‐sulfur interaction.
N‐Acetylglutamate Synthetase from Rat‐Liver Mitochondria
Authors: SHIGESADA Katsuya , TATIBANA Masamiti
Published: March 2005

N‐Acetyl‐l‐glutamate synthetase was purified approximately 300‐fold from the extract of rat liver mitochondria. The enzyme shows a high substrate specificity for l‐glutamate and acetyl‐CoA. No or very low activity is observed with other amino acids and acyl compounds. The reaction velocity fits normal Michaelis‐Menten kinetics with respect to both l‐glutamate (Km, 3.0 mM) and acetyl‐CoA (Km, 0.7 mM).

Acetylglutamate, the reaction product, and some of its structural analogues, such as N‐acetylaminoadipate, N‐propionylglutamate, and N‐acetylglutamine, significantly inhibited the enzyme reaction. The Ki for acetylglutamate was 0.07 mM. The enzyme activity is sensitive to thiol‐blocking agents and is inhibited by divalent cations.

The enzyme activity is markedly stimulated by arginine, presumably by an allosteric mechanism. Other intermediates of the urea cycle and various structural analogues of arginine were practically not effective. Arginine increases the maximal velocity with no influence on Km values for the substrates. The activation shows a sigmoidal dependence on arginine concentrations and reaches a half‐maximal level at 5–10 μM. The enzyme behaves as a mixture of multiple forms upon gel filtration (the range of molecular weight, 30000 to 300000) and its mean value is considerably increased in the presence of arginine.

The specific activity of the enzyme increases with higher enzyme concentrations in the assay. The effect is more marked in the presence of arginine than in its absence. These observations suggest that the enzyme undergoes association and dissociation with concomitant changes in catalytic properties.
A Study of Unwinding of DNA and Shielding of the DNA Grooves by RNA Polymerase by Using Methylation with Dimethylsulphate
Authors: MELNIKOVA Anna F. , BEABEALASHVILLI Robert , MIRZABEKOV Andrei D.
Published: March 2005

The dimethylsulphate method has been used to study the complexes of RNA polymerase (Escherichia coli) with DNA of T7 phage, poly[d(A–T)] and fragments of calf thymus DNA protected against DNase digestion by RNA polymerase. The binding of RNA polymerase to DNA significantly increases the formation of 1‐methyl‐adenine produced by methylation of the single‐stranded DNA region, diminishes by about 10% the formation of 3‐methyl‐adenine by methylation within the minor groove and does not affect the formation of 7‐methyl‐guanine by methylation within the major DNA groove. The presence of nascent RNA decreases the formation of 1‐methyl‐adenine in DNA of the complex by about 30%. The initiation of RNA synthesis or RNA synthesis itself does not influence the methylation of the major groove but shielding of the minor groove increases by about twice as much.

These results suggest that RNA polymerase, upon binding, breaks Watson‐Crick base‐pairing in a DNA region of about 15‐base‐pairs long, that nascent RNA forms a duplex with DNA of about 10‐base‐pairs long; and that the enzyme weakly interacts with DNA along its grooves and preferentially makes contacts with the minor groove.
Interaction of the Acetylcholine (Nicotinic) Receptor Protein from Torpedo marmorata Electric Organ with Monolayers of Pure Lipids
Authors: POPOT Jean‐Luc , DEMEL Rudy A. , SOBEL André , VAN DEENEN Laurens L. M. , CHANGEUX Jean‐Pierre
Published: March 2005

Membrane fragments rich in cholinergic (nicotinic) receptor protein were purified from the electric organ of Torpedo marmorata. Their lipid composition is essentially characterized by the prominence of cholesterol, phosphatidylethanolamine and phosphatidylcholine, long‐chain fatty acyl constituents, and the absence of sphingomyelin. Solubilised receptor was purified from these fragments and the concentrations of sodium cholate lowered by dialysis to 0.01% (w/v). When this preparation was injected under a lipid monolayer, an increase of surface pressure developed, which was not observed with the detergent alone nor in the absence of lipid film. When covalently radiolabelled receptor preparations were injected at a constant surface pressure the radioactivity recovered with the film was proportional to the increase in area. It is concluded that the pressure or area increases are due to the penetration of the cholinergic receptor protein into the lipid film.

Incorporation experiments into films formed from various pure lipids showed that the protein interacts more readily with cholesterol than with ergosterol, phosphatidylcholine, or other phospholipids. Its affinity is also higher for long‐chain phosphatidylcholines than for short‐chain ones. The degree of unsaturation and fluidity of the 3‐sn‐phosphatidylcholine (lecithin) films are of secondary importance. Parallel experiments with covalently and non‐covalently labelled receptor preparations showed that part of the protein recovered with the film lost its α‐toxin binding ability during the penetration.

Similar data were obtained with the receptor purified from Electrophorus electricus electric organ.
Phosphoglycerate Kinase B from Ram Testis
Authors: STEWART Andrew A. , SCOPES Robert K.
Published: March 2005

The testis‐specific isoenzyme of phosphoglycerate kinase (phosphoglycerate kinase B) has been isolated from ram testes using a procedure which separates it from ‘normal’ phosphoglycerate kinase which is also present in testis tissue. The purification procedure is described.

The best preparations had no detectable impurity on electrophoresis, and had specific activities comparable with the same enzyme from other sources.

Kinetic studies indicated that the two isoenzymes have identical properties, within experimental error, for substrate affinity (for MgATP, 3‐phosphoglycerate and MgADP), energy of activation and thermal denaturation.

The molecular weights of both isoenzymes were not distinguishably different from those previously reported, as measured by polyacrylamide/dodecylsulphate electrophoresis. The amino acid compositions showed only slight differences, and tryptic peptide maps showed that there was close homology of sequence. Starch gel electrophoresis at pH 6.5 indicates that the B isoenzyme has 1–2 more positive charges than the A.

Phosphoglycerate kinase A isolated from sheep muscle was shown, within experimental error, to be identical to the phosphoglycerate kinase A isolated from testis.

The results further substantiate the suggestion that the B isoenzyme is coded by a gene which was duplicated from the phosphoglycerate kinase A gene.
Effect of Methylamine on Trypsin Catalysis
Authors: VAJDA Tamás , SZABÓ Tibor
Published: March 2005

The effect of the basic group of specific substrates of trypsin on the kinetics of the enzyme was studied. The basic group was substituted by methylamine added to a non‐specific substrate. The kinetic constants of tryptic hydrolysis of N‐acetyl‐l‐alanine ethyl ester involving methylamine, at pH 6.3–8.5 were determined. For comparison, the constants of trypsin and α‐chymotrypsin were also determined.

It can be concluded that methylamine increases the rate of hydrolysis catalyzed by trypsin. The relative increase of the pH‐independent limiting value of (kc/Km)max is shown to be fivefold and the pKa decreases by 0.4 unit. The increase of kc is nearly fourfold. The rate constants of the hydrolysis steps catalyzed by trypsin and trypsin · methylamine, respectively, were assayed by means of the nucleophile 1,4‐butanediol at pH 6.6. There is a sixfold increase of the original rates caused by methylamine for acylation (k2) and a threefold increase for deacylation (k3) but the substrate dissociation constant (Ks) was almost unchanged.

A comparison of the individual rates of the methylamine‐activated tryptic hydrolysis of nonspecific substrates with the appropriate data of the trypsin‐catalyzed hydrolysis of specific substrates indicates that there is an approximately 1000‐fold difference between the rate constants for the acylation step, while in deacylation the difference is insignificant.

The methylammonium ion bound to trypsin is supposed to keep the enzyme through the catalysis in a more active conformation than the original one. This indirect effect might bear only a small part of the trypsin‐substrate interaction.
Particulate Tyrosinase of Human Malignant Melanoma
Authors: NISHIOKA Kenji
Published: March 2005

Studies of mammalian tyrosinase have been conducted principally with soluble forms (T1 faster, T2 slower migrating by gel electrophoresis). However, a majority of tyrosinase from mouse and human melanoma tissue is of a particulate (T3) nature, being bound to melanosomes. In addition, T1 and T2 enzymes have been considered isozymes with T3 being a separate enzyme.

Using human malignant melanoma tissue, solubilization and purification of particulate tyrosinase have been attempted. Melanosome‐bound tyrosinase was solubilized with sodium cholate and purified 1500‐fold by ammonium sulfate fractionation, DEAE‐cellulose column chromatography, concanavalin A affinity chromatography, and Sephadex G‐150 gel filtration. Tryptic cleavage of this solubilized tyrosinase yielded a fast migrating tyrosinase by gel electrophoresis. This trypsin‐cleaved tyrosinase was purified by Sephadex G‐150 and DEAE‐cellulose columns with an apparent final yield of 56% and an approximate 10000‐fold purification. This preparation gave a single band by 7% gel electrophoresis and 5% sodium dodecyl sulfate gel electrophoresis. In addition, electrophoresis indicated that this tyrosinase was a glycoprotein showing positive staining by periodic acid/Schiff stain. This enzyme oxidized both l‐tyrosine and l‐3,4‐dihydroxyphenylalanine at a similar rate. The molecular weight was approximated at 66 700 by sodium dodecyl sulfate gel electrophoresis. This enzyme migrated to the T1 position by gel electrophoresis; however, upon treatment with neuraminidase, it shifted to the T2 position. Sialic acid content was determined as 4.8%. Copper content was estimated as 2 atoms/molecule. These results tend to support the dynamic conversion of T3→ T1→ T2 rather than their existence as separate enzymes.
The Electrochemical Proton Gradient Génerated by Light in Membrane Vesicles and Chromatophores from Rhodopseudomonas sphaeroides
Authors: MICHELS Paul A. M. , KONINGS Wil N.
Published: March 2005

The electrochemical proton gradient, , generated upon illumination of membrane vesicles and chromatophores from Rhodopseudomonas sphaeroides. has been determined. The components, the transmembrane pH gradient, ΔpH, and the membrane potential, ΔΨ, were measured with several methods. The ΔpH, determined from the pH changes in the membrane suspensions, is in good agreement with the ΔpH obtained from the distribution of acetate or methylamine. The ΔpH values obtained from the fluorescence quenching of 9‐aminoacridine in chromatophores are more than two‐fold larger. The ΔΨ was determined from the distribution of the permeant ions triphenylmethylphosphonium and thiocyanate. The ΔΨ values in chromatophores obtained from the absorbance changes of carotenoids were several‐fold larger.

Illumination of membrane vesicles results in a ΔpH, inside alkaline and a ΔΨ inside negative. The ΔpH, determined from the distribution of acetate, depends on the composition of the medium and is optimally 0.6 at an external pH 7; the ΔΨ, measured from the distribution of triphenyl‐methylphosphonium, is about 70 mV. The in membrane vesicles is thus at the most 110 mV.

Illumination of chromatophores results in a ΔpH, inside acid, and a ΔΨ inside positive. The ΔpH, calculated from the distribution of methylamine, depends on the external pH and increases from 0.98 at pH 6 to 1.36 at pH 8. The ΔΨ is not affected and remains about 50 mV. The in chromatophores is thus at the most 140 mV.

The ΔpH and ΔΨ are in both membrane preparations strongly dependent on the composition of the medium. So is for instance the ΔΨ decreased by the presence of chloride ions and the ΔpH increased. Phosphate ions decrease the ΔpH in membrane vesicles. The ΔpH is generated at a lower rate than the ΔΨ; once the has reached a maximum the ΔpH continues to increase at the expense of the ΔΨ.
Steady‐State Kinetic Studies on Adenylate Cyclase from Brevibacterium liquefaciens
Authors: ÖZER Inci , SCHEIT Karl Heinz
Published: March 2005

Kinetic data of basal adenyiate cyclase activity were found to be in agreement with a negatively cooperative model for this enzyme. The model assumes a dimeric structure for the enzyme with two binding sites per dimer. The binding of the first substrate molecule increases the dissociation constant for the substrate molecules increases the dissociation constant for substrate molecules at the second site. However, the doubly occupied enzyme has a greater catalytic efficiency per site than the singly occupied species. The effect of various nucleoside 5′‐triphosphates on the basal enzymatic activity was investigated. At low concentrations of substrate and analogue relative to Ks and Ki, compounds closely related to ATP, like 3′‐deoxy‐adenosine 5′‐triphosphate, 2‐amino‐adenosine 5′‐triphosphate and 3′‐deoxy‐3′‐amino‐adenosine 5′‐triphosphate activate adenylate cyclase. An increase in the concentration of either substrate or analogue reverses the activation effect. GTP, UTP, CTP and 2‐aminopurine‐riboside 5′‐triphosphate are purely competitive inhibitors of basal adenylate cyclase activity at low substrate concentrations. This behaviour can be explained by the assumption that coexistence of substrate and inhibitor fails to induce the increase in catalytic efficiency. Kinetic analysis of the activation of adenylate cyclase by pyruvate suggested that the primary effect of the activator is to abolish the negative cooperativity between subunits, at the same time inducing a better fit between the enzymes and its substrate. A tentative model, which is in reasonable agreement with the kinetic data is presented. Mixed inhibition of pyruvate‐stimulated adenylate cyclase was observed with GTP. Kinetic analysis revealed that this was the result of normal competitive inhibition of ATP binding, on which an increase of the apparent affinity of the enzyme for pyruvate was superimposed.
Isolation of Soluble Proteins Capable of Stimulating Aerobic Plasmalogen Biosynthesis
Authors: PALTAUF Fritz
Published: March 2005

The terminal step during aerobic plasmalogen biosynthesis is catalyzed by a microsomal desaturase system which converts 1‐O‐alkyl‐2‐acyl‐sn‐glycerophosphoethanolamine to 1‐O‐alk‐1′‐enyl‐2‐acyl‐sn‐glycerophosphoethanolamine (ethanolamine plasmalogen). The reaction depends on oxygen and NAD(P)H and is stimulated 3–10‐fold by soluble activating factors contained in the 100 000 X g supernatant. Two stimulating proteins have been isolated from pig kidney; the partially purified proteins have identical molecular weights (27000) but differ in their respective isoelectric points (protein 1, 5.1 and protein 11, 4.9). Both proteins behave identically in the biochemical studies conducted. Exogenous substrate binds to the stimulating proteins; the transfer of ethanolamine, but not of choline phospholipids, from liposomes to microsomes is enhanced by the stimulating proteins. They stimulate plasmalogen synthesis from either exogenous or endogenous substrate (synthesized from alkylglycerophosphoethanolamine by microsomal transacylases).

The stimulating proteins have no enzymatic activity themselves; it is suggested that they affect events within the membrane and function as specific mediators between the membrane‐bound enzyme system and the lipophilic substrate.
Ferredoxins from Nitrogen‐Fixing Bacteria
Authors: YATES M. Geoffrey , O'DONNELL Michael J. , LOWE David J. , BOTHE Hermann
Published: March 2005

Two ferredoxins have been purified from N2‐fixing Mycobacterium flavum. Ferredoxin I had a minimum molecular weight (by amino acid analysis) of 11230 and contained 7.4 mol Fe and 7.0 mol S2− per mol. The ratio of the absorbance at 400 nm to that at 275 nm was 0.7. Ferredoxin II had a minimum molecular weight of 13478 and contained 3.4 mol Fe and 3.2 mol S2− per mol. The ratio of the absorbance at 400 nm to 268 nm was 0.42.

Both ferredoxins were paramagnetic when oxidised by potassium ferricyanide and diamagnetic when reduced by sodium dithionite. This suggests that, like Azotobacter ferredoxin, they operate solely between the – 2 and – 1 oxidation states. A ferredoxin fraction of similar properties was isolated from Spirillum lipoferum. The electron paramagnetic resonance (EPR) spectra of both ferredoxins had features at g= 2.023, 2.017 and 2.00. The signals were affected by pH: below pH 6.0 that at g= 2.023 predominated, whereas above pH 6.0 the g= 2.017 features was clearly present. The g= 2.00 features was broader between pH 6.0 and 9.0 than outside this pH range. The change to the low‐pH form was not reversible.

Ferredoxin I had redox centres with mid‐point potentials at – 420 mV and + 230 mV; ferredoxin II had one redox centre of mid‐point potential at approximately – 420 mV. Above + 300 mV ferredoxin I suffered irreversible oxidative damage and lost the g= 2.023 feature of the EPR spectrum. Both ferredoxins were damaged by prolonged exposure to potassium ferricyanide. Ferredoxin I took up 1.8 electrons per molecule when reduced by sodium dithionite but the EPR signal of the oxidised form integrated to only 0.35 electron per molecule. Ferredoxin II took up 0.9 electron per molecule on reduction and the EPR signal of the oxidised form integrated to 0.66 electron per molecule.

It was concluded that M. flavum ferredoxin I resembles Azotobacter ferredoxin and is thus the second ferredoxin isolated that has two redox centres with mid‐point potentials approximately 700 mV apart. Ferredoxin II has no exact counterpart in other systems. Aerobic nitrogen‐fixing bacteria apparently contain ferredoxins which operate between the – 2 and – 1 oxidation states.
Study of the Sulfhydryl Groups of β‐d‐Xylosidase from Bacillus pumilus
Authors: SAMAN Eric , CLAEYSSENS Marc , DE BRUYNE Clement K.
Published: March 2005

β‐d‐Xylosidase from Bacillus pumilus contains one intramolecular disulfide bridge and four cysteinyl residues per monomer (Mr 60000). Under denaturing conditions two of these ‐ SH residues react with 5.5′‐dithiobis(2‐nitrobenzoic acid) (Nbs2) with concomitant formation of a second cystine.

Under native conditions one cysteinyl group per polypeptide chain reacts with Nbs2 or p‐chloromercuribenzoate (ClHgBzOH) and this results in complete loss of enzymic activity. This inactivation can completely be reversed by reductive treatment (dithiothreitol) of the β‐d‐xylosidase.

Kinetic analysis of the inactivation reaction points to a saturation effect, occurring when the concentration of ‐ SH reagent is increased. A reaction model, which includes prior association of the regent with the enzyme, is proposed.

Competitive inhibitors protect against inactivation of the enzyme with Nbs2. These experiments prove that two classes of ligands, with two different binding modes, exist.

Inactivation with Nbs2 and iodoacetamide as a function of pH reveals the presence of two ionizing groups on the enzyme; their pK values (6.0 and 9.0 respectively) are consistent with those of a protein‐imidazol and protein‐cysteine group.

Cumulative evidences obtained with other ‐ SH reagents, such as 5,5′‐dithio bis(2‐nitro‐N‐trimethylbenzylammonium iodide) and iodoacetate, indicate a negatively charged reaction site. Identity with the active site or direct participation of a histidine and/or a cysteinyl group in the enzymic proces can only be suggested.
The Binding Site of Ribosomal Protein L1 from Escherichia coli on the 23‐S Ribosomal RNA from Bacillus stearothermophilus
Authors: STANLEY John , SLOOF Paul , EBEL Jean‐Pierre
Published: March 2005

The region of Bacillus stearothermophilus strain NCA 1503 23‐S ribosomal RNA protected from T1 ribonuclease digestion by the 50‐S ribosomal subunit protein L1 from Escherichia coli has been established. The sequence of 115 nucleotides is compared to the analogous region in E. coli. The similar behaviour of the RNA towards the recognition of protein L1 may be explained in terms of secondary base‐pairing, even though there exists almost 40% difference between the primary nucleotide sequences.
Anion Binding to Yeast Phosphoglycerate Kinase
Authors: WROBEL Jean A. , STINSON Robert A.
Published: March 2005

The single thiol of yeast phosphoglycerate kinase was labelled with the chromophoric sulfhydryl reagent, 2‐chloromercuri‐4‐nitrophenol. Sequential additions of individual anions to this modified enzyme brought about a decrease in absorbance at 410 nm that reflected the degree of saturation of the enzyme with anion. The binding curves were analyzed to determine the dissociation constants of a number of anions with charges varying from – 1 to – 4.1. A linear relationship was found between the charge of the anion and the negative logarithm of the dissociation constant for the labelled enzyme‐anion complex. The highly charged anions, such as ATP, bound more tightly than did anions with less charge, such as Cl−. The average number of binding sites for those anions for which accurate results could be obtained was 1.06 mol per 47000g of enzyme. Several lines of evidence suggested that titration of the active center was not being monitored.

Anions bound to phosphoglycerate kinase decreased the rate of reaction between the enzyme thiol and 5,5′‐dithiobis(2‐nitrobenzoic acid). The relationship between the degree of saturation of the anion binding site and the reaction rate constant was used to calculate the dissociation constant between anion and enzyme. Dissociation constants determined in this manner were in good agreement with those determined by titration of the enzyme‐mercurial complex.
Cerulenin‐Induced Changes in the Lipopolysaccharide Content and Phospholipid Composition of Proteus mirabilis
Authors: ROTTEM Shlomo , MARKOWITZ Ora , RAZIN Shmuel
Published: March 2005

Inhibition of Proteus mirabilis growth by cerulenin, a specific inhibitor of fatty acid biosynthesis, was reversed by exogenously supplied fatty acid mixtures containing oleic acid and palmitic or pentadecanoic acids. The growth rate of the cells treated with cerulenin in the presence of the fatty acid mixtures was slower, however, than that of untreated cells, and their Iipopolysaccharide content was decreased by 30–50%, resulting in an increased sensitivity of the organisms to rifamycin and vancomycin. Polyacrylamide gel electrophoresis of the lipopolysaccharide fraction from cerulenin‐treated cells revealed that of the two P. mirabilis lipopolysaccharide types, the relative amount of the higher molecular weight lipopolysaccharide was reduced from 50% to 30% of the total lipopolysaccharide. Fatty acid analysis of the phospholipid and lipopolysaccharide fractions from cells grown with cerulenin, pentadecanoate, and oleate revealed that over 60% of the native even‐numbered fatty acids of the phospholipid fraction was substituted by the odd‐numbered fatty acid, while no incorporation of either the pentadecanoate or oleate could be demonstrated in the lipid A moiety of the lipopolysaccharide. The only change in the lipid A observed was an increase in the content of 3‐hydroxymyristic acid accompanied by a decrease in the nonhydroxylated fatty acids, supporting the highly conserved nature of this molecule.
Translation of Encephalomyocarditis Virus RNA in vitro Yields an Active Proteolytic Processing Enzyme
Authors: PELHAM Hugh R. B.
Published: March 2005

In contrast to other cell‐free translation systems, the mRNA‐dependent reticulocyte lysate can translate encephalomyocarditis virus RNA efficiently and completely when supplemented with heterologous tRNA. Cleavage of the nascent polypeptide chain occurs, and one of the translation products appears to be a specific proteolytic enzyme which correctly processes the primary products. The identity of the proteins made in vitro was verified by comparison with infected cell proteins on dodecylsulphate/polyacrylamide gels, and by mapping their coding sequences on the viral genome.
Nucleotide Torsional Flexibility in Solution and the Use of the Lanthanides as Nuclear‐Magnetic‐Resonance Conformational Probes
Authors: GERALDES Carlos F. G. C. , WILLIAMS Robert J. P.
Published: March 2005

In this article the single family of conformations of adenosine 5′‐monophosphate (5′AMP) in aqueous solution previously obtained using the lanthanide ions as nuclear magnetic resonance (NMR) probes is shown to disagree with the conformational data available from independent NMR methods, such as coupling constants and nuclear Overhauser enhancement analysis. The original lanthanide experimental data is reinterpreted assuming the contribution of different conformers to the observed magnetic perturbations. The average conformation obtained for 5′ AMP agrees quite satisfactorily with the other independent conformational information.
Conformational Studies of Some 2′:3′‐Cyclic Mononucleotides in Solution by Different Nuclear‐Magnetic‐Resonance Methods
Authors: GERALDES Carlos F. G. C. , WILLIAMS Robert J. P.
Published: March 2005

The conformations of the 2′:3′‐cyclic mononucleotides of adenosine and cytidine in deuterium oxide has been studied at pH 2.3, using lanthanide ions as paramagnetic nuclear magnetic resonance (NMR) probes. It was not possible to find any single conformation for these molecules which accounts for the observed shift and relaxation data. This situation is in agreement with the interpretation of vicinal 1H‐1H and 1H‐31P coupling constants, which indicate that the ribofuranose and cyclic phosphate rings are in rapid equilibrium between different puckered forms. The interpretation of the lanthanide data in terms of an equilibrium between different conformations give average rotamer populations in good agreement with the coupling constant analysis. The conformations of these systems in aqueous solutions were found to be more flexible than in the solid state, where rigid planar ribofuranose rings have been observed. Adenosine 2′:3′‐monophosphate differs from cytidine 2′:3′‐monophosphate at the glycosidic link.
Studies on the Control of Development
Authors: RHAESE Hans‐Jürgen , GROSCURTH Reinhard
Published: March 2005

Ribosomes from vegetative cells (vegetative ribosomes) of Bacillus subtilis synthesize guanosine 3′(2′)‐diphosphate 5′‐diphosphate (p2Gp2) and guanosine 3′(2′)‐diphosphate 5′‐triphosphate (p3Gp2). In contrast, ribosomes isolated from sporulating cells (sporulation ribosomes) synthesize adenosine 3′(2′)‐diphosphate 5′‐diphosphate (p2Ap2) and adenosine 3′(2′)‐diphosphate 5′‐triphosphate (p3Ap2). The products obtained with the aid of vegetative ribosomes were characterized by comparison with authentic substances in two chromatographic systems. The reaction mechanism is identical with that found in Escherichia coli, since [3H]GTP or GDP is pyrophosphorylated by ATP to form p2Gp2 and p3Gp2. Omission of ATP completely stops the formation of any reaction products. Furthermore, no reaction products are observed when GTP or GDP are omitted in a reaction mixture containing vegetative ribosomes. In contrast, incubation of sporulation ribosomes with ATP alone or ADP and ATP results in the formation of two products identified as p2Ap2 and p3Ap2 by their mechanism of synthesis as well as chemical and enzymatic degradation. Sporulation ribosomes (isolated at least an hour after the end of logarithmic growth) synthesize drastically less (by a factor of 100) p2Gp2 and p3Gp2. Vegetative ribosomes, on the other hand, synthesize only 1/100 the amount of p2Ap2 and p3Ap2 compared to sporulation ribosomes when isolated at least an hour before the end of logarithmic growth. By using washed ribosomes and ribosomal wash from vegetative and sporulation ribosomes, we were able to show that changes in the composition of ribosomes (probably in ribosomal proteins and/or ribosome‐associated factors) must occur during the transition from vegetative to sporulating cells. Washed ribosomes from vegetative cells cannot replace washed ribosomes from sporulating cells to synthesize p2Ap2 and p3Ap2 in the presence of ribosomal wash from sporulating cells and vice versa. During the transition from one type of ribosomes to the other, as far as the unusual nucleotide‐synthesizing activity is concerned, both types of nucleotides can be synthesized. While the ability to synthesize p2Gp2 and p3Gp2 decreases approximately at the end of logarithmic growth, the ability to synthesize p2Ap2 and p3Ap2 increases at the same time. The reason behind this obvious change in ribosomal structure and associated factors is discussed in the light of our finding that these changes are not observed in some early blocked asporogenous mutants.

The observation that ribosomes from sporulating cells synthesize traces of p2Gp2 and p3Gp2 (by a factor of almost 100 less than vegetative ribosomes) is probably due to the presence of some non‐sporulating cells in any B. subtilis culture. Likewise, there are always some spores contained in a vegetatively growing culture, which are probably responsible for the observed small amounts of p2Ap2 and p3Ap2 synthesized.
The Plastid Membranes of Barley (Hordeum vulgare)
Authors: APEL Klaus , KLOPPSTECH Klaus
Published: March 2005

Illumination of dark‐grown barley plants induces a massive insertion of the light‐harvesting chlorophyll a/b protein into the developing thylakoid membrane. In addition to the onset of chlorophyll synthesis, light induces specifically the appearance of a prominent mRNA species which codes for a polypeptide of Mr 29500. This component was identified as a precursor of the apoprotein of the light‐harvesting chlorophyll a/b protein. The precursor has an Mr larger than the authentic protein by approximately 4000. Studies of the chlorophyll‐b‐less mutant chlorina f2 of barley offer the first clue to the mechanism which controls the light‐dependent mRNA formation. The induction of the mRNA coding for the apoprotein of the light‐harvesting chlorophyll a/b protein does not seem to be linked directly to the assembly process of the light‐harvesting structure and does not require chlorophyll b. It is proposed that light exerts its influence on the mRNA formation by a reaction which is different from the phototransformation of protochlorophyll(ide) to chlorophyll(ide).
An Adenosine 3′:5′‐Monophosphate/Adenosine Binding Protein from Mouse Liver
Authors: UELAND Per Magne
Published: March 2005

Synthetic and naturally occurring adenosine derivatives were tested for their ability to inhibit the binding of cyclic [3H]AMP and [3H]adenosine to the activated cyclic AMP/adenosine binding protein from mouse liver [Ueland, P. M. and Døskeland, S. O. (1977) J. Biol. Chem. 252, 677–689]. The structural requirements for inhibition of the binding of the two ligands were different, as demonstrated by the existence of adenosine derivatives that inhibited the binding of [3H]adenosine but not the binding of cyclic [3H]AMP and vice versa. The interaction of the binding protein with AMP, ADP and adenine was investigated in more detail. The binding of radioactive AMP and ADP to the protein was determined under equilibrium conditions. Competition studies indicate that AMP binds to the cyclic AMP binding site with an apparent dissociation constant of 2 × 10−6 M and to a low‐affinity site. The double‐reciprocal plot for the binding of ADP deviated slightly from linearity suggesting the possibility of heterogeneity also for the ADP binding sites. Competition studies indicate that this nucleotide binds mainly to the cyclic AMP binding site.

Cyclic AMP has previously been shown to activate its own sites under certain conditions (30°C, in the presence of KCl and Mg2+). This phenomenon has been termed homologous activation of binding sites [Ueland, P. M. and Døskeland, S. O. (1978) J. Biol. Chem., in the press]. Similar results were obtained with AMP and ADP.

Displacement of [3H]adenosine binding from the sites specific for this adenine derivative was observed with unlabelled adenosine, adenine, AMP and ADP with decreasing efficiency in the order mentioned. This indicates that adenine, AMP and possibly ADP interact with the adenosine binding sites. Adenine and AMP inhibited the activation of the cyclic AMP binding capacity by ATP as previously shown for adenosine [Ueland, P. M. and Døskeland, S. O. (1978) Arch. Biochem. Biophys. 185, 195–203]. The inhibitory power closely parallelled the displacement of [3H]adenosine binding. Adenine was also demonstrated to inhibit the homologous activation of the cyclic AMP binding site.