bluuebunny/crossref_metadata_2025 huggingface dataset:
Spectroscopic Studies on Effector‐Induced and Substrate‐Induced Conformation Changes of Phosphofructokinase
Authors: GROSSE Richard , ECKERT Klaus , OTTO Matthias , JACOBASCH Gisela , REPKE Kurt R. H.
Published: March 2005

The interaction of phosphofructokinase with NH4+, AMP, ATP, citrate, MgATP or fructose 6‐phosphate, and in part with their mixtures forming either binary or ternary complexes has been studied by means of ultraviolet difference spectroscopy and circular dichroism spectroscopy in the wavelength range 265 – 300 nm with the aim of characterizing the conformational corollaries of the ligand effects on phosphofructokinase.

The positive as well as the negative effectors change phosphofructokinase conformation in different ways, not easily interpretable in terms of one active and one inactive enzyme conformation. The spectroscopic equivalents of phosphofructokinase conformation changes resulting from catalytic activity are similar to those produced by the reaction products. The ligand concentration‐dependent changes of absorption differences in the tryptophyl, tyrosyl and phenylalanyl region parallel each other, i.e. the interactions of the ligands with phosphofructokinase are not confined to specific aromatic side chains, but involve conformation changes of the large domains of the protein. ATP affinity to the enzyme shows temperature‐dependent biphasic changes so that ATP binding appears to be either an entropy‐driven or enthalpy‐driven process. The dissociation constants of the ligands derived from spectroscopic titration of binary complex formation are comparable to those calculated from kinetic experiments. MgATP and fructose 6‐phosphate each alone change phosphofructokinase conformation by binary complex formation in keeping with a random order of reaction sequence.
Isolation of Mutants Conditionally Blocked in the Biosynthesis of the 3‐Deoxy‐d‐manno‐octulosonic‐acid—Lipid‐A Part of Lipopolysaccharides Derived from Salmonella typhimurium
Authors: LEHMANN Volker , RUPPRECHT Eckhard , OSBORN Mary Jane
Published: March 2005

A procedure is described for the selection of conditional 3‐deoxy‐d‐manno‐octulosonic‐acid—Lipid A mutants which depends on temperature sensitivity for both synthesis of complete lipopolysaccharide and for growth. Using this procedure new types of mutants were isolated which cease growth and accumulate lipid A precursors following a shift to nonpermissive temperatures. All precursor molecules differ in their charge as judged by DEAE‐cellulose chromatography. While they all contain glucosamine, phosphate and 3‐hydroxymyristic acid, they lack detectable 3‐deoxy‐d‐manno‐octulosonic acid (dOclA) as well as the nonhydroxylated fatty acids of the complete lipid A structure.

Three mutants proved to be conditionally defective in dOclA metabolism, whereas one seems to be blocked at a relatively early step in lipid A synthesis. The phenotypes of all these mutants appear to be due to single mutations by reversion analysis and by characterization of the temperature‐resistant revertants.

Studies of these mutants may shed light on the essential role of the complete dOclA—lipid A part of lipopolysaccharides in membrane function.
The Purification and Properties of 4‐Hydroxyisophthalate Hydroxylase from Pseudomonas putida NCIB 9866
Authors: ELMORSI Elmorsi A. , HOPPER David J.
Published: March 2005

4‐Hydroxyisophthalate hydroxylase has been purified from cells grown with 2,4‐xylenol and gave a single protein band on polyacrylamide‐gel electrophoresis after a 24‐fold purification.

In the reaction, 4‐hydroxyisophthalate was converted to protocatechuate by an oxidative decarboxylation and the reaction stoichiometry is typical of a monooxygenase with an external electron donor.

The enzyme can function with either NADH (Km 105 μM) or NADPH (Km 71 μM). Besides 4‐hydroxyisophthalate (Km 42 μM), 5‐sulphosalicylate was the only aromatic substrate found for the enzyme.

The enzyme has a molecular weight of about 103000, consisting of two identical polypeptide chains, and contains one mole of FAD per mole of protein. The FAD could not be replaced by FMN in reconstituting active enzyme from native apoenzyme.

The formation of an enzyme‐substrate complex was detected by the change in flavin spectrum when 4‐hydroxyisophthalate was added to the enzyme.

Rapid reduction of the enzyme‐bound FAD by NADPH only occurred in the presence of substrate. No stable flavin semiquinones were observed in the titration with NADPH or during anaerobic photoreduction in the presence of EDTA.

Although a number of substrate analogues inhibit the enzyme no non‐hydroxylatable effectors of the NADPH oxidation were found.

The enzyme was inhibited by sulphydryl‐binding reagents and not protected by substrate. Addition of sulphydryl compounds was essential for stabilization of the enzyme during purification.

Extracts of cells grown with 2,4‐xylenol and 4‐hydroxyisophthalate also contain a separate 4‐hydroxybenzoate hydroxylase.
On the Interrelationship of Prostaglandin Endoperoxide G2 and Cyclic Nucleotides in Platelet Function
Authors: CLAESSON Hans‐Erik , MALMSTEN Curt
Published: March 2005

The prostaglandin endoperoxide G2 caused rapid aggregation and release of ADP and [14C]serotonin in human platelets. Since the presence of the ADP phosphorylating system creatine phosphate creatine phosphokinase markedly inhibited the aggregation caused by the endoperoxide, this effect seemed to be mediated mainly by ADP, which is instantaneously released by the endoperoxide.

The prostaglandin G2 counteracted the increasing effect of prostaglandin E1 on the adenosine 3′:5′‐monophosphate (cAMP) levels in platelet‐rich plasma. This effect of prostaglandin G2 was only observed when ADP was released by the endoperoxide. This finding indicates that the effect of prostaglandin G2 on the cAMP levels in platelet‐rich plasma is principally mediated by ADP. The rapid release of ADP by prostaglandin G2 and the time courses for the effects of the endoperoxide and ADP on the level of cAMP give further evidence for this hypothesis. ADP also caused primary aggregation in the presence of indomethacin, and prostaglandin synthesis inhibitors did not influence the decreasing effect of ADP on the cAMP levels. N2,O2‐Dibutyrylguanosine 3′:5′‐monophosphate did not influence the aggregation and release‐reaction caused by ADP and no changes of the cGMP levels were observed after addition of prostaglandin G2.
Bacteriorhodopsin‐Mediated Photophosphorylation in Halobacterium halobium
Authors: HARTMANN Rainer , OESTERHELT Dieter
Published: March 2005

The rate of halobacterial photophosphorylation was found to be a linear function of light intensity over a wide range (between 1 and 20 mW/cm2). At higher light intensities (above 25 mW/cm2) the ATP‐synthesizing system itself limits the maximal rate of photophosphorylation. The optimal external pH range for this type of photophosphorylation is between pH 6.2 and 7.2 external. The photophosphorylation rate is directly proportional to the bacteriorhodopsin content of the cells. The quantum requirement for photophosphorylation was found to be 22 ± 5 photons per ATP molecule synthesized.

According to Mitchell's chemiosmotic hypothesis of energy coupling phosphorylation can be driven by a membrane potential or a pH gradient or a combination of both. From the results of experiments with drugs which abolish or reduce either one of the two components we conclude that the major driving force for photophosphorylation above an external pH value of 6.5 is the membrane potential, while at more acidic pH value the pH gradient becomes dominating.

We did not observe a correlation between a transient alkalinization of the medium and ATP‐synthesis upon illumination under certain conditions.
Amino Acid Control of Stable RNA Synthesis in Friend Leukemia Cells in Relation to Intracellular Purine Nucleoside Triphosphate Levels
Authors: DEHLINGER Peter J. , HAMILTON Thomas A. , LITT Michael
Published: March 2005

Histidinol is known to cause deacylation of histidyl‐tRNA in cultured mammalian cells, thereby producing a functional deprivation of histidine. Such deprivation of an essential amino acid is known to produce various effects, including inhibition of tRNA synthesis and of nucleolar RNA synthesis and processing. It has been proposed [Grummt, F. & Grummt, I. (1976) Eur. J. Biochem. 64, 307–312] that this response to amino acid deprivation is mediated by decreases in GTP and ATP pool sizes caused by a deacylated‐tRNA‐dependent hydrolysis of GTP. In contrast, we find that Friend leukemia cells treated with histidinol show no significant changes in GTP or ATP pool sizes, although this treatment does produce the expected inhibition of rRNA and tRNA synthesis.
Protein Components of Very Low Density Lipoproteins from Hen's Egg Yolk
Authors: BENGTSSON Gunilla , MARKLUND Sven‐Erik , OLIVECRONA Thomas
Published: March 2005

Egg yolk lipoproteins of very low density were found to contain proteins with cofactor activity for lipoprotein lipase. When delipidated very low density lipoproteins were dissolved in 10 mM HCl and fractionated by gel filtration about two thirds of the protein were in several components with estimated molecular weights of 60000 to more than 170000. The major low‐molecular‐weight proteins were the dimeric and monomeric forms of a previously characterized 9000‐dalton peptide. The cofactor activity was not associated with any of these major proteins.

A large‐scale fractionation method was developed by which two protein fractions with cofactor activity for lipoprotein lipase were purified more than thousand‐fold. One fraction had a molecular size of about 9000 daltons and the other had a size of about 5000 daltons. Both these fractions could be further separated on the basis of charge into several fractions with cofactor activity. The cofactor proteins were relatively soluble both at high and at low pH. They retained their cofactor activity after denaturation in guanidinium hydrochloride and after reduction. During the initial steps in the purification of the cofactor proteins another low‐molecular‐weight protein followed the cofactors. It had a single 17500‐dalton peptide chain and was present in four variants, three of which contained carbohydrate.
A Kinetic Study of Calcium Transport by Heart Mitochondria
Authors: NOACK Eike A. , HEINEN Edgar M.
Published: March 2005

Initial velocities of energy‐dependent Ca2+ uptake in cardiac mitochondria were measured with the ethyleneglycol bis(2‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid/ruthenium red quenching technique. Accurate concentrations of free Ca2+ between 1 and 15μM were generated with ATP as a Ca2+ buffer system. Using the method of iterative approximation, the accurate value of the initial velocity of ATP‐driven 45Ca uptake, and the time (t0.5) for half‐maximal uptake can be calculated.

The rate of initial Ca2+ uptake increased in response to the rising free Ca2+ concentrations in the medium (in the presence of 0.6–5.0 mM ATP). A maximal rate was obtained between 10 and 15 μM of free Ca2+. For different ATP concentrations (0.6–5.0 mM), the half‐maximal rate of Ca2+ transport (Km) was observed between 4.1 and 7.4 μM Ca2+ (with a mean Km of 5.6 ± 0.6 μ Ca2+; n= 5). Hill plots of the data yield straight lines with an average slope n, of 1.93 ± 0.29, indicating the existence of two binding sites for Ca2+ in the transmembrane transport system. Keeping the free Ca2+ concentration on a constant level (6 μM), the rate of initial Ca2+ uptake also increased continually with rising ATP concentrations (maximum velocity v= 7.1 nmol of Ca2+× mg protein−1× s−1).

From energy‐independent calcium‐ion binding studies with cardiac mitochondria, a dissociation constant (Kd) of 26.8 × 10−6M and a total number of external binding sites (n) of 12.6 nmol of Ca2+/mg of protein was obtained.

The data are discussed with regard to the physiological role which cardiac mitochondria may play as a reversible storage site during the contraction relaxation cycle of the heart.
On the Role of Tryptophan in Luteinizing‐Hormone‐Releasing Hormone (Luliberin)
Authors: HAZUM Eli , FRIDKIN Mati , MEIDAN Rina , KOCH Yitschak
Published: March 2005

The tryptophan residue of luteinizing‐hormone‐releasing hormone (luliberin) was chemically modified to produce the following analogs: [Trp(o)3]luliberin, Trp‐(2,4‐dinitrophenylsulfenyl)‐luliberin, Trp‐(2‐hydroxy‐5‐nitrobenzyl)luliberin, (Trp‐S‐luliberin)2, Trp‐CH3S‐luliberin and Trp‐formyl‐luliberin. The luteinizing‐hormone‐releasing activity of these analogs was determined by bioassay in vitro and found to be 0.2%, 0.2%, 0.6%, 1.5%, 1.7% and 7% of that of the natural hormone, respectively. These results demonstrate that alterations in the indole moiety of tryptophan‐3, which lead to a reduction in its electron density or sterically restrict its electron availability, are associated with a dramatic loss of biological activity.
Kinetics of Refolding of Completely Reduced Human‐Serum Albumin
Authors: WICHMAN Anders , SVENSON Anders , ANDERSSON Lars‐Olov
Published: March 2005

The kinetics of refolding of completely reduced human serum albumin has been studied by various methods including immunological techniques. The decrease in thiol content is very rapid in the beginning of the reoxidation process and rather slow in the later stages. Polyacrylamide gel electrophoresis studies show that, in the earlier stages of refolding, the main part of the albumin is present as various oligomers and that a slow conversion to monomer occurs as reoxidation proceeds.

Rocket immunoelectrophoresis shows that the completely reduced protein is devoid of native albumin antigenic determinants but that a rapid regain of immunoprecipitability is obtained upon reoxidation. A new ‘consumption’ rocket immunoelectrophoretic method has been used to estimate the total regain of antigenicity.

The data obtained indicate that there is a preferential rapid folding to native structure in certain parts of the molecule but that areas with wrong or incomplete foldings exist a considerable time after the initial refolding period.
Purification and Further Characterization of an RNA‐Dependent DNA Polymerase from the Allantoic Fluid of Leukosis‐Virus‐Free Chicken Eggs
Authors: BAUER Georg , JILEK Gabriele , HOFSCHNEIDER Peter Hans
Published: March 2005

The purification of an RNA‐dependent DNA polymerase from the allantoic fluid of uninfected. embryonated chicken eggs is described in detail. Comparison to the polymerase of avian myeloblastosis virus shows that the two enzymes are different with respect to ion concentrations for optimal reaction, response to increasing concentrations of substrate, thermal stability and protection from thermal inactivation by viral RNA. It is concluded that the enzymes compared to each other are different proteins, which must have been coded by different genes. The RNA‐dependent DNA polymerase in the allantoic fluid, therefore, does not derive from the partial or complete expression of the endogenous virus genome of the normal chicken cell or from infection by exogenous viruses.
The Effect of Cyclic Nucleotides on Cellular ATP Levels and Ribosomal RNA Synthesis in Ehrlich Ascites Cells
Authors: GRUMMT Friedrich , GRUMMT Ingrid
Published: March 2005

Amino acid starvation of Ehrlich ascites cells leads to a significant decrease of the intracellular ATP concentration concomitant with a marked decrease in nucleolar RNA polymerase activity. Addition of 8‐bromoguanosine 3′:5′‐monophosphate (br8cGMP) to the amino‐acid‐deficient culture medium increased the cellular ATP levels and restored the rRNA synthesis capacity of nucleoli to control levels. Exogenous br8cAMP overcame the effects of br8cGMP. Administration of br8cAMP to exponentially growing ascites cells resulted in a shrinkage of ATP levels and in an inhibition of nucleolar RNA synthesis similar to that observed under shift‐down conditions. These effects of br8cAMP could be antagonized by exogenous br8cGMP or hypoxanthine. Since the br8cGMP‐induced increase in the total adenine nucleotides was abolished in the presence of azaserine (an inhibitor of the amidation of formylglycineamide ribonucleotide) it is concluded that cyclic nucleotides exert at least a part of their regulatory effects on cell proliferation by regulating nucleotide biosynthesis de novo.
Synthesis by Avian‐Myeloblastosis‐Virus RNA‐Dependent DNA Polymerase of Discrete Reverse Transcripts of Bacteriophage RNA Polyadenylated in vitro
Authors: DEVOS René , VAN EMMELO John , CELENN Paul , GILLIS Elie , FIERS Walter
Published: March 2005

After polyadenylation in vitro, several viral RNAs become excellent templates for the synthesis of complementary DNA using RNA‐dependent DNA polymerase from avian myeloblastosis virus, in the presence of oligo(dT) as a primer. If dTTP was the only deoxynucleoside triphosphate present, the reverse transcriptase catalyzed a ‘slippage’ reaction on the poly(A) tail, especially if the dTTP concentration exceeds 1 μM. In the case of MS2 RNA‐poly(A), this poly(dT) synthesis could be specifically inhibited, but not completely abolished, by addition of dGTP. Lowering the dTTP concentration also decreased the average size of the poly(dT) products formed. In the presence of all four dNTPs, but using a low dTTP concentration to avoid slippage of the primer, discrete partial transcripts could be synthesized, complementary to the 3′‐terminal region of the viral RNA. Although the average size of the products increases at higher temperatures of incubation, the pattern of the labeled transcripts, as revealed by gel electrophoresis, was mainly dependent on the nature of the deoxynucleoside triphosphate present in limiting concentration. These results indicate that it is not the secondary structure of the template but its primary sequence that is the major determinant of the discrete partial transcripts. Restricted synthesis, using a combination of only three or less dNTPs. results in very small cDNA products. The successive incorporation of the different dNTPs added to the primer, using MS2 RNA‐poly(A) or Qβ RNA‐poly(A) as a template, was in complete agreement with the known 3′‐terminal sequence. Under the conditions used and with MS2 RNA‐poly(A) or poly(A) as template, a ribosyl primer could not be substituted for the deoxyribosyl primer. Similarly, with MS2 RNA‐oligo(C) only (dG)10 was an efficient primer for reverse transcription. Ribonucleoside triphosphates were very poor substrates.
Change in Location of Ornithine Carbamoyltransferase and Carbamoylphosphate Synthetase among Yeasts in Relation to the Arginase/Ornithine Carbamoyltransferase Regulatory Complex and the Energy Status of the Cells
Authors: URRESTARAZU L. Antonio , VISSERS Stephan , WIAME Jean‐Marie
Published: March 2005

Partial fractionation of Saccharomyces cerevisiae spheroplast lysate shows that arginase, ornithine carbamoyltransferase and carbamoylphosphate synthetase have the same distribution as the cytosol marker enzyme glucose‐6‐phosphate dehydrogenase. The same holds true in stepwise homo‐genization experiments of spheroplast lysates of S. cerevisiae.

This second method applied to Debaryomyces hansenii, Hansenula anomala and Schizosaccharomyces pombe shows, in contrast, that ornithine carbamoyltransferase is particulate in these yeasts. Its solubilization is in line with that of citrate synthase taken as mitochondrial matrix marker, whereas arginase acts like glucose‐6‐phosphate dehydrogenase. In Schizosaccharomyces pombe carbamoylphosphate synthetase is particulate too. Density gradient centrifugation of a particulate fraction of Schizosaccharomyces pombe suggests that ornithine carbamoyltransferase and carbamoylphosphate synthetase are associated with the mitochondria.

In Saccharomyces cerevisiae and in some other yeasts [Messenguy & Wiame (1969) FEBS Lett. 3, 47–49; Wiame (1974) Proc. 9th Meet. Fed. Eur. Biochem. Soc., pp. 161–177] arginase binds to ornithine carbamoyltransferase in the presence of arginine and ornithine, thereby inhibiting its activity but leaving arginase activity unmodified. This regulation by reversible stoechiometric interaction between the two enzymes prevents recycling of ornithine to arginine when the latter is used as a source of nitrogen. Results presented here show a correlation between the shift of ornithine carbamoyltransferase and carbamoylphosphate synthetase from mitochondrial to extramitochondrial fraction and the existence of arginase/ornithine carbamoyltransferase regulatory interaction. This regulation and extramitochondrial ornithine carbamoyltransferase are absent from obligate aerobe yeasts and may reflect an adaptation to the fermentative type of energy production.
Enzyme Activities in Different Cell Compartments of the Involuting Rat Myometrium
Authors: GEYER Helmut , MÜLLER Ulrich , AFTING Ernst‐Günter
Published: March 2005

Marker enzymes for various intracellular fractions were measured in rat uterus myometrium during the post‐partum involution. Following a lag phase of 48 h after delivery, the activities of the cytoplasmic enzymes aldolase, glucose‐6‐phosphate dehydrogenase and creatine kinase, decreased at equal rates. The plasma membrane marker 5′‐nucleotidase, again after a lag phase of 48 h, showed a decrease in activity which was parallel to that of the cytoplasmic enzymes when allowance was made for the geometry of the cell.

The mitochondrial glutamate dehydrogenase and succinate dehydrogenase activities decreased in parallel together after a lag phase of 72 h. The significantly longer lag phase of the mitochondrial enzymes can be explained in terms of the importance of the mitochondria as a source of the energy which is necessary for intracellular protein degradation.

The activity of the lysosomal acid phosphatase showed no significant decrease until the involution of the tissue was essentially complete.

The activities of cytosol, mitochondria and plasma membrane enzymes all fell during involution to levels below those measured in non‐pregnant control animals. This can be explained by the absence of estrogenic stimulation of the uterus during the post‐partum period, which results from suppression of ovarian function by lactation.
Negatively Charged Reactants as Probes in the Study of the Essential Mercaptide‐Imidazolium Ion‐Pair of Thiolenzymes
Authors: HALÁSZ Piroska , POLGÁR László
Published: March 2005

The reactive mercaptide‐imidazolium ion‐pair at the active site of papain and thiolsubtilisin was alkylated with negatively charged reactants. The reactivities of the two thiolenzymes differ considerably. The iodoacetate reaction is faster by more than 1000 times with papain than with thiolsubtilisin. On the other hand, towards 3‐iodopropionate thiolsubtilisin is more reactive than papain by about a factor of 7. These findings, which are interpreted in terms of the different geometries of the two ion‐pairs, offer an explanation of the basic difference between the catalytic abilities of papain and thiolsubtilisin.
The Mechanism by which Heparin Stimulates Transcription in Isolated Rat Liver Nuclei
Authors: COUPAR Barbara E. H. , CHESTERTON C. James
Published: March 2005

The polyanion heparin, whilst inhibiting free RNA polymerase, stimulates the rate of RNA synthesis in eucaryotic nuclei; it has been suggested that this process involves either the inactivation of R Nase or the activation of blocked transcription complexes. These hypotheses have been investigated in isolated rat liver nuclei at 22 °C. We confirm that heparin inhibits free form A and B RNA polymerases but has little effect on the transcribing enzymes. The sensitivity of these enzymes to α‐amanitin is unchanged by the agent and the stimulation is dependent on the presence of (NH4)2SO4 and Mn2+ ions. The size of the RNA product was analysed on denaturing formamide sucrose gradients. It shows only a small increase when heparin is included in the incubation. In addition, no significant size changes are observed if the incubation is continued in the presence of actinomycin D and α‐amanitin which together inhibit 97% of all RNA synthesis. These results show that inactivation of R Nase cannot be an explanation of the stimulatory effect in this system.

Polynucleotide elongation rates and numbers of transcribing RNA polymerase molecules were determined by alkaline degradation of RNA labelled from [3H]UTP during 1‐min incubations. The [3H]UMP and [3H]uridine, derived from internal nucleotides and 3′‐terminal nucleosides respectively, were separated by thin‐layer chromatography and their tritium content measured. From this data the above parameters can be calculated. We show that the activity of R Nase and phosphatase do not significantly affect the data and that levels of endogenous UTP are negligible. Data from eight separate experiments show that isolated rat liver nuclei contain 25–35×103 transcribing RNA polymerase molecules per diploid genome comprising 18–20×103 form A and 5–15×103 form B. Form C was not detected. Addition of heparin has no effect on these values. Polyribonucleotide elongation rates in nuclei are approximately 0.15 and 0.4 nucleotide per second for form A and B respectively. In the presence of heparin the latter rate is increased by 2–4‐fold but that of the form A enzyme remains essentially unchanged. Similar results were obtained using [3H]GTP in place of [3H]UTP indicating that polyribonucleotide termination was random. Our findings show that heparin exerts its stimulatory effect by increasing the form‐B‐mediated polyribonucleotide elongation rate. It does not activate previously inactive RNA polymerase molecules.
The Calcium‐Induced and Sodium‐Induced Effluxes of Calcium from Heart Mitochondria
Authors: CROMPTON Martin , KÜNZI Marianne , CARAFOLI Ernesto
Published: March 2005

Addition of Na+, Li+, Ca2+ or Sr2+ induces efflux of the intramitochondrial 45Ca2+ from rat heart mitochondria. These reactions are inhibited by La3+ but not by ruthenium red. The ability of La3+ to inhibit has been exploited to develop an inhibitor‐stop technique to study the effluxes of 45Ca2+ induced by Ca2+ and other cations. The Ca2+‐induced efflux of 45Ca2+ shows Michaelis‐Menten kinetics (Km, 13 μM) and is attributable to a Ca2+‐Ca2+ exchange across the inner mitochondrial membrane with a stoichiometry of 1:1. The Ca2+‐Ca2+ exchange and the Na+‐induced efflux of Ca2+ show similar sensitivities to inhibition by La3+. Moreover, the Ca2+‐Ca2+ exchange is inhibited by Na+. The Na+‐dependent efflux of Ca2+ is stimulated by mitochondrial energisation without changing the affinity of the system for Na+ or Ca2+. In contrast, the Ca2+‐Ca2+ exchange is not influenced by the mitochondrial energy state. The data agree with the existence of an exchange‐diffusion carrier in the inner membrane able to catalyse an exchange between intramitochondrial Ca2+ and extramitochondrial Ca2+, Sr2+, Na+ and Li+. The energy‐dependence of the exchanges is discussed.
Isolation and Characterization of Presynaptically Acting Neurotoxins from the Venom of Bungarus Snakes
Authors: ABE Teruo , ALEMÀ Stefano , MILEDI Ricardo
Published: March 2005

1. Five presynaptic toxins have been isolated in pure from from the venom of Bungarus multicinctus and Bungarus caeruleus and named β1, β2, β3, β4, and β‐ceruleotoxin.

2. They differ in electrophoretic mobility and amino acid composition, while all have the same molecular weight (22000) and are composed of two subunits of molecular weight 9000 and 12000.

3. The toxins have phospholipase A activity when assayed with both natural and synthetic phospholipids, and this activity requires the presence of Ca2+ ions.

4. β‐Bungarotoxin (β3) binds 1 mol of Ca2+ per mol of protein and this binding induces a conformational change as detected by fluorescence measurements in the presence of the dye 8‐anilino‐1‐naphthalene sulfonic acid.

5. The phospholipase activity of all the toxins is lost when a critical histidine residue is modified with p‐bromophenancyl bromide.

6. As a result of the modification the lethality of the toxins is greatly reduced.

7. Native toxin causes a rapid decrease in amplitude of end‐plate potentials, followed by a transient increase and subsequent decrease, until transmitter release is completely abolished. The modified toxin still causes the early decrease in release but toxin action does not progress to complete block.

8. The rate of blockage of transmitter release by native toxin is reduced in the presence of modified toxin.

9. It is concluded that phospholipase activity plays an important role in the action of this class of toxins at the neuromuscular junction.
The Effect of Various Substituted p‐Benzoquinones on Electron Transport in Spinach Chloroplasts
Authors: BARR Rita , CRANE Frederick L. , BEYER Gail , MAXWELL Larry A. , FOLKERS Karl
Published: March 2005

Four different substituted p‐benzoquinones resembling the natural chloroplast quinone, plastoquinone A, in structure [2,3‐dimethyl‐5‐bromo‐6‐n‐octadecylmercapto‐1,4‐benzoquinone (compound 6712); 2,3‐dimethyl‐5‐n‐propylamino‐1,4‐benzoquinone (compound 6711); 2.3‐dimethyl‐5‐oleylamino‐1,4‐bezoquinone (compound 6710); and 2.3‐dimethyl‐5‐n‐hexadecylamino‐1,4‐benzoquinone (compound 6709)] were tested for their effect on electron transport in spinach chloroplasts. 2,4‐Dimethyl‐p‐benzoquinone reduction by photosystem II was inhibited best by compound 6712 (I50= 27 μg/ml, or 54 μM conc.), followed by compounds 6709 and 6710 (I50= 63 μg/ml or 125 μM conc. of either); compound 6711, a short‐chain analog, was least effective (I50= 75 μg/ml or 0.3 mM conc.). (I50 is the dose which inhibits 50%.) Inhibition by compounds 6712 and 6710 could be reversed by the addition of exogenous plastoquinone A indicating that these two benzoquinones interfered with the reduction of plastoquinone A before the well‐known 2,5‐dibromo‐3‐methyl‐6‐isopropyl‐p‐benzoquine inhibition site between the two photosystems (Trebst and Reimer, 1973, Z. Naturforsch. 28c, 710). A negative synergistic effect between exogenous plastoquinone A and the four benzoquinones studied was observed on silicomolybdate reduction by photosystem II in the presence of 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea. Another benzoquinone inhibition site, not reversed by exogenous plastoquinone A. occurred in photosystem I on the ascorbate plus N,N,N′,N′‐tetramethyl‐p‐phenylenediamine → methylviologen pathway. The relevance of these benzoquinone inhibition sites to photosynthetic electron transport is discussed.
Glutathione Reductase from Human Erythrocytes
Authors: KROHNE‐EHRICH Gisela , SCHIRMER R. Heiner , UNTUCHT‐GRAU Renate
Published: March 2005

1. From 90 l erythrocytes 200 mg glutathione reductase was purified in four steps with an overall yield of 40%. The specific activity rose from 3.71 U glutathione reductase per g hemoglobin in the erythrocytes to 235 U/mg protein in the isolated enzyme. The purification procedure which was completed in 5 days, included (a) denaturation of hemoglobin by a mixture of butan‐1‐ol and chloroform [Scott, E. M. (1976) Prep. Biochem. 6, 147–152], (b) precipitation of glutathione reductase with acetone, (c) extraction of the precipitate and fractionation of the extract with ammonium sulfate, (d) affinity chromatography on adenosine‐2′,5′‐bisphosphate–Sepharose 4B.

2. The 280/460 nm absorbance ratio was found to be 5.9 for the purified enzyme; all other studies on physical and chemical properties of the enzyme confirmed the results of Worthington and Rosemeyer [Eur. J. Biochem. 67, 231–238 (1976) and earlier papers].

3. The new purification procedure provided crystals suitable for X‐ray diffraction analysis at high resolution. Furthermore sufficient amounts for sequencing the enzyme were isolated. As a first step in the determination of the protein's primary structure, CNBr‐produced fragments of the carboxymethylated enzyme were fractionated.

4. A chain segment containing the catalytic disulfide of the native protein was isolated. Its sequence was determined to be . An identical sequence is present in yeast glutathione reductase, and lipoamide dehydrogenases contain an active site peptide with very similar sequence [Williams, C. H. Jr(1976) The Enzymes 13, 89–173]. Thus the long‐recognized mechanistic similarities between glutathione reductases and lipoamide dehydrogenases are reinforced by sequence similarities around the two redox‐active cysteine residues. The sequenced regions are, however, too short to allow speculations on phylogenetic relationships within this category of proteins.
Studies by Small‐Angle X‐Ray Scattering of the Quaternary Structure of the β‐Haemocyanin of Helix pomatia
Authors: BERGER Johann , PILZ Ingrid , WITTERS Raphaël , LONTIE René
Published: March 2005

Helix pomatiaβ‐haemocyanin was studied in solution by small‐angle X‐ray scattering. The following molecular parameters were determined: molecular weight = 9.02 × 106, volume = 14000 nm3, radius of gyration = 18.4 nm, radius of the spherical subunits = 2.5 ± 0.2 nm.

With these data, and with information of dissociation products in a former paper, a model of the molecule was built whose theoretical scattering curve showed good agreement with the experimental one. The model consists of 160 spherical subunits of a radius of 2.5 nm; 12 rings each built up of 10 spheres form the outer wall of a hollow cylinder; 20 subunits are situated at the inner side of each end.
Separation of Saccharomyces cerevisiae tRNAs on Two‐Dimensional Polyacrylamide Gels as Applied to Investigations on the Mutational Alterations of tRNA that Produce Nonsense Suppressors
Authors: PIPER Peter W. , WASSERSTEIN Marilyn
Published: March 2005

Nonsense suppressors of the yeast Saccharomyces cerevisiae have been found that cause the translational suppression of UAA (ochre) or UAG (amber) nonsense mutants of iso‐1‐cytochrome c by inserting tyrosine, serine or leucine at the site of the nonsense codon. This report describes the fractionation of tyrosine, serine and leucine tRNAs from yeast on two‐dimensional polyacrylamide gels, as well as a partial characterisation of these species. There is only one tyrosine tRNA in yeast, whilst the serine and leucine tRNAs identified in this study can between them decode all the serine and leucine codons which differ in only one base from either UAA or UAG. We did not characterise those serine and leucine tRNAs whose cognate codons differ in more than one base from the amber or ochre codon.

Separation of the tRNA of individual yeast strains on two‐dimensional gels can be used in conjunction with RNA fingerprinting techniques to investigate whether the amber or ochre suppressor mutations of yeast determine structural changes in the tyrosine, serine or leucine tRNAs. This approach has already allowed an elucidation of the tRNATyr structural change determined by tyrosine‐inserting amber suppressor mutations at the SUP5 locus. Although it has, to date, only been successfully applied to the sequencing of yeast amber suppressors, it has also a potential application in structural studies of ochre suppressors. One of the leucine tRNAs analysed in this study contains an unidentified modified nucleotide in its anticodon, a minor component of interest as it may also be present in the anticodon of yeast ochre suppressor tRNAs and cause the restrictive recognition of UAA but not UAG codons. The possible anticodon structures of yeast ochre‐suppressing tRNAs are discussed in the light of present knowledge of codon‐anticodon pairing.
Inhibition of Fructose‐1,6‐bisphosphate Aldolase from Rabbit Muscle and Bacillus stearothermophilus
Authors: LEWIS Dennis J. , LOWE Gordon
Published: March 2005

Phosphoglycollohydroxamic acid and phosphoglycollamide are inhibitors of rabbit muscle fructose‐1,6‐bisphosphate aldolase. The binding dissociation constants determined by enzyme inhibition and protein fluorescence quenching suggest that two distinct enzyme inhibitor complexes may be formed.

The binding dissociation constants of the two inhibitors to Bacillus stearothermophilus cobalt(II) fructose‐1,6‐bisphosphate aldolase have also been determined. The hydroxamic acid is an exceptionally potent inhibitor (Ki= 1.2 nM) probably due to direct chelation with Co(II) at the active site. The inhibition, however, is time‐dependant and the association and dissociation constants have been estimated.

Ethyl phosphoglycollate irreversibly inhibits rabbit muscle fructose‐1,6‐bisphosphate aldolase in the presence of sodium borohydride, presumably by forming a stable secondary amine through the active‐site lysine residue.

A new condensation assay for fructose‐1,6‐bisphosphate aldolases has been developed which is more sensitive than currently used assay procedures.
Structure of the Cell Wall of Bacillus Species C.I.P. 76‐111
Authors: LEDUC Mireille , ROUSSEAU Micheline , van HEIJENOORT Jean
Published: March 2005

An unusual type of bacterial cell wall was encountered in a Bacillus strain referred to as Bacillus sp. C.I.P. 76‐111. The major constiuent of this cell wall is a high‐molecular‐weight anionic protein non‐covalently associated to a peptidoglycan‐polysaccharide complex. The cell wall appeared as a multilayered structure when sections of whole cells or of isolated cell walls fixed with glutaraldehyde and postfixed with osmium tetroxide were examined by electron microscopy. The correlation between the observed morphological features and the biochemical data suggested that a thin central electron‐dense layer identifiable with the peptidoglycan‐polysaccharide complex is located between two similar thick layers of protein. Furthermore, negative staining of isolated cell walls revealed that the outer protein layer has a regular surface array of subunits with hexagonal symmetry. Several structural properties of the cell wall peptidoglycan were investigated and were found to resemble those of other bacilli. Further characterization of the autolytic system showed that an N‐acetylmuramyl‐L‐alanine amidase and a glycosidase, presumably a N‐acetylmuramidase, were associated with the peptidoglycan‐polysaccharide complex. It was also established that this strain is devoid of membrane teichoic acid.
Differences in the Modulations of the Soluble or Plasma‐Membrane‐Bound 5′‐Nucleotidase
Authors: LELIEVRE Lionel , ZACHOWSKI Alain , MAGET‐DANA Régine , AUBRY Jacques , JONKMAN‐BARK Gitty
Published: March 2005

The treatment of plasma membranes by a French pressure cell in sucrose medium devoid of detergents solubilized 20% of the total protein and 95–100% of 5′‐nucleotidase activity. The soluble enzyme was 40–90‐fold purified by centrifugation in a sucrose gradient with a 10–20% yield with respect to the original lysate.

The purified fraction retained the same high specificity for 5′‐AMP (Km= 20 μM) as in the plasma membranes and was enriched in sphingomyelin. Whereas 5′‐AMP at high concentration inhibited the membrane‐bound enzyme, it had no effect on the solubilized form. The soluble enzyme was stimulated by 2 × 10−13–2 × 10−15 g concanavalin A without any inhibition with higher doses of lectin. The plasma‐membrane‐bound stimulated and inhibited 5′‐nucleotidase was modulated by concanavalin A concentrations higher than 0.1 μg. Inhibition of the activity of the soluble enzyme by antiphosphorylcholine antibodies was not observed with membranes. The regulation of 5′‐nucleotidase activity in plasma membranes might be associated with a supramolecular organization.
Large‐Scale Purification of the Acetylcholine‐Receptor Protein in Its Membrane‐Bound and Detergent‐Extracted Forms from Torpedo marmorata Electric Organ
Authors: SOBEL André , WEBER Michel , CHANGEUX Jean‐Pierre
Published: March 2005

A method is described for the large‐scale purification of membrane fragments very rich in acetylcholine (nicotinic) receptor from the electric organ of Torpedo marmorata. The preparations of purified membrane fragments have a specific activity of more than 4000 nmol α‐toxin binding sites/g protein and give only four main polypeptide bands by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Observations by electron microscopy show that the purified preparation of receptor‐rich membrane fragments is composed of only one class of membrane fragments covered with 8‐nm rosettes identified as acetylcholine receptor molecules.

This preparation is used as a starting material for the detergent solubilization and the large‐scale purification of the acetylcholine receptor protein, without using affinity chromatography. A sucrose gradient centrifugation of a Triton X‐100 extract of receptor‐rich membranes done in the presence of 2‐mercaptoethanol yields large quantities of receptor protein in a homogeneous form as indicated by polyacrylamide gel electrophoresis, isoelectric focussing and electron microscopy.

Polyacrylamide gel electrophoresis of the purified protein in the presence of sodium dodecylsulfate reveals three bands of apparent molecular weights 40000 ± 1000, 50000 ± 2000 and 66000 ± 2000, the two heaviest ones being present in significantly lower amounts than the 40000‐Mr one. The comparison of several samples of purified protein with different specific activities reveals a striking variability of the ratios of the 40000‐Mr band to the 50000 and 66000‐Mr ones.
Sugar‐Specific Antibodies Reactive towards Cell‐Surface Carbohydrates
Authors: BLOCH Robert , MACCECCHINI Maria Luisa , JUMBLATT James , BUTTRICK Peter , BURGER Max M.
Published: March 2005

Antibodies generated against sugar‐azoproteins have been purified by affinity chromatography using elution with monosaccharide. They have high specificity for monosaccharide and they react with terminal, non‐reducing sugars on the surfaces of human erythrocytes. Some of them, either native or as Fab fragments, inhibit mitogenic stimulation of lymphocytes by concanavalin A.
Topology of the Peptides in Free and Membrane‐Bound Complex III (Ubiquinol — Cytochrome c Reductase) as Revealed by Lactoperoxidase and p‐Diazoniumbenzene [35S] sulfonate Labeling
Authors: GELLERFORS Pär , NELSON B. Dean
Published: March 2005

The arrangement of the peptides of free complex III and complex III incorporated into phospholipid vesicles was investigated by labeling with 125I + lactoperoxidase and with p‐diazonium‐benzene [35S]sulfonate (N = N – PhSO3H). The peptides were separated by sodium dodecylsulfate acrylamide gel electrophoresis and scanned for radioactivity. In free complex III, the most extensive iodination occurred in peptides II and IV, whereas all polypeptides (I – VI), except peptide VII, could be coupled to N = N – PhSO3H. Peptide VII was, however, labeled in the sodium‐dodecyl‐sulfate‐dissociated enzyme. These results show that, with the possible exception of peptide VII, all peptides are at least partially exposed on the surface of the isolated complex. Complex III incorporated into phospholipid vesicles showed a labeling pattern which differed from the free enzyme, both with respect to the total incorporation and to the relative incorporation between the peptides. Total incorporation of N = N – Ph35SO3H and 125I was decreased by 60 – 70% in complex III‐vesicles, suggesting that a large number of the binding sites on the free enzyme become buried within the membrane. In addition, peptides I, II, IV and V were relatively less labeled in complex III‐vesicles than were peptides III and VI, suggesting a deeper location of the former peptides within the membrane. This is supported by the finding that labeling of deoxycholate‐treated complex III‐vesicles with N = N – Ph35SO3H results in an incorporation pattern similar to that in free complex III. Binding of N = N – PhSO3H and its effects on enzymatic activity was also studied in free and membrane‐bound complex III. Both the binding of N = N – Ph35SO3H and inactivation of duroquinol – cytochrome c reductase were significantly slower in complex III‐vesicles. These results indicate that many of the N = N – PhCO3H‐binding sites on the free complex are inaccessible when the enzyme is incorporated into a vesicle. This approach was also used to ascertain intactness of the vesicles, and to show that N = N – PhSO3H labeled only those peptides exposed on the outer surface.
Kinetic Studies of the Interaction between Rifampicin and DNA‐Dependent RNA Polymerase of Escherichia coli
Authors: WEHRLI Walter
Published: March 2005

Rifampicin specifically inhibits bacterial RNA polymerase by forming a tight complex with the enzyme in a stoichiometric ratio of 1:1. In the present work, dextran‐coated charcoal was used as a tool to quantify the kinetic rate constants of the association and dissociation of this complex. With this simple and sensitive assay, the rate of dissocaition (kd) of the enzyme · antibiotic complex was shown to follow first‐order kinetics, with a half‐life of 12 h at 0°C, which decreases to 9 min at 37°C. The rate of association follows second‐order kinetics, with a rate constant (ka) of 6 × 104 M−1 s−1 at 0°C, which increases to 1.2 × 106 M−1 s−1 at 37°C. From the kd/ka ratio, the equilibrium constant K has been calculated to be 3.5 × 10−10 M at 0°C and 10−9 M at 37°C. The physical nature of the bonds involved in the rifampicin · RNA‐polymerase complex seems to be mainly lipophilic, since high ionic strength had no influence on ka and even decreased kd, whereas dimethylsulfoxide (Me2SO), a less polar solvent than water, decreased ka and increased kd, resulting in an up to 20 times higher K. The close correlation between binding of rifampicin to RNA polymerase and enzyme inhibition by the drug can be demonstrated directly: at ascending concentrations of Me2SO there is a good correlation between the rise in K and the increase in the amount of rifampicin required for 50% enzyme inhibition.
Effects of Chloroquine and Colchicine on the Degradation of Chyle Cholesteryl Ester and Phospholipids in vivo
Authors: FLORÉN Claes‐Henrik , NORDGREN Hans , NILSSON Åke
Published: March 2005

1. Both chloroquine and cholchicine given intraperitoneally delayed the hydrolysis of chyle cholesteryl ester after the uptake by the liver. The cholesteryl ester radioactivity per mg protein of hepatocytes isolated from the drug‐treated rats was 97.2 and 96.5%, respectively, of that in the intact liver. The decrease could be accounted for by hydrolysis of cholesteryl ester during cell isolation. Most of the unhydrolyzed cholesteryl ester was thus intracellular or firmly bound to the cells. Also in perfused rat livers chloroquine inhibited the hydrolysis of chyle cholesteryl ester that had been taken up in vivo.

2. After subcellular fractionation of livers from chloroquine‐treated rats, that had been injected with cholesterol‐labelled chyle 60 min earlier, 55% of the radioactive cholesteryl ester was in the large granule fractions (mitochondrial plus light mitochondrial pellet). The 600 ×g and 100000 ×g pellets contained a higher proportion of the unesterified radioactive cholesterol. Similar findings were made with colchicine‐treated animals.

3. 60 min after injection of chyle labelled with 32P and [3H]cholesterol only 6% of the injected [32P]phospholipid radioactivity was in the liver in normal rats and 7.3% and 7.8% in the liver of rats treated with chloroquine and colchicine, respectively. Whereas the cholesteryl ester radioactivity in the large granule fractions in rats treated with chloroquine and colchicine was increased by 2.4 and 3.1 times, there was no such increase in the 32P radioactivity of any of the phospholipid classes.

4. Three conclusions are drawn. Chylomicron remnant cholesteryl esters are likely to be interiorized by the hepatocytes before their degradation. The data are compatible with a role of the acid cholesterol esterase in their hydrolysis. There was no positive evidence that any significant part of the chylomicron phospholipids are taken up by the liver and metabolized by lysosomal enzymes.